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1 CENTRAL REGULATION OF BLOOD PRESSURE IN HYPERTENSION By JOO YUN JUN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Joo Yun Jun
3 To my parents and husband, for their unconditional love and support throughout the years
4 ACKNOWLEDGMENTS First of all, I would like to thank my mentor, Dr. Mohan R aizada, who has provided me wonderful training and enlightening experiences for my PhD I have come to respect Mohans brilliance as a scientist as well as his knowledge and understanding of things beyond. He also carries a great sense of humor with him, which has made my graduate study much enjoyable and valuable. I also appreciate the immense patience he has exercised with me through the years and the support he has provided as an advisor. He has been an amazing teacher, leader and motivator in my graduate years. Moreove r, he taught me a lot about knowledge of life and it is always pleasure to have a chat with him. I would also like to acknowledge my committee members Dr. Sumners, Dr. Katovich, Dr. Waters and also lab members Dr. Zubcevic, Dr. Shan, Dr. Shi, Dr. Shenoy, D r. Bradford, Jessica Marulanda, Fan Lin Alish a Dunn, Sara Croft for offering their expertise, suggestions and support through my graduate study. Most importantly, I cannot express enough gratitude to my parents and my two sisters who have supported me throughout these school years M y husband Arnold for helping me finish all the work with his experiences. I could never have come this far without them.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 NEUROGENIC HYPERTENSION ................................ ................................ .......... 12 Central Regulation of Blood Pressure ................................ ................................ ..... 12 Oxidative Stress and Inflammation in Neurogenic Hypertension ............................ 15 Endothelial Progenitor Cells and Vascular Dysfunction in Hypertension and Cardiovascular Diseases ................................ ................................ ..................... 19 2 ROLE OF BRAIN MITOCHONDRIAL OXIDATIVE STRESS IN NEUROGENIC HYPERTENSION ................................ ................................ ................................ ... 24 Increased Oxidative Stress in Neural Mechanisms of Hypertension ....................... 24 Role of Brain Mitochondrial ROS in Neurogenic Hypertension ............................... 25 Methods ................................ ................................ ................................ .................. 26 Animal ................................ ................................ ................................ .............. 26 Primary Neuronal Culture ................................ ................................ ................. 26 Measurement of ROS Production ................................ ................................ ..... 27 Te lemetry Recordings of Arterial Pressure and Heart Rate .............................. 27 Measurement of Full Spectral Analysis ................................ ............................ 28 Implantation of Subcutaneous Osmotic Minipumps ................................ .......... 28 Intracerebroventricular mitoTEMPO Infusion ................................ .................... 29 Immunohistochemistry ................................ ................................ ...................... 29 Cardiac Pathology ................................ ................................ ............................ 30 RNA Isolation and Real Time PCR ................................ ................................ .. 30 Results ................................ ................................ ................................ .................... 31 ICV Infusion of mitoTEMPO Attenuates Ang II induced Neurogenic Hypertension ................................ ................................ ................................ 31 mitoTEMPO Scavenges Ang II induced Mitochondria ROS in Neuronal Cells in Primary Cultures ................................ ................................ ............... 32 Central Mitochondrial Superoxide Inhibition Influences Autonomic Nerve Activity in AngII induced Neurogenic Hypertension ................................ ....... 32 IC V mitoTEMPO Inhibits AngII induced Microglia Activation in the PVN .......... 33 ICV mitoTEMPO Inhibits Ang II induced Increase in NADPH Oxidase mRNA and Decrease in nNOS mRNA in the PVN ................................ ........ 34 ICV mitoTEMPO Prevents Ang II induced Cardiac Hypertrophy and Interstitial Fibrosis ................................ ................................ ......................... 34 Discussion ................................ ................................ ................................ .............. 35
6 3 BRAIN MEDIATED DYSREGULATION OF THE BONE MARROW ACTIVITY IN NEUROGENIC HYPERTENSION ................................ ................................ ...... 48 Inflammation and Endothelial Progenitor Cells in Neurogenic Hypertension .......... 48 Methods ................................ ................................ ................................ .................. 50 Animal ................................ ................................ ................................ .............. 50 MNC Isolation from Blood and BM ................................ ................................ ... 51 Isolation of EPCs from MNCs ................................ ................................ ........... 51 Direct Flow Cytometry (FACS) Analysis ................................ ........................... 52 DiLDL and Lectin Staining ................................ ................................ ................ 53 Results ................................ ................................ ................................ .................... 53 Dysfunctional Endothelial Progenitor Cells in Chronic Ang II induced Hypertension ................................ ................................ ................................ 53 BM derived EPCs are dysfunctional in Ang II induced hypertension ......... 53 Tube formation ability of cultured MNCs from Ang II infusion rats is diminished ................................ ................................ ............................... 54 Ang II induced Imbalance of EPCs and ICs ................................ ..................... 54 BM derived EPCs are reduced by chronic Ang II infusion ......................... 54 Inflammatory cells are elevated in the BM and circulation by chron ic Ang II infusion ................................ ................................ ......................... 55 MitoTEMPO ICV Treatment Inhibits Elevated BM Inflammatory Cells and Reduced EPCs ................................ ................................ .............................. 55 Discussion ................................ ................................ ................................ .............. 56 4 ROLE OF NDUFA10 IN NEUROGENIC HYPERTENSION ................................ .... 65 Proteomic Approach of Hypertension Utilizing Spontaneous Hypertensive Rat ..... 65 Methods ................................ ................................ ................................ .................. 66 Animal ................................ ................................ ................................ .............. 66 Primary Neuronal Culture ................................ ................................ ................. 67 Western Blot Assay ................................ ................................ .......................... 67 2D Gel and Ima ge Analysis ................................ ................................ .............. 68 Ndufa10 Overexpression ................................ ................................ .................. 68 Measurement of Mitochondrial and Cellular ROS Production .......................... 69 Data and Statistical Analysis ................................ ................................ ............ 69 Results ................................ ................................ ................................ .................... 70 2D Gel Analysis Revealed Differential Protein Expressions in PVN of WKY Rat and SHR ................................ ................................ ................................ 70 Ndufa10 Expression is Increased in Cardiovascular Regulatory Regions of SHR Compared to WKY ................................ ................................ ................ 71 N dufa 10 Over expression Induces Cellular and Mitochondrial Oxidative Stre ss in HEK 293 Cells ................................ ................................ ................ 72 Discussion ................................ ................................ ................................ .............. 72 5 CONCLUDING REMARKS AND FUTURE DIRECTIONS ................................ ...... 84 LIST OF REFERENCES ................................ ................................ ............................... 91
7 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 104
8 LIST OF FIGURES Figure page 1 1 Central nervous system mediated circulatory regulation.. ................................ .. 21 1 2 Hypothalamic and brain stem areas in cardiovascular regulatory brain regions. ................................ ................................ ................................ ............... 22 1 3 Neuronal mechanisms of blood pressure regulation in the brain. ....................... 23 2 1 Animal experimental design.. ................................ ................................ .............. 38 2 2 The chemical str uctures of mitoTEMPO and TEMPOL ................................ ...... 39 2 3 The representati ve software image of spectral analysis using Hey Presto. ........ 40 2 4 Effects of mitoTEMPO on Ang II Induced Hypertension and heart rate. ............. 41 2 5 Scavenging of AngII induced superoxide in mitoTEMPO treated neurons. ........ 42 2 6 Effects of ICV mitoTEMPO on autonomic nerve activity in Ang II induced neurogenic hypertension. ................................ ................................ ................... 43 2 7 Effects of ICV mitoTEMPO on microglia activation and cytokine mRNA in the PVN.. ................................ ................................ ................................ .................. 44 2 8 Effects of ICV mitoTEMPO on NADPH oxidase and nNOS mRNA. ................... 45 2 9 Effects of ICV mitoTEMPO on cardiac hypertrophy and myocyte diameter. ....... 46 2 10 Effects of ICV mitoTEMPO on interstitial fibrosis of left ventricles. ..................... 47 3 1 DiLDL uptake and lectin binding of CD90 + /CD4 5 8 EPCs. ................................ 59 3 2 Dysfunctional BM EPCs by chronic Ang II infusion. ................................ ............ 60 3 3 Decreased ability of tube formation by chronic Ang II infusion. .......................... 61 3 4 Reduced number of BM EPCs in Ang II induced hypertensive rats. ................... 62 3 5 Elevated ICs and imbalance of EPC/IC in Ang II induced hypertension. ............ 63 3 6 Effects of mitoTEMPO ICV treatment on BM EPCs and ICs in Ang II induced hypertension. ................................ ................................ ................................ ...... 64 4 1 Experimental design of 2D gel analysis.. ................................ ............................ 76 4 2 Representative 2D DIGE gel from PVN of SHR. ................................ ................ 77
9 4 3 The list of differentially expressed proteins from PVN of SHR compared to WKY. ................................ ................................ ................................ .................. 78 4 4 Distribution of Ndufa10 in cardiovascular relevant brain regions of WKY and SHR. ................................ ................................ ................................ ................... 79 4 5 Increased expression of Ndufa10 mRNA and protein in SHR compared to WKY. ................................ ................................ ................................ .................. 80 4 6 ncreased expression of Ndufa10 in cultured neurons from SHR compared to neurons from WKY.. ................................ ................................ ........................... 81 4 7 Ndufa10 overexpression vector and its effi ciency. ................................ .............. 82 4 8 Increased cellular and mitochondrial oxidative stress by Ndufa10 overexpression. ................................ ................................ ................................ .. 83 5 1 AngII AT 1 R mediated ROS production in neurons and glia cells. ....................... 88 5 2 Proposed hypothesis of activated microglia neuron astrocyte interaction in the CNS of hypertension. ................................ ................................ ................... 89 5 3 Proposed regulatory mechanism of neural microglial BM connection in neurogenic hypertension ................................ ................................ ................... 90
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CENTRAL REGULATION OF BLOOD PRESSURE IN HYPERTENSION By Joo Yun Jun May 2012 Chair: Mohan K. Raizada Major: Medical Sciences Physiology and Pharmacology Hypertension is the underlying pathology in many cardiovascular diseases including stroke and renal failure. In past decades, neurogenic component of hypertension, defined as high blood pressure with elevated sympathet ic drive, and angiotensin II (Ang II) mediated oxidative stress, have been suggested as major contributors in the pathogenesis of hypertension. We hypothesized that i. oxidative stress is primarily generated by mitochondria in the brain that contributes to Ang II induced neurogenic hypertension via the increase in sympathetic drive ii. there is a neural bone marrow (BM) connection that regulates endothelial progenitor cells (EPCs) and inflammato ry cells (ICs) in hypertension iii. an increased expression of Ndufa10, a subunit of mitochondrial electron transport chain in cardiovascular (CV) brain regions of the spontaneously hypertensive rat (SHR) is genetically linked to the development of neurogenic hypertension. First we observed beneficial effects of mitoTEMPO, a sc avenger of mitochondrial superoxide, on hypertension, autonomic function, BM derived EPCs and ICs. Chronic Ang II infusion in Sprague Dawley (SD) rats resulted in an elevation of blood pressure and sympathetic vasomotor activity, decrease in spontaneous ba roreceptor reflex gain, and increase in activated microglia in the PVN. Intracerebroventricular (ICV)
11 administration of mitoTEMPO attenuated these changes. To further elucidate potential role of central nervous system on EPCs cardiovascular protective fun ction, the numbers of circulating and BM EPCs compared to those of IC were determined. Ang II induced hypertension was associated with ~46% decrease in EPCs and ~250% increase in ICs, resulting in ~5 fold decrease of EPCs/ICs ratio in the BM. ICV mitoTEMPO treatment normalized Ang II induced imbalance of EPC/IC ratio in the BM. Finally, 2D gel analysis and Western blot assay demonstrated a mitochondrial Ndufa10 is ~3 fold up regulated in the PVN of SHR compared to WKY and that is associated with mitochondri al oxidative stress. In summary, these observations demonstrated that brain mitochondrial ROS play an important role in Ang II induced hypertension that is associated with an imbalance in EPC/IC. Increased mitochondrial ROS may be a result of an increased expression of Ndufa10 in the brain. These data suggest that mitochondria ROS mediated imbalance in EPC/IC is responsible for the pathophysiology of hypertension.
12 CHAPTER 1 NEUROGENIC HYPERTENS ION Central Regulation of Blood Pressure Hypertension is a chronic elevation of the blood pressure (BP) that is associated wit h cardiovascular (CV) diseases such as stroke, heart failure, and kidney disease. 1, 2 About 90 95 % of cases are characterized as primary hypertension that has no clear medical cause, and there is no single factor underlying this multi factorial disease. 3 In the past decades, significant progress has been made in the treatment of hypertension using inhibitors of rennin angiotensin system including a ng iotensin converting enzyme (ACE) inhibitors Ang II receptor type 1 ( AT 1 R ) blockers, and diuretics and calcium channel blockers. 4 7 However, there have been extreme difficulties to manage hypertension in about 40% of hypertensive patients. These unresponsive patients frequently exhibit increased sympathetic outflow and dysfunctional bar oreflex 8, 9 D ysregulation of neural signal within the central nervous system (CNS) an d autonomic nervous system (ANS) have been suggested as major c ontributors to the development of hypertension and those are characterized as neurogenic components. 10 12 There are numerous studies linking the increased sympathetic outflow with elevation of BP in rat and human suggesting that neurogen ic hypertension is a cardiovascular disease with dysfunctional ANS 8, 13, 14 T he significance of sympathetic nervous system in the regulati on of BP via the modulation of peripheral vascular tone and cardiac output is well established, however the brain involved mechanism in the pathophysiology of neurogenic hypertension associated with elevated sympathetic nerve activity (SNA) needs to be fur ther investigated.
13 Autonomic nervous system is a powerful modulator of blood pressure, which is controlled by a network of brain nuclei mainly localized in the hypothalamus and brainstem (Figure 1 1) 15 These specialized nuclei include the paraventricular nucleus (PVN) adjacent to the third ventricle in hypothalamus, the subfornical organ (SFO) in the roof of the third ventricle, the organumvasculosum of the lamina termina lis (OVLT) in the forebrain, the nucleus of the solitary tract (NTS), and the rostral ventrolateral medulla (RVLM) in the brainstem (Figu re 1 2 and 1 3 ). 16, 17 The nuclei are activated by not only peripheral circulating Ang II signal but also local activation of brain re nin angiotensin system (RAS). 18 19 The RAS is an enzymatic cascade by which angiotensinogen is cleaved by renin and further cleaved by ACE to produce Ang II R AS is well known systemic BP control mechanism 20 and the potent vasoconstrictive action of Ang II is a main contributor to the development of hypertension 21, 20, 22 23 via the AT 1 R 6, 24, 25 The brain expresses genes that encode all components of the RAS 26 and the central actions of Ang II on B P regulation and fluid homeostasis are mediated via this receptor 27, 28 Accordingly, high densities of this receptor within the brain are distributed in CV regulatory brain regions including the SFO, PVN, RVLM and the NTS 29 30, 31 Systemic infusion of Ang II increases AT 1 R mRNA in the SFO leading high blood pressure, and pharmacological blockade of AT 1 R attenuates the pr essor response to Ang II in the RVLM and PVN. 32, 33 AT 1 R expressing PVN neurons integrate peripheral Ang II signals from circumventricular organs such as SFO where blood brain barrie r are incomplete ( Figure 1 3) 34 Arginine vasopressin (AVP) is synthesized in the PVN and released from posterior pituitary (PP), which increase bl ood volume. These projections of SFO are innervated to PVN where its parvocellular neurons are transmitted to
14 sympathetic preganglionic motor neurons influencing sympathetic activity. 35 RVLM and NTS are in the brainstem area that provides a majo r input for maintaining blood pressure by regulating sympathetic nervous system as shown in Figure 1 1. 36 Increased RAS in t he brainstem may also contribute to alterations in baroreflex function. A fferent baroreceptor input to the NTS manifests its action via directly mediating vagal control of heart rate, and involving a multisynaptic pathway with an excitatory projection from the NTS to the CVLM, a subsequent inhibitory projection to the RVLM, In the NTS, AT 1 R blockade facilitates baroreflex control of heart rate, and conversely, AT 1 R activation by peripheral Ang II depressed both sympathetic and vagal components of baroreflex induced bradycardia, indicating that An g II decreases baroreflex gain. 37 39 The greatest understanding of brain RAS dysfunction in hypertension has been achieved from studies of s pontaneously hypertensive rat ( SHR ), a rat model of primary hypertension In this animal model, the increase in brain angiotensinogen contributes to development of hypertension 40, 41 Increased RAS components within the PVN SFO, and NTS 41 43 as well as increased cellular levels of AT 1 R within the RVLM of SHR versus WKY animals 44 have all been documented Furthermore, studies utilizing pharmacological blockers of the RAS have provided evidence of the brain RAS mediated centrally induced hypertension 45, 46 It was shown that the anti hypertensive effect following the treatment of brain RAS blockers in hypertensive rats is mainly due to a de crease in sympathetic activity 45, 47 suggesting that the mechanism by which increased brain RAS induces hypertension involves increased sympathetic vasomotor tone. In support of this, it has been shown that AT 1 Rs are associated with presympathetic vasomotor neurons in the RVLM 48 and blockade of Ang II signal within
15 the RVLM decreased BP in the SHR but not in the WKY 49 Thus, in contrast to normotensive rats, it appears that AT 1 R stimulation within the CV presympathetic brain areas contributes to the enhancement of sympathoexcitatory activity of the vasomotor neurons in hypertensive rats. This suggested that hypertension can be produced by up regulation of the brain RAS via a mechanism involving enhanced sympathetic outflow. However the contribution of brain RAS to BP regulation in different areas or nuclei, and its precise mechanism in hy pertension is still not fully understood. Additionally t he effect of Ang II on the CV regulatory regions including PVN and RVLM neurons involves several potential mechanisms, for example an increase in r eactive oxygen species (ROS), neuroinflammatory signa ls from microglia, and a decrease in the concentration of NO in the brain. 50, 51 Oxidative S tress and Inflammation in Neurogenic Hypertension Increased o xidative stress in the vasculature, the heart, the kidney, and the brain is associated with cardiovascular disease including hypertension. 52 56 The increased production of cellular oxidative stress by Ang II is well docum ented in the peripheral organs. Ang II ac tivated N ADPH oxidase ( nicotinamide adenine dinucleotide phosphate oxidase) enzyme and subsequent production of cellular ROS were first inve stigated by Griendling et al 57 59 Ang II infusion induced hypertension shows increased vascular superoxide production 60 and adenoviral vector mediated superoxide dismutase (SOD) in neuronal cells scavenged Ang II induced increase in cellular super oxide 61 Also, pharmacological blockade of the NADPH oxidase complex by apocynin (NADPH oxidase i nhibitor) attenuates Ang II induced vascular superoxide production and further prevents hypertension. 62, 63 Among the target organs of hypertensive vascular disease, the brain is most affe cted by oxidative stress. 64, 65 Although there have been many
16 studies regarding target organ damages in hypertension, relat ively few studies have addressed the role of oxidative stress in the activation of the sympathetic nervous system. One of the critical roles of Ang II in the CNS is the activation of NADPH oxidase and the production of ROS resulting in the increase of ce ntral sympathetic outflow. 66, 67 Ang II stimulates cellular ROS production in culture d neurons isolated f rom the cardiovascular relevant brain areas and this was prevented by the Ang II type 1 receptor (AT 1 ) antagonist losartan or superoxide dismutase mimetic TEMPOL treatment 68, 69 In addition to NADPH oxidase induced production of ROS in cytosol, mitochondria are another major source of ROS production in many cell types. 70 74 M itochondria appear to be in the main loop of a vicious cycle of oxidative damage since mitochondria produce the majority of intra cellular oxidants providing main target s of redox sig naling 72, 75 This increase s mitochondria induced cellular oxidative stress and further induces oxidative damages in the organs Therefore the si gnificance of increase d oxidative stress in the CNS and that is involved with mitochondria induced ROS in the brain needs to be examined Recent study from Nozoe et al ha s demonstrated that Ang II increases mitochondrial ROS production in the RVLM, leading to sympathetic activation. 76 Chan et al also demonstrated the role of mitochondrial electron transport chain complex in the RVLM of SHR and found that the impairment of mitochondrial ETC complexes contributes to chronic oxidative stress in the R VLM of SHR, leading to enhanced central sympathetic drive and hypertension. 77 Interestingly studies have demonstrated that Ang II induced mitochondri a derived ROS production perpetuates NADPH oxidase mediated production of ROS. This suggests a feed forward activatio n
17 mechanism of cellular ROS production between NADHP oxidase and mitochondria 78 It is tempting to suggest that NADPH oxidase deri ved ROS first trigger Ca 2+ accumulation within mitochondria and t his subsequently induce mito chondrial superoxide generation which fu rther activates NADPH oxidase and mitochondria However additional studies need to establish a more direct link between AngII NADPH oxidase mitochondria in neuronal redox signaling and the pathophysiology of neurog enic hypertension. Both human and animal studies have provided strong evidence that increase in inflammatory modulators and overall inflammatory status are critical in cardiovascular diseases and hypertension 79 The levels of plasma inflammatory cytokines and other markers of inflammation are increased with the progression of hypertension, and suppressing immune response produces beneficial effects. 80 S tudies have demonstrated that circulating levels of TNF IL6, and adhesion molecules such as P selectin are increased in patients with primary hypertens ion. 81, 82 Inhibitors of the re nin angiotensin system, which are effective antihypertensive treatment in some patients, are shown to decrease C reactive protein, IL6 and TNF levels and inflammation. 83 In animal studies, T cells play an important role in Ang II induced inflammation and vascular dysfunction in hyperte nsion. 84, 85 The SHR also exhibits increased levels of activated monocytes and that is associated with high blood pressure. 86 Ang II promotes leukocyte endothelial interaction contributing to vascular inflammation, along with increases in the expression of cytoki nes IL 6, IL 1 and TNF 87 Although the participation of peripheral cytokines and proinflammatory modulators in hypertens ion has been established there is no clear evidence available for their involvement i n neuroinflammation in neurogenic hypertension Cytokines modulate neuronal activity
18 and receptors for cytokines are found in different cell types in the brain. 88 Re cent study from Cardinale et al demonstrated that bilateral NF kB blockade in the PVN using NK kB decoy oligodeoxynucleotid e and serine mutated inhibitory kB (AdIkB) reduced proinflammatory cytokines such as TNF IL 1 IL 6, MCP 1 and ROS production in Ang II induced hypertension. 89 In addition, nNOS mRNA level was increased by bilateral NF kB inhibition, resulting in increase in NO bioavailability and decrease in plas ma NE levels. 89 NO is a well known sympathoinhibitory neurotransmitter, and the expression of nNOS and plasm a NE are indirect indicator of neuronal activity and sympathetic outflow. Another study provided the evidence that TNF increase Ca 2+ influx int o hippocampal pyramidal neurons 90 and IL 1 has similar effect on neuronal activity that increase Ca 2+ curre nt via phosphorylation of N m ethyl D aspartic acid receptor. 91 These findings indicate that central cytokines levels are associated with sympathetic drive and hypertension. Inflammatory cytokines are p olypeptides and peripherally produced cytokines are not able to cross BBB. However, there are multiple ways of influencing CNS to modulate ca rdiovascular system when cytokines are transported from plasma. As discussed above, brain has a specific area called circumventricular organ (SFO, OVLT AP ), in which the BBB is not present. Variety of signals including AngII and cytokines are transmitted through these areas. Also, CNS produce cytokines locally by activated monocytes, macrophages and activated mictoglia. 88, 92 mRNAs and proteins of IL 6 and IL 1 have been identified in different region of brain including hippocampus and brainstem. 93 It seems that both peripheral cytokines and centrally generated cytokines contribute to the neuroinflammatory activation in neurogenic hypertension. The inflammatory process in the CV regulatory regions of the brain is
19 associated with the modulation of autonomic nervous system and microglia is one of the importa nt cell types for triggering neuro inflammation in the brain. Microglia, as local microphages in the brain are activated and increased in response to brain injury, and observed in the patients with neurodegenerative disease including Alzheimer and Parkinsons disease. 94, 95 Activated microglia produce inflammatory cytokines such as NF kB IL 6, TNF within th e brain 96 and these cytokines are the important stimu la tor s of sympathetic activation. Shi et al has demonstrated that c hronic Ang II infusion induced hypertension which is associated with an increase in the activated microglia in the PVN. 97 R ecent study from Miyoshi et al also provided the evidence indicating that neurons and microglia express AT 1 R, that are primarily resp onsible for Ang II induced TGF production in the CNS 98 Systemic administration of centrally acting AT 1 R block er ameliorates these responses. 99 However the involvement of microglia activation in peripheral inflammatory proces s including increase in bone marrow (BM) derived inflammatory cells in neurogenic hypertension is not yet clear. Endothelial Progenitor Cells and Vascular Dysfunction in Hypertension and Cardiovascular Diseases Accumulating st udies have shown that improve ment of t he ability of endothelial repair function with BM stem cells is important in hypertension induced vascular pathophysiology 100, 101 Endothelial progenito r cells (EPCs) are BM derived endothelial stem cells and released in to the circulation to maintain va scular homeostasis. 102 In order to replace damage d endothelium, EPCs are mobilized to the site of injury and involved in the repair process. However, dysfunctional and reduced number of EPCs are unable to perform blood vessel regeneration in damaged vasculature in hypertension Decreased function and num ber of EPCs are correlated in patients with
20 hypertension, obesity, chronic kidney disease, and immune diseases. 103, 104 Patients with diabetes or pulmonary hypertension also showed dysfunctional EPCs are associated w ith endothelial dysfunction. 105, 106 Animal studies have demonstrated that dysfunctional EPCs are responsible for the increase in EPC ROS, NADPH oxidase and th e decrease in NO availability. 106 Endtmann et al have demonstrated that Ang II decreases EPC number s and function by increasing oxidative stress and apoptosis and this effect is blocked by AT 1 antagonist, demonstrating Ang II is an important signaling trigger. 105 As discussed above, Ang II induced c entral production of neuroinflammatory process including PVN microglia activation which directly or indirectly raises cytokines, chemokines, ROS et c ., stimulates neuronal activity. Considering that BM is efficiently innervated by cholinergic nerve fibers from the sympathetic nervous system, altered sympathetic drive induced by Ang II from the brain to the BM may impai r EPCs function and increases per ipheral inflammatory cells from BM to the circulation resulting in a compromised vascular repair mechanism in hypertension. Based on the available evidence, it is tempting to suggest that peripheral inflammatory signals including circulating Ang II propagates across the BBB into the CV relevant brain regions and mediate sympathetic activation participating further generation of inflammatory modulators in the periphery such as BM. Therefore, CNS may play a key role in the regulation of endothelial dysfunction and inflammation in hypertension This suggests the plausible evidence of the existence of a functional neural inflammator y BM communication that is responsible f or the pathophys i ology of neurogenic hypertension
21 Figure 1 1 Central nervous system mediated circulatory regulation The figure illustrates simplified schematics of neural control of circulation. Peripheral c irculating Ang II signal activates hyp othalamic CV regulatory regions and signals transmits to brain stem regions. Activated aortic and carotid afferent bodies also send a neuronal signal to the brainstem, which send an output signal to the effector organs (heart, arteries, veins, adrenal gland and bone marrow ) via both the sympathetic and parasympathetic efferents, thereby regulating cardiac output (by regulating rate and force of heart contractions) and peripheral resistance (by regulating the contractili ty of the arteries and veins, and adrenaline synthesis). The preganglionic neurones mostly signa l vie acetylcholine (Ach) while the postganglionic neuronal transmitter is mainly noradrenaline (NA).
22 Figure 1 2 Hypothalamic and brain stem areas in car diovascular regulatory brain regions. Saggital diagram of the rat brai n indicatesthe c ardiovascular regulatory brain regions with high densities of AT 1 receptors. Circumventrical organs such as SFO that lack a BBB and exposed to influences of the periphera l renin angiotensin system. The area in the boxes highlights the specific nuclei within the hypothalamus and medulla oblongata involved in cardiovascular homeostasis. SFO, OVLT, PVN in the blue dotted box, NTS, R VLM, CVLM in the red dotted box.
23 Figure 1 3 Neuronal mechanisms of blood pressure regulation in the brain. Arginine vasopressin (AVP), posterior pituitary (PP), Intermediolateral cell column (IML) Arginine vasopressin (AVP) is synthesized in the PVN and released from posterior pituitary (PP) that results in increase of blood volume. Increase in baroreceptor afferent activity is transmitted to NTS to inhibit efferent sympathetic nerve activity Increase in oxidative stress in these areas is associated with increas ed neuronal activity resulting in high blood pressure.
24 CHAPTER 2 ROLE OF BRAIN MITOCHONDRIAL OXIDAT IVE STRESS IN NEUROG ENIC HYPERTENSION Increased Oxidative Stress in Neural Mechanisms of Hypertension Accumulating evidence indicates that i ncreased oxidative stress in the vasculature, the heart, the kidney, and the brain is associated with cardiovascular disease including hypertension. 52 56 Indeed, excessiv e production of reactive oxygen species (ROS) in the brain nuclei not only is an important signaling trigger but also plays a crucial role in the regulation of sympathetic nerve activity 55, 108, 109 Ci rculating Ang II has been shown to increase cellular superoxide production in the brain and that is mediated by NADPH oxidase 69, 78 NADHP oxidase is multisubunit membrane bound enzyme complex and Ang II is the trigger of NADPH oxidase a ctivation Ang II induced stimulation of ROS production may compromise sympathoinhibitory mechanisms in the CNS, thereby contributing to chronic increase in blood pressure. Importantly, it has been reported that elevated ROS production in the key CV regula tory nuclei such as the PVN and the RVLM leads to increase in sympathetic activity and BP. 109 The basal level of O 2 and H 2 O 2 in the RVLM that generates and maintains sympathetic vasomotor tone, is elevated in animal models of hypertension. 110 For example, a s tudy by Kimura e t al showed that overexpression of inducible nitric oxide synthase in the RVLM produced hypertension by increasing ROS levels in normotensive WKY rats 111 Davissons group demonstrated that increased O 2 I n the forebrain of mice is associated with cardiac dysfun ction in myocardiac infarction. 112 Our previous studies have demonstrated that sol uble epoxide mediated BP regulation in the SHR is mediated by NADPH oxidase derived generation in forebrain nuclei. 69 Taken together t hese
25 observations suggest that accumulating ROS and NO availability in the CV brain regions play a key role in the development of neurogenic hypertension. Role of B rain Mitochondrial ROS in Neurogenic Hypertension Although Ang II activated NADPH oxidase is known to be the major source of ROS production in hypertension 66, 67 the contribution of mitochondrial superoxide generation by Ang II in these regions of the brain is relatively unclear in neurogenic hypertension. M itochondria are another major source of cell ular superoxide generation and it has been evidenced that mitochondrial ROS is involved in the pathogenesis of cardiovascular, metabolic and neurodegenerative diseases. 113, 114 Recently, Chan et al investigated the role of mitochondrial electron tr ansport chain in the brain and dysfunctional electron transport chain in the RVLM of SHR results in oxidative stress by inhibiting chain complex activity III or I. 77 Recent study by Dikal ova et al also demonstrated that mitochondrial superoxide is important for the development of hypertension by using mitochondria targeting antioxidant mitoTEMPO. 115 Systemic infusion of mitoTEMPO in Ang II induced hypertensive mice improved endothelial function and NADPH oxidase activity 115 However, the contribution of the brain mitochondrial ROS in the regulation of sympathetic activation and hypertension cannot be deduced from these studies since mitoTEMPO, like TEMPOL is like ly to cross the BBB (Figure 2 2 ). Given that targeting mitochondria for intracellular ROS and excessive oxidative stress may be significantly effective in the regulation of blood pressure it is possible that mitochondrial derived oxidative stress in the b rain alter s neural redox state, and initiates development of neurogenic hypertension. However, potential mechanisms by which brain mitochondrial oxidative stress alters autonomic nervous system and
26 cardiovascular parameters in neurogenic hypertension have not been tested yet. Hence, we hypothesized that mitochondrial ROS in the brain is responsible for dysfunctional neural signals in pa thophysiology of hypertension. W e investigated the effects of central administration of mit ochondria targeted antioxidant, mitoTEMPO on autonomic function, cardiac hypertrophy, and brain microglia activation including cytokine release in Ang II induced neurogenic hypertension. Methods Animal Adult male Sprague Dawley (SD) rats aged 6 to 7 weeks were purchased from Charles Ri ver Laboratories (Wilmington, MA). Rats were individually housed in a temperature controll ed room (22C to 23C) with a 12 : 12 hour light dark cycle. Rat chow (Harlan Tekland) and water were provided by Animal Care Services. All experimental procedures were approved by the University of Florida Institute Animal Care and Use Committee. Primary Neuronal Culture Neuronal cells in primary culture from the brainstem and hypothalamus of one day old SD were est ablished Brains were isolated from neonatal rats and hypothalamic and brain stem areas were dissected and trypsinized (Worthington Biochemical, cat# 3667) for 15 min at 37 C to dissociate individual neurons. Cells were then plated in poly L lysine (Sigma, cat# P 1274) pre coated 6 or 12 well culture dishes. After 48 hours, c ells were treated with anti mitotic agent, arabinoside (Cytosine 1 B D, cat# C 1768)for 2 days and media was replaced with DMEM +10% FBS. Cultures were established for 12 14 days prior to use in the experiments Previous studies have shown that the culture contain <95% neuronal cells and remaining being astrocytes. 116
27 Measure ment of ROS Production Eleven to thirteen days of c ultured neurons were treated with AngII (500nM), or co treatment with mitoTEMPO (2 and 5 nM, Enzolifescience, ALX 430 150 ) for 4 hours. Cellular superoxide w as measured by DHE (dihydroethidium, Invitrogen) fluoresc ent staining a nd mitochondrial superoxide was measured by MitoSOX Red staining (Invitrogen). DHE (1nM) was added to neurons for 30 min at 37 C and cells were washed with PBS three times. For the detection of mitochondrial ROS, neurons were incubate d with 5uM of mitoSOX r ed dye for 10 min at 37 C and washed with PBS three times. Additionally, mitoTrac ker gree n (Invitrogen) staining was used for the mitochondrial subcelluar location of MitoSOX. Cells were fixed with 4% PFA to be examined. Images were obtained from Zeiss Axio plan 2 Fluorescent Microscope. Telemetry Recordings of Arterial Pressure and Heart Rate Male SD rats (6 to 7 weeks old ) were anesthetized with a mixture of O 2 (1 L/min) and isoflur ane (2 % to 4%) during the surgery A radiotransmitt er (TA11PAC40, Data Sciences International) was implanted to record arterial pressure and hear t rate from the abdominal aorta About ~1 inch long i ncision was made in the middle of abdominal skin and aorta was exposed and isolate d carefully. The catheter from the telemetry transducer was inserted using curved needle into the vessel toward the heart and sealed with 3M vetbond. Transducer was suture d (3 0 nylon, non absorbable) with inner skin to se cure its position under the skin. Second suture was placed to close up the outer skin and additional surgical clips were used. To minimize post operational pain, bolus injection of buprenorphine (0.03 mg/kg SC) was administered after the surgery. Rats were allowed to recover for 7 to 10 days before bas eline telemetric measurements
28 were taken. Telemetry recording was performed every 3 4 days. A full spectral analysis was performed on the blood pressure signal. Measurement of Full Spectral A nalysis The variance of blood pressure, heart rate and pulse inte rval variance was calculated from the dark period (12:00am 5:00am) data collected for 10 min every hour Pulse intervals were calculated in miliseconds (ms) by inversion of heart rate values. The spectral analysis was performed using the software Hey Prest o The low frequency component of the power spectrum (LF(SBP) ) indicating sympathetic drive included the power from 0.04 to 0.15 Hz, and the high frequency component the power spectrum (HF (PI) ) indicating parasympathetic drive included the power from 0.15 Hz to 0.25 Hz. The power of each band was calculated as integral of power spectral density under the curve in the frequency range. LF ( SBP ) and HF (PI) were computed in absolute units in mmHg 2 and in normalized units expressed as a percentage of the total power. sBRG(PI) was computed in unites of ms/mmHg indicating spontaneous barareflex gain. Figure 2 3 shows the representative image of the software. Implan tation of Subcutaneous Osmotic M inipump s Rats were further assigned to subgroups (n=5 8) to receive Ang II (200 ng/kg/minute), 0.9% saline or mitoTEMPO ( Enzo Life Science 100 or 170 ng/kg/min) delivered via an osmotic minipump (No. 2004, ALZET) .Pumps were prepared and filled with drugs day before the implantation and incubated at 37 C for overnight. A s mall incision was made between the scapulae and pumps were placed subcutaneously Skin was clipped a nd pumps were secured. Each pump lasts for 4 weeks from the day of drug preparation and animals we re euthanized before day 28 (Figure 2 1 )
29 Intracerebroventricular mitoTEMPO Infusion Ten to fourteen days after implan tation of telemetry transducers rats were subjected to second surgery of intracerebroventricular (ICV) cannulae implantation. As shown i n figure 2 1 experimental design, either the ICV or SC infusion of mitoTEMPO started on day 0. In brief, rats were anesthetized with a 4% isoflurane/O 2 mixture, and the head was positioned in a Kopf stereotaxic apparatus (Ha r vard apparatus) using the earplugs Incision was made and the bregma was exposed. Surgical drill (Complete bone micro drill system, 724950, Harvard apparatus) was used to make a hole to place a cannula inside to the brain. An infusion cannula (Brain infusion kit 1 3 5mm, ALZET) was implanted into the left cerebroventricle (1.3 mm caudal to bregma, 1.5 mm lateral to the midline, and 4.5 mm ventral to the dura). Small amount of dental cement was added to secure the cannular for the period of experiments. A four week osmotic minipump was connected to the infusion cannula via the catheter tube to deliver mitoTEMPO (Enzo Life Science, 100 or 170 ng/kg/min). Immunohistochemistry Brains were post fixed with 10% PFA for one hour and placed into 30% sucrose for 2 3 days until they are ready. OCT embedded brains were frozen prior to sectioning and cut into 10~20 m coronal sections including PVN Sections were then incubated with 0.1% triton X 100 followed by serum incubation for one hour. Primary anti Iba 1 antibody ( Waco, cat# 019 19741), a specific marker for microglia was diluted into 1:500 and incubated for 3 hours at room temperature. Af ter washing three times with PBS anti rabbit IgG (1:200, VEGTOR, cat# BA 1000) antibody was used as a secondary antibody that is conjugated wit h 3,3' diaminobenzidine (DAB). Activated microglia were
30 visualized To obtain the images of PVN secti ons an Olympus BX41 microscope was used. Cardiac Pathology Hearts were collected from the rats at the end of the experiment, and processed for cardiac morphology and histological examination as described previously. 118 Briefly, left ventricles were first separated and rinsed with PBS to remove residual blood before weighing and fixing with 10% paraformaldehyde Later ventricles were embedded in paraffin and cross sectioned into 4 m Sections were stained with either hematoxylin eosin for the myocyte diameter measurement or with p ico sirius red dye for interstitial fibrosis measurement. Twenty five to thirty images were taken from each section using Olympus BX41 mic roscope and analyzed with the image J software from NIH. RNA Isolation and Real T ime PCR To analyze mRNA levels from the PVN, hypothalamic tissue s including PVN were dissec ted. C oronal segments were first sliced according to Paxinos and Watson ( The Rat Br ain: In Stereotaxic Coordinates) and s mall blocks of each area were excised (2.0mm wide and high). Total RNA was prepared using RNeasy kit (Qiagen) according to the manufacturer s instruction. About 200 to 300 ng of purified RNA were reverse transcribed using high capacity cDNA reverse transcription kit (Bio Rad Laboratories). Quantitative Real Time PCR was performed with specific primers and probes of IL1 ( Rn0058043_m1 ) TNF ( Rn00562055_m1 ) and CD11b ( Rn007 09342_m 1 ), NADPH oxidase ( Rn00577357_m1; p22 phox, Rn00576710_m1; Gp9 1phox ), n NOS ( Rn00561646_m1; NOS2 ) by using PRISM 7000 sequence detection system (Applied
31 Biosystem). Data were normalized to 18s ribosomal RNA ( Hs99999901_s1 ) and GAPDH (9 Rn00566603_m1 ) Results ICV Infusion of mitoTEMPO Attenuates Ang II i nduced Neurogenic Hypertension Despite recent studies demonstrated the role of oxidative stress and inflammation in Ang II induced hypertension, the role of brain mitochondrial ROS associated with pathophysiology of neurogenic hypertension has not been examined To test whether scavenging of mitochondrial ROS in the brain can prevent Ang II mediated development of hypertension, Ang II infused SD rats were co treated with mitoTEMPO either subcutaneou sly or intrace re broventricularly for 4 weeks. Two di fferent doses of mitoTEMPO (100ng/kg/min and 170ng/kg/min) were administrated based on previous study 119 Over the experimental period chronic subcutaneous ( SC ) infusion of Ang II (200ng/kg/min) in creases mean arterial pressure ( control: 982mmHg ; n=8 Ang II: 1776 mmHg ; n = 8). Although SC mitoTEMPO did not prevent the Ang II induced increase in MAP intrace re broventricular ( ICV ) infusion of mitoTEMPO significantly attenuated the increase of MAP A dose of 100ng/kg/min caused ~30mmHg decrease (Ang II: 1776 mmHg; n = 8 vs. 146 12 mmHg; n=6 ) while the dose of 170ng/kg /min resulted in ~60mmHg decrease in MAP (Ang II: 1776 mmHg; n = 8 vs. 11213 mmHg; n=8) (Figure 2 4A and B ) ICV mitoTEMPO alone had no effect on MAP ( control: 982mmHg ; n=8 mitoTEMPO alone: 104 ng/kg/min; n=4). Heart rate in control group has decreased as the rats aged, however they did not show significance changes between the mitoTEMPO treatment groups (Figure 2 4C)
32 mitoTEMPO Scavenges Ang II i nduced Mitochondria ROS in Neuron al Cells in Prima ry Cultures To further verify mitoTEMPOs superoxide scavenging function in neurons Ang II treated (500nM) primary neuronal cultures were co treated with mitoTEMPO (2 or 5nM) for 4 hours followed by staining with mitoSOX ( a mitochondrial specific superoxi de detector ) M itochondrial localized actions of mitoTEMPO were confirmed by co staining with a mitochondrial specific fluorescence dye (i.e., M itoTracker G reen ) Figure 2 5B shows Ang II increased mitochondrial superoxide oxidized by mitoSOX was completely diminish ed in the neuron. Additionally levels of total cellu lar ROS as detected by DHE (dihydroetidium excitation 490 nm/emission 585 nm ) staining were normalized by mitoTEMPO treatment (Figure 2 5A). This suggests that mitoTEMPO specifically decrease not only mitochondrial ROS but also to tal cellular ROS in Ang II treated neurons The neuronal cultures that is used in the experiment contains >90% of neurons, howe ver still has <10% of glia and astrocytes The possibility of gila and astrocytes induced mitochondrial oxidative stress stained by mitoSOX cannot be ruled out. Central Mitochondrial Superoxide Inhibition Influences Autonomic Nerve Activity in AngI I i nduced Neurogenic H ypertension F ull spectral analysis was performed based on the data obtained from 24 hours of telemetry recording t o investigate whether mitochondrial ROS influence s autonomic functions (Figure 2 3) Spectral analysis using telemetry data is non invasive classical methods of quantification of the cardiovascular variability, and the variance of systolic blood pressure and heart beat intervals that provide an insight into autonomic control of the circulation in hypertensive subjects 120 The LF component o f systolic blood pressure power spectrum, LF ( SBP ) is considered as a marker of oscillations of the sympathetic
33 activity addressed to resistant arterie s, and HF(PI), the high frequency pulse interval variability indicates a marker of cardiac parasympathetic drive Also, sBRG (PI) is considered spontaneous cardiac baroreceptor reflex gain LF (SBP) and sBRG (PI) indicate ~6 fold increase in sympathetic vasomotor drive 2 /mmHg 2 AngII: +2.2010.3 ms 2 /mmHg 2 ] and ~3 fold decrease in cardiac spontaneous baroreflex gain (PI) control: +0.1480.1 ms/mmHg, AngII: 0.2470.06 ms/mmHg)] respectively after 4 weeks of Ang II infusion (Figure 2 6) However, ICV mitoTEMPO could normalize these changes to the c ontrol level [100ng/kg/min, 170ng/kg/min 2 /mmHg 2 (PI): 0.070.04, 0.0680.1 ms/mmHg] Cardiac parasympathetic drive measured by HF (PI) did not show significant chan ges in any of these groups (Fig ure 2 6 C), ye t the ratio of LF to HF, which is a n indication of vasovagal balance, was ~2.5 fold elevated by Ang II (Figure 2 6D). ICV mitoTEMPO treatment was able to attenuate Ang II mediated change in the vasovagal balance (Figure 2 6 D). ICV mitoTEMPO Inhibits AngII i nduced Microglia Activation in t he PVN Shi et al have shown that chronic Ang II infusion results in microglia l activation and increases mRNA levels of various inflammatory gene s in the PVN. 121 To test if mitochondrial ROS inhibition would influence microglial activation, we determined the effects of ICV mitoTEMPO treatment on mRNA levels cytokines and CD11b, a marker of activated microglia in Ang II induced hypertensive rats Consist with previous finding s Ang II increased Iba1 positive cells by ~85% and CD11b expressing cells (i.e., activated microglia) by ~1.6 fold in the PVN ICV mitoTEMPO significantly reduced the number of Iba1 expressing microglia and the level of CD11b mRNA in the PVN to the control levels (Fig ure 2 7A and B) In addition, Ang II increased mRNA levels of IL1 and TNF in the
34 PVN as ~1.6 folds and~ 2.3 fold s, respect ively, while ICV mitoTEMPO completely abolish ed the increase of IL1 and TNF transcript levels (Fig ure 2 7 C E ). ICV mitoTEMPO Inhibits Ang II induced Increase in NADPH Oxidase mRNA and D ecrease in nNOS mRNA in the PVN Although Ang II induced increase in ROS production in the brain is medi ated by NADPH oxidase, the involvement of mitochond ria in the activity of NADPH oxidase in neuronal ROS production is not yet tested. mRNAs were prepared from PVN and Real tim PCR was performed to determine the levels of NADPH oxidase subunit p22 phox, gp91 and nNOS mRNA. Figure 2 8 shows Ang II infusion significantly increases p22 m RNA by ~1.9 fold and decreases nNOS mRNA by ~2 fold in the PVN. ICV mitoTEMPO treatment prevents these changes and normalized to the control levels. These data suggests that mitocondria ROS inversely affect NADHP oxidase mRNA as well as nNOS mRNA, resultin g in the imbalance of cellular redox state, and excessive oxidative stress. ICV mitoTEMPO Prevents Ang II i nduced Cardiac Hypertrophy and Interstitial Fibrosis At th e end of the experiments (27days after telemetry recording) rats were euthanized and hearts were collected to determine the effects of ICV mitoTEMPO treatment on Ang II induced cardiac pathology. Chronic Ang II infusion resulted in an increase in the heart weight/body weight ratio (control: 3.0 0.2, AngII: 4.15 0.1) and cardiac myocyte diameter (control: 12.48 0.9 m, AngII: 15.8 1.2 m) two indicator s of cardiac hypertrophy Ho wever, the change of heart/body weight ratio and hypertrophy by Ang II infusion was normalized with the mitoTEMPO ICV treatment (100ng/kg/ min: 3.4 0.8, 170ng/kg/min: 3.5 0.3) ( Figure 2 9A ). Also, ICV mitoTEMPO treatment
35 prevented increase in myocyte diam eter (13.1 1.1 m) (Figure 2 9 B) Interestingly, SC infusion of mitoTEMPO that did not affect MAP, had no beneficial effects on cardiac hypertrophy (14.8 0.8 m) To determine cardiac interstitial fibrosis, left ventricles were stained with p ico s irius Red dye and examined with Olympus BX41 microscope Consistently, interstitial fibroti c area is significantly increased with Ang II infusion (control: 4 0.5, Ang II: 15 1.1 area %) but inhibited with mitoTEMPO ICV treatment (ICV mitoTEMPO: 6 0.8, SC mitoTEMPO: 16 0.9) ( Figure 2 10 A ). F igure 2 10 B shows quantific ation graph from Image J software (NIH) Discussion The most significant f inding of this study is that increase in brain mitochondrial ROS is responsible for the changes in autonomic function and microglial activation in Ang II induced neurogenic hypertension. O xidative stress in the CV relevant regions in the brain has been a ssociated with the pathogenesis of the hypertension and observed in experimental rat model. Studies hav e shown that SOD mimetic, TEMPOL or adenoviral mediated deliver of SOD gene in the SFO or RVLM decrease blood pressure and prevented hypertension. 56, 122, 123 It is well established that Ang II increases NAD PH oxidase mediated o xidative stress in the brain and that is associated with neurogenic hypertension 61, 124, 125 NADP H oxidase is the enzyme complex, mainly activated by Ang II. Although NADPH oxidase mediated ROS production is well doc umented, the involvement of mitochondria as another source of ROS production in the brain is not completely understood Antioxidant treatment such as vitamin E in clinical trial s has not been very successful in hypertensive patients, 126 which might be explained by the failure of
36 targeting ROS in the subcellular level such as mitochondria. Recent study demonstrated the protect ive effects of mitochondrial targeting antioxidant on different animal models of hypertension 77, 127, 128 The mitochondrial targeting antioxidant, mitoTE MPO infusion improved endothelial function by preventing loss of en dothelial nitric oxide in Ang II or D OCA salt induced hypertension. 119 Also, the possibility of NADPH oxidase activation by mitochondrial ROS has been suggested. 119 As shown in figure 2 8A, in fact, Ang II mediated increase in NADPH oxidase mRNA was blocked by ICV mitoTEMPO treatment in the PVN. It is possible to suggest th at mitochondria superoxide production can be triggered by NADPH oxidase activation through Ca 2+ accumulation within the mitochon dria, and mitochondrial ROS would regulate NADPH oxidase vice versa Therefore, it is tempting to propose that targeting mitochondr ia to inhibit cellular ROS can be more effective in hypertensive disease In this study, we used ICV administration of mitoTEMPO to examine the central role of mitochondria l ROS and to demonstrate its beneficial effects on neurogenic hypertensio n and brain microglia. ICV infusion of mitoTEMPO prevents hypertension via the reduced microglia activation and sympathetic outflow. However, comparable dose of SC infusion of mitoTEMPO did not prevent hypertension. This observation is in contrast to the p revious observation of Dikalova et al who determined an attenuation of hypertension by SC administration of mitoTEMPO in mice. 119 Th is discrepancy in the results may likely be due to an increased accessibility of mitoTEMPO in the brain s of mice, as a result of the previously reported altered permeability of the blood brain barrier in mice subjected to twice the concentration of Ang II compared to the one used in the rats in the present study. 129 This is particularly relevant in view of evidence that
37 autonomic regions of the brain are highly vascularized. 87 However, differences in other humoral responses and metabolic processes between the two species cannot be ruled out at this poi nt Finally, our findings establish that Ang II induced mitochondrial ROS in the brain triggers neuronal activity that results in increased sympathetic drive, decreased baror eceptor r eflex gain PVN microglia activation and cardiac hypertrophy Additiona lly increased cytokines in the PVN is a marker of proinflammatory signal in the CNS that is associated with increased sympathetic drive. Thus, we s peculate that central production of mitochondrial ROS is an important signaling mechanism mediating autonomi c function in pathophysiology of Ang II induced neurogenic hypertension.
38 Figure 2 1 Animal e xperi mental design. Radiotelemetry was implanted on day 10 and subcutaneous Ang II with eigher ICV or subcutaneous mitoTEMPO are administrated at day 0. Blood pressure and heart rate were monitored twice a week for 24 hours until the end of expreriment. Brains and hearts were collected for further histolo gy and bone marrow cells were isolated to enrich EPCs and ICs.
39 Figure 2 2 The chemical structures of mitoTEMPO and TEMPOL. An antioxidant TEMPOL is a superoxide dismutase mimetic and mitoTEMPO is mitochondria targeting antioxidant. Their structure is similar but conjugation of a lipophilic triphenylphosphonium cation to the structure of TEMPOL allows targeting of an antioxidant to the mitochondria TEMPOL is known to cross BBB and based on the structural similarity mitoTEMPO has a great chance to pass BBB.
40 Figure 2 3 The representative software image of spectral analysis using Hey Presto. Spectral analysis is non invasive classical methods of quantification of the cardiovascular variability, and the varianc e of systolic blood pressure and heart beat intervals that provide an insight into autonomic controls The variance of blood pressure, heart rate and pulse interval was calculated from the telemetry data collected for each 10 min every hour until 24 hours Pulse intervals were calculated in miliseconds (ms) by inversion of heart rate values.
41 Figure 2 4 Effects of mitoTEMPO on Ang II Induced Hypertension and heart rate. A, ICV mitoTEMPO significantly attenuated MAP in Ang II induced hypertension in a dose dependent manner (100 and 170 ng/kg/min). P <0.05, ** P <0 .01 vs control. B, SC mitoTEMPO did not attenuate MAP. C, Heart rate did not show differences between mitoTEMPO treatment groups. Bar graph is meanSEM. ** P <0.01 vs control.
42 Figure 2 5 Scavenging of AngII induced superoxide in mitoTEMPO treated neurons. A, C ellular superoxide stained by di hydroethidium. Relative fluorescence units (RFUs) were detected by microplate reader. P <0.05 vs control, # P <0.05 vs Ang II. B, Representative images of neurons. mitoSOX red staining is used to detect mitochondrial superoxide and mitoTracker green is used for mitochondrial localization.
43 Figure 2 6 Effects of ICV mitoTEMPO on autonomic nerve activity in Ang II induced neurogenic hypertension. P <0.05 vs control, # P <0.05 vs Ang II.
44 Figure 2 7 Effects of ICV mitoTEMPO on microglia activation and cytokine mRNA in the PVN. A, Ang II induced Iba 1 positive activated microglia within PVN were reduced to control level by ICV mitoTEMPO treatment. B, Quantification of the number of Iba 1 positive microglia in the PVN. C, mRNA of CD11b, a marker of activated microglia. D, mRNA of mitoTEMPO alone group was not included in the data since we did not see any significant changes in MAP and HR. P <0.05 vs control, # P <0.05 vs Ang II.
45 Figure 2 8 Effects of ICV mitoTEMPO on NADPH oxidase and nNOS mRNA A, p22 phox and gp99 phox mRNA. B, nNOS mRNA mitoTEMPO treatment normalized p22phox and nNOS mRNA levels. P <0.05, vs c ontrol, # P <0.05 vs Ang II.
46 Figure 2 9 Effects of ICV mitoTEMPO on cardiac hypertrophy and myocyte diameter. A, The Ratio of heart weight to body weight and body weight did not change between groups B, Cardiac myocyte diameter measured from H&E stained left ventricle section. There were no significant changes in their body weight between groups. P <0.05, ** P <0.01 vs control, # P <0.05 vs Ang II. C, Representative pic tures of hearts from each group.
47 Figure 2 10 Effects of ICV mitoTEMPO on interstitial fibrosis of left ventricles A, Representative left ventricle sections of sirius red staining positive fibrotic areas. B, Quantification gr aph generated from Image J software. ICV mitoTEMPO treatment prevented the increase in interstitial fibrotic areas in left ventricles induced by Ang II. P <0.05 vs control, # P <0.05 vs Ang II.
48 CHAPTER 3 BRAIN MEDIATED DYSREGULATI ON OF THE BONE MARRO W ACTIVI TY IN NEUROGENIC HYPERTENS ION Inflammation and Endothelial Progenitor Cells in Neurogenic Hypertension It is well established that hypertension is associated with increase in inflammatory modulators in the circulation as well as in the central nervous system. 130 132 Chronic inhibition of inflammatory markers prevents ischemic induced vascular path ology in type II diabetic mice, 133 and i ncreased inflammation in the circumventricular organs and brainstem is associated with incr ease in the sympathetic drive. 134 Vascular inflammation in the brainstem of the SHR, the rat model of neurogenic hypertension was increased with elevated inf lammatory cytokines such as IL 1 IL 6, and TNF 132 T he treatment of Ang II ty pe 1 receptor (AT 1 R) blocker with access to the brain showed anti hypertensive effects and inhibited cerebrovascular inflammation including reduced macrophage infiltration and decreased cytokine expressions such as TNF a, IL1b 135, 136 These data and Felder s work 137 allow us to propose that vascular inflammation i n cardiovascu lar disease including hypertension may be CNS regulated, and inflammatory process in the CV regulatory regions of the brain is associated with modulation of the autonomic function. In particular, there is supportive evidence that the vascular inflammation and brain cytokines play an important role in the pathogenesis of hypertension. 138 Furthermore, e xpression of i nflammatory cytokines is increased in the cardiovascular relevant brain regions of various animal model of hypertension. 132, 139 For example adeno associated viral mediated IL 10 overexpressi on in the PVN attenuates Ang II induced hypertension 121 and IL 6 mic roinjection in the NTS attenuates the barorceptor reflex gain function in rats 140 In addition, ICV infusion of IL1 a
49 proinflammatory cytokine increase sympathetic nerve activity resulting in high BP. In previous study, shi et al have demonstrated that mi croglia in the PVN are activated in response to chronic Ang II infusion and produce a variety of inflammatory mediators, including cytokines. 121 Microglia produces neurotoxic responses through production of cytokines and ROS. This da mages the BBB integrity resulting in the infiltration of inflammatory cells in the brain. 141 It is reported that a ctivated microglia cells are increased in the central nervous system from the patient s with neurodegenerative disease, such as Alzheimer and Parkinsons disease. 94, 95 However, the involvement of brain in peripheral BM derived inflammatory signaling and the regulation of endothelial regeneration in hypertension has not been studied extensively EPCs are BM derive d stem cells that are important components in the endothelial repair process after vascular injury in cardiovascular disease. It is well established that endothelial dysfunction is a relatively early event in hypertension induced vascular pathogenesis therefore preventing endothelial d amage from high blood pressure could be a critical therapeutic strategy for hypertension Since various cardiovascular disorders are found to be associated wit h loss of intact vasculature and vascular inflammation, well functioning E PCs might not only be i mportant for maintaining intact vasculature in healthy individuals but also for preventing various cardiovascular disorders including diabetes and hypertension. There are reports that d ysfunctional EPCs induce oxidative stress and inflammatory cytokines t hat could result in vasoconstriction, inflammation, and vascular fibrosis. 106, 142, 143 Moreover, the number of circulating EP Cs are d ecreased, and the ability of their functions are im paired in both experimental animal models and human patients of cardiovascular dis ease 104 106, 144
50 Considering the dysfunctional sympathetic and parasympathetic drive in neurogenic hypertension, it is tempting to suggest that altered sympathetic drive to BM may contribute to EPC impairment. This contention is s upported by recent experiment in diabetes with retinopathy, a pathophysiology that is also associated with increase in proinflammatory cytokines and microglia cells in the eye. 145 A reduction in the number of nerve terminal ending in the BM and impaired sympathetic drive has been associated with diabetic retinopathy. 146 This study was designed to investigate the hypothesis that there is central regulation of BM EPC s and a functional balance between inflammatory cells ( ICs) and EPCs in neurogenic hypertension We obser ved the effects of Ang II infusion on the numbers and functions of BM derived EPCs and the ratio of EPCs/ ICs in Ang II induced rat model of hypertension. Additionally, mitochondrial targeting superoxide scavenger, mitoTEMPO ICV treatm ent induced antihypertensive model ( descried in C hapter 2) was utilized to investigate the functional connection of BM and brain. Methods Animal Adult male Sprague Dawley (SD) rats aged 6 to 7 weeks were purchased from Charles River Laboratories (Wilmingt on, MA). Rats were individually housed in a temperature controll ed room (22C to 23C) with a 12 : 12 hour light dark cycle. Rat chow (Harlan Tekland) and water were provided by Animal Care Services. All experimental procedures were approved by the Universit y of Florida Institute Animal Care and Use Committee.
51 MNC Isolation from B lood and BM Rats were subjected to anesthesia with iso f l urane and about 10 ml of blood was drawn from ab dominal vein. Collected blood was dilute d with PBS+ 2%FBS+1mM EDTA followed by the addition of 10ml of Ficoll Paque (GE Healthcare, Cat# 17 1440 02) .Y ellow buffy coat was obtained after centrifugatio n at 1200rpm for 25min and was transferred to a new tube to isolate mononuclear cell (MNC) pallet. To remove residual red blood cell s in the pellet, ammonium chloride (STEM CELL technology, Cat# 07850) was added and incubated for 10min on ice. Cells were then centrifuged twice after was hi ng with PBS+2% FBS+1mM EDTA to remove residual ammonium chloride. White MNC pellet was obtained after second centrifugation at 1200rpm For BM MNCs, intact femur and tibia were isolated from rats and rinsed with PBS+2% FBS+1mM EDTA buffer followed by cleaning and removing muscle an d fat. The tips of the bones were cut to flush out bone marrow cells using sy ringe s into 50ml tube. MNC pellets were obtained by spinning down at 1200rpm for 15mins at room temperature. Ammonium chloride was added to remove RBCs for 10 min on ice same as blood MNC isolation followed by 2 times washing with PBS+2% FBS+1mM EDTA. I solation of EPCs from MNCs MNCs were prepared in the 5ml tubes at a concentration of 5 10 7 cells/ml in PBS+2% FBS+1mM EDTA. 50ul of EasySep negative selection cocktail ( STEM CELL technology ) is added to the cells and incubated for 10 min at room temperatu re followed by incubation with 50ul of EasySep Magnatic Nanoparticles ( STEM CELL technology ). Tubes were placed into the EasySep M agnet for 5min at room temperature for the negative selection The EasySep Magnet generates a high gradient magnetic field i n the interior cavity that is strong enough to separate cells labeled with EasySep
52 Magnetic Particles The magnetically labeled cells remain bound inside the tube, held by the magnetic field when unlabeled cells within the magnet are transferred to the new tube placed outside. This step was repeated for three times After centr ifugation for 10min at 1200rpm, cell pellet s were re suspended in 100ul of PBS+2% FBS+1mM EDTA at a concentration of 1 10 7 cells or fewer with 5ul of mouse ser um for positive selection ( STEM CELL technology ) CD90 + antibody cocktail and Magnetic nanoparticles from the positive selection kit ( STEM CELL technology ) were added to the cell suspension. T ubes containing cell mixture were placed in the magnet for 5min incubation. The ma gnetically labeled cells remained in the tube, held by magnetic field of the EaseSep M agnets while the rest of unlabeled cells were removed. This step was repeated for three times to isolate less contaminated CD90 + cells. Cells were plate d in 96well plate in Stem Span media (STEM CELL technology Cat# 09650) and functional assay. Direct Flow Cytometry (FACS) A nalysis To profile the level of inflammation MNCs from BM and blood were prepared in a concentration of 0.5 1 x 10 6 cells/ 100ul in 2% FBS, 1mM EDTA and 1xPBS mixture media Antibodies were from AbD Serotec (Alex647 conjugated CD4/5/8/3/68, RPE conjugated CD25, FITC conjugated CD45, Percp cy5.5 conjugated CD90) as recommended by the company. Cells are incubated with antibodies for 45 minu te at 4 o C Individual antibodies were prepared in each cell suspension as control. After twice of washing, cells were fixed with 2% paraformaldehyde for later analysis. All samples were read on LSR II (BD Biosystems) in University of Florida Interdisciplinary Center for
53 Biotechnology Research (ICBR) and data were analyzed withFACS Diva software version 6.1.2. DiLDL and Lectin S taining CD90 + cells are examined for DiLDL ( 1,1 dioctadecyl 3,3,3,3 tetramethylindocarbocyanine low density lipoprotei n ) uptake and lectin binding to confirm its EPC characteristics. First, isolated cells are incubated with DiLDL (final con. 10 g/mL, Invitrogen) at 37C for 1 h followed by twice of washing with PBS. Then cells were fixed in 2 % paraformaldehyde for 10 min and counterstained with 20 g/ml FITC labelled lectin from Ulexeuropaeus ( L9006, Sigma) for 1 h at 37C in the dark. After washing cells with PBS, double positive Dil LDL/Lectin cells were observed from Olympus BX41 fluorescence microscope. Res ults Dysfunctional Endothelial Prog enitor Cells in Chronic Ang II i nduced Hypertension BM derived EPCs are dysfunctional in AngII induced hypertension CD90 + (Thy 1 + ) and CD4 /CD5 /CD8 markers were used to isolate EPCs. They are known to be human CD34+ cel ls in rats. 102, 147 In order to separate CD90 + / CD4 /CD5 /CD8 cells from mononuclear cells (MNCs), magnetic beads with antibodies were utilized. These cells stained positive for LDL uptake an d Lectin binding (Figure 3 1 ). Double positive staining confirmed the characteristics of EPCs. Second, functiona l assay s were performed to determine if CD90 + / CD4 /CD5 /CD8 cells demonstrate EPCs characteristics, and the effects of chronic Ang II infusion on BM derived EPCs. Cells were plated onto 96 well for 24 hours and t he abilities of proliferation and migration toward SDF 1 were measured by fluorescence and luminescence, respectively. Both
54 proliferation and migration abilities are significantly reduced after 6 (~70%) and 12 weeks (~40%) of Ang II infusion compared to control but not after 4 weeks in fusion (Figure 3 2 A and B).However, Ang 1 7 pretreatment with EPCs for 24 hour did not improve these abilities toward SDF (Figure 3 2 C and D ). T ube formation ability of cultured MNCs from Ang II infusion rats is diminished Isolated MNCs were plated in fibronecti n pre coated 6 well plates and maintained with endothelial basal medium for 3 weeks until they differentiate into endothelial cells. Cells were then transferred to 96 well Matrigel matrix plate (BD BioCoat TM Angiogenesis System Endothelial Cell Tube Format ion, Cat #: 354149) at 2.5~3x10 4 cells/ml a nd incubated for 12 hours at 37 5% CO 2 Then cells were monitored under microscope (bright field) every 2 3 hours to identify the ability of tube formation (Figure 3 3 A and B) The length of tube s and the nu mber of branches from each cell were measured. Cells continued to form tubes until 10 hours after plati ng. Analysis with Image J demonstrated that the length of tubes and the number of branches were significantly reduced in cells from Ang II infusion rats comp ared to cel ls from control rats (Figure 3 3 C and D ). Ang II i nduced Imbalance of EPCs and IC s BM derived EPCs are reduced by chronic Ang II infusion To investigate the effects of Ang II on the EPC numbers, BM derived MNCs were isolated from femur and tibia of control rats and Ang II infused rats (4, 6 and 12 weeks infusion) BM EPC number s were measured from MNCs sorted by FACS analysis. CD90 + /CD4 5 8 cells were counted as EPCs. Increased mean arterial pressure confirmed hypertension induction by Ang II infusion in SD rats (4 12 weeks infusion: 182 207mmHg of MAP) (Figure 3 4A ). As discussed above, t he number of EPCs was significantly reduced by 50% from 4 weeks to 12 weeks of Ang II infusion (4 week:
55 73 4, 6 week: 36 5, 12 week: 38 8 % of control) (Figure 3 4 B ), when blood pressure increased. B l ood EPCs showed similar trend but were not significantly reduced in neither of the Ang II infusion rats. I nflammatory cells are elevated in the BM and circulation by chronic Ang II infusion To further investigate the involvement of inflammatory cells in chronic Ang II infusion model of hypertension, BM derived MNCs were isolated to profile the change s in the number of inflammatory cells ( IC s) Inflammatory cells such as CD4 + /8 + (T lymphocytes), CD4 + /8 + /25 + (T regulatory cells), CD45 + /3 + (T lymphocytes), and CD68 + (macrophages) were measured by FACS analysis to compare the changes of its numbers from MNCs Chronic Ang II infusion resulted in ~250% increases in BM derived IC s and similar trend in blood I Cs (Figur e 3 6B) As a result, there were significant decrease s in the EPCs/ IC s ratio s ( control: 1 0.2, Ang II: EPCs/ CD4 + /8 + ( 0.75 0.06 to 0.2 0.02) EPCs/ CD4 + /8 + /25 + (0.6 0.08 to 0.23 0.01), EPC/ CD45 + /3 + (0.79 0.1 to 0.21 0.03), EPC/ CD68 + (0.62 0.08 to 0.3 0.05 ) in the BM and blood by 4 to 12 week s of Ang II infusion (Figure 3 6 ). MitoTEMPO ICV Tre atment I nhibits Elevated BM I nflammatory Cells and Reduced EPCs The effect of ICV m itoTEMPO on BM EPCs and ICs was studied to determine if central alteration of hypert ension correct the imbalance in EPC/IC Ang II induced decrease in the number of BM EPCs was completely restored by ICV mitoTEMPO treatm ent ( Ang II: 52 4 % ICV mitoTEMPO: 88 6 % SC mitoTEMPO: 49 8 % of control ) (Fig ure 3 6 A). In addition, BM IC levels were nor malized by ICV mitoTEMPO treatment (Fig ure 3 6 B). As a result, there was a ~5 fold decrease in the EPCs/ICs ratio in the BM and ICV but not SC mit oTEMPO normalized this (Fig ure 3 6 C). These results
56 demonstrate the existence of functional neural BM in teraction in Ang II induced neurogenic hypertension Discussion This study demonstrates that Ang II induced hypertension that exhibits neurogenic component is associated with a decrease in the number of EPCs, the function of EPCs and EPCs/ICs ratio. This m ay compromise the ability of EPCs to repair vasculature and contribute to the establishment of hypertension linked pathophysiology. This significant finding suggest s that there is a neural BM connection that regulate s inflammatory status and help s to maintain healthy endothelium in normal animals. Furthermore, brain mitochondrial oxidative stress plays an important role in the regulation of the neural BM communication It is shown that d ysfunctional EPCs are associated with human metabo lic and cardiova scular diseases, increase in NADPH oxidase activity and decrease in NO production in hypertension. 105, 148, 149 Recent s t udy from Jarajapu et al described that blockade of NADPH oxidase restores vasoreparative function in diabetic EPCs. 150 The protective role of EPCs and its regulatory mechanism in neurogenic hypertension h as become attractive in past years. Although damaged endothelium and imbalanced vascular homeostasis in cardiovascular disease are associat ed with dysfunctional EPCs, it has not been evidenced that there is a central regulation of EPCs. Present study suggest s that Ang II induced dysfunctional EPCs and the role of central mitochondrial ROS in the regulation of EPCs and IC s in hypertension. We demonstrate d that there is a dysfunctional regulatory link between brain and BM in Ang II induced hypertension and brain mitochondrial RO S plays an important role in neural BM connection. As shown in Figure 3 4 A, chronic Ang II infusion reduced BM EPC numb er in 4, 6, and 12 weeks of infusion in a t ime dependent manner. T he functional
57 ability of migration and proliferation toward SDF was also decreased in 6 and 12 weeks infusion of Ang II (Figure 3 2 A and B ) These detrim ental effects of Ang II on EPCs from t he hypertensive rats could be induced by either vasoconstrictive signals from circulating Ang II levels or brain mediated sympathetic drive or both. Further experiments will be require d to elucidate more precise mechanisms regarding this matter. The possibility of direct Ang II signals into BM sympathetic nerve endings causing EPCs to dysfunctional cannot be ruled out from this study I ncreased vascular inflammation in hypertension and c ardiovascular disease are well established based on both clinical and animal studies. 104, 105 Harrisons group was among the first to demonstrate the role of T cells and adaptive immune system in the pathogenesis of hypertension. 85 In the present study, Ang II infusion increased BM inflammatory cells (ICs) and reduced EPCs/ICs ratio accordingly. mitoTEMPO, mitochondria targeting antioxidant ICV treatment significantly restored the Ang II induced cha nges of EPCs and ICs in the BM, demonstrating the functional connec tion of neural BM interaction. As discussed above, increased circulating Ang II may directly affects the increase in BM ICs resulting in vascular inflammation. However, t he novel ty of the p resent study is that inhibition of the brain ROS is able to attenuate changes in BM activity and inflammatory status, which supports a brain BM communication. T he functional balance of inflammatory cells and EPCs needs to be maintained in normal vascular physiology. However in hypertensive disease increased sympathetic drive and oxidative stress within the brain may increase BM ICs and decrease EPCs resulting in impaired vascular pathophysiology. M itochondrial ROS contributes to increase in sympathetic ac tivation and neuroinflammation by activating microglia in the
58 PVN. This concept is supported by the following evidence. First, Ang II hypertension increases activated microglia and proinflammatory cytokines in the PVN 97 (a nd the study in chapter 2 ). Second, interruption of microglia activation by either minocycline 97 or by mi toTEMPO attenuates hypertension and third, hypertension induced imbalance in BM activity is prevented by central mi toTEMPO treatment indicating the existence of regulatory link between the autonomic brain regulatory regions and the BM. Finally these data demonstrate that dysfunctional neural BM communicatory pathway may be the critical mechanism in pathophysiology of Ang II induced neurogenic hypertension.
59 Figure 3 1 DiLDL uptake and lectin binding of CD90 + /CD4 5 8 EPCs. Isolated EPCs from BM were stained with lectin (green, excitation wavelength 477 nm) or DiLDL (red, excitation wavelength 543 nm) Double positive cells appearing yellow in the merge were identified as EPCs
60 Figure 3 2 Dysfunctional BM EPCs by chronic Ang II infusion. A, Migration assay toward SDF. B, Prolifertation assay toward SDF. C, Ang 1 7 pretreatment before migration as say. D, Ang 1 7 pretreatment before proliferation assay. P <0.05 # P <0.01 vs control n=5 6.
61 Figure 3 3 Decreased ability of tube formation by chronic Ang II infusion. A and B, Representative images of tube formation from control rat and Ang II infusion rat C, The length of tubes from each group were analyzed using Image J. D, The number of branches were counted and quantificated by graph. P <0.05 vs control n=3 4.
62 Figure 3 4 Reduced number of BM EPCs in Ang II induced hypertensive rats. A, Tail cuff measured mean arterial pressure (MAP) Systemic Ang II infusion elevates MAP. B, Decreased EPC numbers by Ang II infusion. P <0.05 # P <0.01 vs control n=5 6.
63 Figure 3 5 Elevated IC s and imbalance of EPC/ IC in Ang II induced hypertension CD4 + /8 + (T lymphocytes), CD4 + /8 + /25 + (T regulatory cells), CD45 + /3 + (T lymphocytes), CD68 + (macrophages) P <0.05 # P <0.01 vs control n=5 6.
64 Figure 3 6 Effects of mitoTEMPO ICV treatment on BM EPCs and IC s in Ang II induced hypertension. A, ICV mitoTEMPO normalized the decreased number of EPCs (CD4 /5 /8 /90 + ) by Ang II infusion to control level B, ICV mitoTEMPO normal ized increases in BM derived inflammatory cells. C, The ratio of EPCs to IC s. P <0.05 vs control, # P <0.05 vs Ang II by 1 way ANOV A followed by Bonferoni. n=7=8.
65 CHAPTER 4 ROLE OF NDUFA10 IN N EUROGENIC HYPERTENSI ON Proteomic A pproach of Hypertension Utilizing Spontaneous Hypertensive Rat Hypertension is a primary cause in many cardiovascular disease including stroke, diabetes, ische mic heart disease, and renal failure The onset of hypertension is also a prod uct of a natural aging process, however i ts severity and timing is usually associated with certain lifestyle relating fact ors such as diet, exercise level, and body weight. From the past decades it is hypothesized that certain gene expression profiles are altered in hyper tensive pati ents compared to healthy individ uals 151 and some of which may b e inheritable. 152 However, whether this gene expression alteration is pro hypertensive or simply a secondary effect of high blood pressure remains unclear. In the majority of case s there is no single factor underlying the onset of hypertension and such cases are considered to be primary hypertension. Primary hypertension is a complex polygenic trait with underlying genetic components, and many of them are still unknown. This contr ibutes to 90 95% of all hypertensive ca ses 153, 154 In past decades the central role s of sympathetic/parasympathetic drive in the development of hypertension have been emphasized Treatments with drugs blocking the re nin angiotensin system (e.g. angiotensin conver ting enzyme inhibitors angiotensin receptor type 1 blockers) are significantly effective in hypertensive patients, however in a considerable number of patients they fail to control high blood p ressure. 155 158 These pharmacotherapy resistant cases have been indicated to exhibit neurogenic component with increased sympathetic drive and altered neural cardiovascular mechanism. Thus, understanding of the cellular and molecular mechanisms of neural control of cardiovascular function is of extreme important in order to establish more effective treatment strategies.
66 Autonomic nervous system is a powerful modulator of blood pressure regulation which is controlled by a complicated network of brain nuclei mainly localized in the hypothalamus and brainstem. 15 These specialized nuclei include the paraventricular nuc leus (PVN) adjacent to the third ventricle in hypothalamus, the subfornical organ (SFO) in the roof of the third ventricle, the organum vasculosum of the lamina termina lis (OVLT) in the forebrain, the nucleus of the solitary tract (NTS), and the rostral ve ntrolateral medul la (RVLM) in the brainstem 16, 17 Circumventricular organs such as SFO and OVLT are BBB incompletes areas within the brain, which allow circulating Ang II signals from periphery to act iva te these nuclei and to transmit to PVN and RVLM resulting in stimulation of sympathetic drive. Collectively, the evidence led us to hypothesize that altered ex pression of key regulatory gene/associated gene ( s ) in CV relevant regions is linked to neurogenic hypertension. We decided to investigate this hypothesis by the use of proteomic profiling of the PVN from hypertensive rats. SHR, an animal model of human primary hypertension and age matched WKY rats were utilized The rational for de tecting the PVN of the brain for protein profiling was that it is a key cardiovascular regulatory nuclei located in the hypothalamus, transmitting signals from circumventricular organ to brainstem such as RVLM and NTS. Methods Animal S pontaneously hyperte nsive rats (SHRs) aged 12 weeks and age matched WKY rats were purchased from Charles River Laboratories (Wilmington, MA). Rats were individually housed in a temperature controll ed room (22C to 23C) with a 12 : 12 hour light dark cycle. Rat chow (Harlan Tek land) and water were provided by Animal Care
67 Services. All experimental procedures were approved by the University of Florida Institute Animal Care and Use Committee. Primary Neuronal Culture Neuronal cells in primary culture from the brainstem and hypothalamus of one day old SD were est ablished Brains were isolated from neonatal rats and hypothalamic and brain stem areas were dissected into separate dishes. Tissues were trypsinized for 15 min at 37C to dissociate individual neurons. Cells are then plated in poly L lysine pre coated 6 or 12 well culture dishes. DMEM with 10% FBS were used as cultural media and neurons are maintained for 12 14 days prior to use in the experiments Anti mitotic agent, arabinoside C was treated 3 days after plating culture and changed media 3 days later to increase the purity of the neuronal culture. Western Blot Assay PVN p rotein s were extracted from 3 WKY control rats and SHR experimental rats at 20 we eks in age Protein concentration was measured in the supernatant with the Bradford assay kit (BioRad), using BSA (bovine serum albumin) as standard protein Fifteen to twenty micrograms of protein s were run on a 12% SDS PAGE, and the proteins were transferred onto a nitrocellulose membrane. After1 hour blocking with 5% milk in Tris buffered saline Tween 20 the membrane was probed with the rabbit polyclonal a ntibody against Ndufa10 ( 1 g/ml in 3 % milk/Tris buffered saline Tween 20 XW 7882, ProSci ) overnight at 4C The m embrane was washed 3 times for 10 minutes in Tris buffered saline Tween 20 and incubated with anti chicken IgG horseradish peroxidase conjugated secondary antibody (1:5000) for 1 hour at room temperature After final washes, the membrane was incubated with chemiluminescent agent for 1
68 minute and the n exposed to film to visualize protein bands. actin was used as a loading control. 2D Gel and Image Analysis The experimental scheme is described in Figure 4 1. PVN p rotein s were extracted and prepared same way as Western blot assay. The fi rst dimension was performed in an EttanIPGphor (GE Healthcare) using DryStrips pH 3 11 NL, 24 c m (GE Healthcare). Samples were loaded on the acid end of the strip with loading device. The isoelectro focusing conditions were those recommended by the manufac turer for the type of strip used. The SDS PAGE (12 %) was developed in an EttanDALTsix(GE Healthcare) at 257C and 1 W/gel for 12 h. Proteins were visualized by staining with SYPRO Ruby Protein Gel Stain The image analysis was performed with DeCyder v.6.5 s oftware (GE Healthcare). The differently labeled protei n spots in the same gel were detected with the Differential In gel Analysis (DIA) mode of the software using its proprietary algorithms. Spot images from 3 different gels were matched with the Biologi cal Variation Analysis (BVA) mode. The normalized ratios of each protein spot from each gel of all 3 replicates were standardized according to internal standard Proteins were selected when the average fold difference of SHR/WKY was greater than 1.5 or low er than 1.5, and when the p value of Student T test of was less than 0.05. Ndufa10 Overexpression For Ndufa10 overexpression in neuronal cells, AAV plasmid vector inserted with ndufa10 cDNA were produced. Full length cDNAs cloned in the pExpress 1 mamma lian expression vector are commercially available from Open Biosyst em. First, ndufa10 cDNAs was cloned to AAV plasmid vector pTR UF22, which is regulated by the 381 bp CMV immediate early gene enhancer, 1352 bp chicken actin promoter and the
69 exon1 intron1 woodchuck post transcriptional regulatory element (WPRE). The plasmids were amplified and purified by using CompactPrep Plasmid Midi Kit (Qiagen 12843) and then transfected into human HEK 293 cells using standard procedures 159 pTR UF22 ndufa10 and GFP control vectors were trans fected to neuronal HEK 293 cells for 24 and 48 hou rs mRNA were collect ed at various time points ( 24 h, 48 h) to measure Ndufa10 expression. Efficiency will be checked by GFP expression under fluorescence microscope. Measurement of Mitochondrial and C ellular ROS Production Eleve n to thirteen days neuronal cultures were treated with Ang II (500nM), or overexpressed with pTR UF22 ndufa10 Cellular superoxide production was measured by DHE (dihydroethidium, Invitrogen) fluoresc ent staining and mitochondrial superoxide were measured by MitoSOX Red staining (In vitrogen). DHE (1nM) was added to neurons for 30 min at 37 C and cells were washed with PBS three times. For the detection of mitochondrial ROS, neurons were incubated with 5uM of mitoSOX r ed dye for 10 min at 37 C and washed with PBS three times DAPI was used as nucleus counter staining before cells were fixed Images were obtained from Zeiss Axioplan 2 Fluorescent Visualization Microscope. Data and Statistical Analysis D ata were expressed as meanSEM 2 way ANOVAs or 1 way ANOVAs, and the Bonferroni pos t test was used to allow multiple comparisons of cardiovascular variables across time and between different groups. Paired/unpaired Student t test was used for further comparisons between 2 groups where applicable, with P <0.05 considered significant.
70 Resu lts 2D Gel A nalysis Revealed Different ial Protein E xpressi ons in PVN of WKY Rat and SHR First, in collaboration with Dr. Sally Yuan, we c ompared 2D gel profiles of the PVN protein from WKY and SHR carried out at the ICBR Core F acility A representative p rotein gel image is shown in Figure 4 2 PVN p rotein s were extracted from 3 WKY control rats (MAP=95 100 mmHg) and 3 SHR experimental rats (MAP=150 160 mmHg) at 20 we eks in age A t least 1600 spo ts were obtained in each gel but only 1000 spots were matched in all three gels (Figure 4 3 ) The image analysis was performed with DeCyder v.6.5 software (GE Healthcare). The differently labeled protei n spots in the same gel were detected with the Differential In gel Analysis (DIA) mod e of the software using its proprietary algorithms. Spot images from 3 different gels were matched with the Biological Variation Analysis (BVA) mode. The normalized ratios of each protein spot from each gel of all 3 replicates were standardized by the inte rnal control The standardized and averaged ratio between differentially expr essed proteins was calculated. BVA was also used for statistical analysis. Proteins were selected when the average fold difference of SHR/WKY was greater than 1.5 or lower than 1 .5, and when the p value of Student T test was less than 0.05. Figure 4 3 shows the list of proteins that has different ly expressed between WKY rat and SHR PVN Glutathione transferase omega 1, Glutathione S transferase Mu 1, and Nucleoside diphosphoate ki nase A were shown to increase in the SHR by ~3.22, ~3.89, and ~5.57 fold, respectively compared to WKY. Lamin B1 protein, in contrast was decreased by ~4.15 fold in the SHR. W e selected NADH dehydrogenase 1 alpha subcom plex 10 encoded by Ndufa10 since it s howed the highest expression difference between the two strains. This protein showed
71 in a two different spots with differ ent molecular weights The ratio of SHR/WKY was 6.17 fold increased in the spot 720 whereas it was 5.75 fol d decreased in the spot 753 (Figure 4 3 ). We hypothesized that post translational modification is process ed in the SHR responsible for the precursor of two different size s of Ndufa10 protein s. However, further experiments would be required to confirm this Ndufa10 Expression is Incre ased in C ardiovas cular Regulatory Regions of SHR Compared to WKY Western blot analysis was carried out to verify the result of the 2D analysis with an anti Ndufa10 antibody (Pro sci XW 7882). Figure 4 4 shows a comparison of Ndufa10 protein levels in different CV relevant brain regions in WKY rats and SHR Ndufa10 was present in all CV relevant brain regions tested: PVN, RVLM, OVLT, SFO and NTS. In addition, there were significant increas e s in Ndufa10 protein l evels in the SHR compared to WKY rats (PVN: ~2, RVLM : ~1.2 NTS : ~1.3, SFO: ~1.5, OVLT : ~3 fold) (Figure 4 5 ) Ndufa10 from SHR showed greater molecular weight compared to one from WKY There is a study by Meng et al also showing that Ndufa10 expressed in left ventricular of SHR had greater molecular we ight than in left ventricular of WKY rats suggesting that Ndufa10 might be involved in develop ment of cardiac hypertrophy 160 The exact mechanism regarding Ndufa10 post transcriptional modification needs to be further examined N euronal cells cultured from hypothalamic brain of prehyp ertensive SHR were use d to determine if Ndufa10 is genetically linked to hypertension. In the SHR, mRNA and protein levels of Ndufa10 were 1.3 fold and 3.5 fold increased respectively compared to WKY neurons (Figure 4 6 ).
72 N dufa 10 O ver expression Induces Cel lular and Mitochondrial Oxidative Stress in HEK 293 Cells We determined if Ndufa10 is involved with cellular oxidative stress with the use of a human cell line HEK293. Full length cDNAs of ndufa10 was cloned in the pExpress 1 mammalian expression vector. n dufa10 cDNAs was first cloned to AAV plasmid vector pTR UF22, which is regulated by the 381 bp CMV immediate early gene enhancer, 1352 bp chicken actin promoter and the exon1 intron1 woodchuck post transcrip tional regulatory element (WPRE) as shown in Figure 4 7 A The plasmids were amplified and transfected into human HEK 293 cells using standard procedures mRNA s were collect ed at various time points ( 24 h, 48 h) to measure Ndufa10 t ranscription. Ndufa10 mRNA was 2 ~2.2 fold higher after 24 48 hours of transfection with Ndufa10 pl asmid compared with GFP plasmid (Figure 4 7 B) Cellular superoxide was measured by DHE red fluorescence and mitochondrial superoxide was measured by mitoSOX red staining. Cellular ROS was ~2 fold increased with nd ufa10 overex pression (Figure 4 8 A). Additionally, the amount of H 2 O 2 produce d by Ndufa10 overexpression was ~2 fold increased after 48 hours of transfection as shown in Figure 4 8B Mito SOX staining confirmed the increase in oxidative stress is generat ed from mitocho ndria (Figure 4 8 C). Discussion The purpose of the present study was to dete rmine if there are hypertension linked protein(s) are present in the PVN of the SHR. These proteomic studies have revealed that NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (Ndufa10) is significantly up regulated in the PVN and other CV regulatory regions of the SHR In addition, overexpression of ndufa10 significantly induced cellular and mitochondrial
73 ROS generation in HEK 293 cells. Our observation of increase d cellula r oxidative stress by Ndufa10 overexpression is supported by Jin X et al who suggested that Ndufa10 expression in the left ventricular of SHR heart is involved in the increased production of ROS leading to hypertrophic heart. 160 It is tempting to suggest that abnormally re gulated Ndufa10 expression may cause excessive ROS production in the CV regulatory regions, leading to high blood pressure and neurogenic hypertension. Ndufa 10 is one of the 45 subunits in electron transport chain complex I and has dehydrogen ase activity Recent study from Olsson et al found that Ndufa10 is one of decreased genes involved in oxidative phosphorylation in human pancreatic islets from patients with type 2 diabetes 161 Another study demonstrated that Ndufa10 mutations disrupted the start codon and amino acid substitution cause complex 1 deficiency i n a patient with Leith disease 162 However i ts involvement of cellular signali ng pathway in neuronal activity and sympathetic drive in neurogenic hyperten sion is not yet tested. It is possible that differentially regulated Ndufa10 would induce the mitochondria electron transport chain malfunction that leads electrons to leak out of the transport chain leading to excessive ROS production and neural firing in the bra in It is well established that cellular ROS are important for normal signaling pathway in the heart, the kidney and the brain 74, 163, 164 Chronic increases of ROS production especially in CNS are strongly implica ted in development of hypertension 109, 165 167 C irculating Ang II induced hypertension also involves an increase in intracellular superoxide levels in neurons. 56 Ang II induced stimulati on of ROS production may stimulate sympathetic nerve activity in the CNS, thereby contributing to chronic increase in blood pressure. Cellular oxidative stress mediated by NADHP oxidase, an enzyme
74 complex that is mainly activated by Ang II is well document ed. However, a s an important source of cellular ROS production, mitochondrial mediated oxidative stress in hypertension has not been tested. T he leak age of electrons from electron t ransport chain complexes results in the cellular accumulation of ROS includ ing superoxide anion and hydrogen peroxide. Mitochondrial ROS is produced when the enzymatic activities of electron transport chain complex I are reduced and dysfunctional resulting in the accumulation of electrons, which facilitates the direct transfer o f electrons to molecular oxygen 77, 168 However, it has been suggested the possibility of NADPH oxidase activation by mitochondrial ROS. 119 It is possible tha t mitochondria superoxide production can be first triggered by NADPH oxidase activation through Ca 2+ accumulation within the mitochondria, and subsequently produced mitochondrial ROS could inversely activate NADPH oxidase. Studies have shown that m itochond ria derived ROS in CNS mediated elevated sympathetic nerve activity i n hypertension and that is induced by Ang II infusion 169 A r ecent Study by Chan et al also showed that impaired mitocho ndrial activity increased ROS production in the RVLM, and the administration of coenzyme Q10, which is an anti oxidative enzyme restored electron transport chain function and attenuated hypertension. 77 In a view of this, Ndufa10 as the one of the electron transport chain complex subunit may be the key regulator of mitochondrial and cellular ROS generation in CNS and the contributor to neural mechanism of neurogenic hypertension. Altered expression of Ndu fa10 in pre hypertensive stage and its effects on mitochondrial ROS production in the brain of SHR suggests that there might be a genetic correlation between Ndufa10 and development of neurogenic hypertension. However the exact
75 mechanisms in which cellular signaling involving the protein expression regulation are not fully identified.
76 Figure 4 1 Experimental design of 2D gel analysis PVN proteins are isolated from the brain of SHR and WKY rats and two dimentional difference gel electrophoresis (2D DIGE) was performed to compare protein expression level between SHR and WKY rats. Additionaly, protein and mRNA were prepared from both tissue and primary neuronal culture for Westen blot analysis and real time PCR.
77 Figure 4 2 R epresentative 2D DIGE gel from PVN of SHR. Each boxed spot shows significantly up or down regulated protein in SHR compared to WKY. PVN p rotein s were extracted from 3 WKY rats and SHR rats at 20 we eks in age A t least 1600 spo ts were obtained in each gel but only 1000 spots wer e matched in all three gels n=3.
78 Figure 4 3 The list of differentially expressed proteins from PVN of SHR compared to WKY. The image analysis was performed with DeCyder v.6.5 software (GE Healthcare). The differently labeled protei n spots in the same gel were detected with the Differential In gel Analysis (DIA) mode of the software using its proprietary algorithms. Spot images from 3 different gels were matched with the Biological Variation Analysis (BVA) mode. The normalized ratios of each protein spot from each gel of all 3 replicates were standardized by internal control The standardized and averaged ratio between differentially expressed proteins was calculated. BVA was also used for statistical analysis. Proteins were selected when th e average fold difference of SHR/WKY was greater than 1.5 or lower than 1.5, and when the p value of Student T test of was less than 0.05. n=3.
79 Figure 4 4 Distribution of Ndufa10 in cardiovascular relevant brain regions of WKY and SHR. A, Ndufa10 pr otein expression from different CV regulatory regions from WKY. B, Ndufa10 protein expression from different CV regulatory regions from SHR. C and D, Quantification of the bands density. n=3 4
80 Figure 4 5 Inc reased expression of Ndufa10 mRNA and protein in SHR compared to WKY Ndufa10 expression is upregulated in the PVN, RVLM, OVLT, SFO and NTS of SHR. There were significant increas e s in Ndufa10 protein levels in the SHR compared to WKY rats (PVN: ~2, RVLM : ~1.2 NTS : ~1.3, SFO: ~1.5, OVLT : ~3 fold). *P<0.05 vs WKY. n=3 4
81 Figure 4 6 Increased expression of Ndufa10 in cultured neurons from SHR compared to neurons from WKY In the SHR, mRNA and protein levels of Ndufa10 was ~1.3 fold and ~3.5 fold increas ed respectively compared to WKY neurons. *P<0.05 vs WKY by t test. n=5 6.
82 Figure 4 7 Ndufa10 overexpression vector and its efficiency. A, AAV plasmid vector inserted with ndufa10 cDNA or hGFP ndufa10 cDNAs was first cloned to AAV plasmid vector pTR U F22, which is regulated by the 381 bp CMV enhancer, chicken actin promoter and the exon1 intron1 woodchuck post transcrip tional regulatory element (WPRE) The plasmids were amplified and transfected into human HEK 293 cells. B, pTR UF22 ndufa10 and GFP vectors were transfected to HEK 293 cells. Ndufa10 mRNA is 2 2.3 fold increased in ndufa10 vector transfected cells after 24 and 48 hours *P<0.05 vs vector control. n=3 5.
83 Figure 4 8 Increased cellular and mitochondrial oxidative str ess by Ndufa10 overexpression A, Cellular superoxide is measured by DHE fluorescence (dihydroetidium excitation 490 nm/emission 585 nm ) In the presence of Ndufa10 vector transfection for 24 hours, DHE is ~2 fold increased. B, The production of h yd rogen peroxide is increased after 48 hours of Ndufa10 overexpression. C, Mitochondrial specific fluorescenc dye MitoSox staining comfirmed that Ndufa10 overexpression induced ROS is from mitochondria Images were taked from Zeiss Axioplan 2 Fluorescent microscop e. *P<0.05. n=5.
84 CHAPTER 5 CONCLUDING REMARKS A ND FUTURE DIRECTIONS We propose the following hypothesis based on our data and availabl e evidence from the literature. First, neurogenic hypertension is associated with increased oxidative stress in the brain and mitochondrial ROS are the major contributor for the pathophysiology of neurogenic hypertension. Second, dysfunctional EPCs and elevated level s of ICs and the imbalance of EPC/IC ratio are regulated by central nervous system, and there exist a n eural BM communication in hypertension. Third, circulating Ang II activates neuron and microglia that generate cel lular and mitochondrial ROS, stimulating central proinflammatory cytokines, especially in the PVN. T hese modulators further increase neuronal activi ty in CV regulatory regions resulting in elevated sympathetic nerve activity and neurogenic hypertension. Oxidative stress in the brain plays an important role i n the sympathetic nerve activation. NADPH oxidase is a well accepted major player of cellular ROS production. 63, 170 However, the role of mitochondria derived ROS and its contribution to the cellular ROS in neuron and microglia within CNS has not b een tested. Figure 5 1 describe s NADPH oxidase and mitochondria medi ated ROS production in neurons and glia cells. Ang II signal stimulates AT 1 R, which activates NADPH oxidase subunits such as gp91phox and Nox1/2 subsequently leading to superoxide product ion. Rece nt findings indicate that NADPH oxidase derived cellular ROS triggers Ca 2+ accumulat ion within the mitochondria resulting in mitochondrial ROS generation 76 171 They also suggested that mitochondria derived ROS mediate neuronal activity and sympathetic activation 76 171 In the present study we have demonstrated that ICV treatment with mitoTEMPO completely abolish Ang II induced development of neur ogenic
85 hypertension and mitochondrial protein Ndufa10 expression is increased in the brain of SHR We propose that mitochondrial ROS triggered by Ang II or alte red expression of Ndufa10 may contribute to increase in NADPH oxidase activity and cellu lar ROS within CNS, resulting in neuronal sympathoexcitation (Figure 5 1). However, the mechanism by which mit ochondrial ROS and NADPH oxi dase induced ROS are linked in feed forward fashion remains to be investigated in neurogenic hypertension. Previous study has evidenced the importance of activated microglia in Ang II induced neurogenic hypertension and increase in the number of Iba1 positiv e microglia in the PVN. 97 Activated microglia in the brain known to mediate neurotoxicity by producing pro inflammatory cytokines such as IL 1, IL 6, and TNF in neurodegenerative disorders, aging, and diabetic retinopathy. 145, 172 174 As shown in our data, chronic Ang II infusion activates PVN microglia and increase cytokine mRNA levels that is attenuated by mitoTEMPO treatment. These suggest that oxi dative stress plays an import role in microglia activation and cytokine release. Microglia and astrocyte express AT 1 R which mediate Ang II signals and produce cytokines. 175, 176 In Figure 5 2, we propose that neurons are activated by these cytokines and produce mitochondrial and cellular ROS that is further activate microglia. The possibility of microglia generated ROS affecting neuron and microglia itself cannot be ruled out. Stimulation of proinflammatory cytokines and other mediators within the brain induces a neuroinflammatory response that is associated with incre ase in p eripheral inflammatory respon se in cardiovascular disease s such as hypertension. Under normal conditions, healthy endothelial cells contribute to maintain ing blood vessel integrity and vascular tone along with smooth muscle cells. EPCs participate in the repair and
86 regeneration of end othelial cell injury induced during the course of normal stress and environmental insult to maintain vascular homeostasis. 177 We propose that an imbalance in EPC/IC ratio and associated EPC dy s fun c ti on compromise their repair capacity leading to vascular pathophysiology, the hallmark of chronic hypertension. Thus, we believe that c hanges in BM or circulating EPC numbe r and function may serve as a biomarker for cardiovascular endothelial dysfunction. A ccording to the Department of Human Health and Services R eport in 2009, a reduction in EPC numbers and decreased functional ability are currently being used to predict potential cardiovascular diseases in c linical studies. Patients with i schemic cardiomyop athy exhibit increased number of EPCs in early disease stages but decreased in the latter stages of congestive heart failure. 178 Also diabetes, chronic kidney disease, pulmonary hypertension and aging showed decreased number of EPCs associated with endothelial dysfunc tion. 102, 103 105 100, 179 T o increase the number and function of endogenous EPCs or to infuse exogenous healthy EPCs could be the pharmacological or genetic targets for cardiovascular disease treatments. It is well established that da maged endothelium and imbalance in vascular homeos tasis are associated with decreased number and dysfunctional EPCs in hypertension. However, there has been no evi dence if there is a central regulation of EPCs in hypertension. Endothelial dysfunction is an early event in high blood pressure induced vascul ar pathophysiology and that is associated with increased sympathetic outflow. It is tempting to suggest that there is a neural regulatory mechanism for EPC mobilization by sympathetic stimulation to BM. Our res ults from mitoTEMPO ICV administration demonst rated the possible regulatory mechanisms of central BM
87 communication in neurogenic hypertension. mitoTEMPO ICV infusion prevents Ang II in duced decrease in BM EPCs and increase in BM ICs Figure 5 3 summarizes the hypothesis of regulatory mechanism of neur al microglial BM connection in neurogenic hypertension There is no clear evidence in the literature to support the involvement of CNS in the regulation of EPC s and IC s in cardiovascular disorder such as hypertension so far. Therefore, the exact mechanism i n which CNS regulates EPC s and IC s by the innervating nerves to BM via sympathetic outflow is uncertain. Additionally the identification and characterization of EPC are still controversial. There are number of different surface markers of EPCs that are bei ng used in the number of studies. It is required to establish a specific cell surface marker to unambiguously distinguish EPCs from closely related other cells. Utilizing different neurogenic hypertensive model such as SHR needs to be required to confirm n eural BM communicatory connection. Moreover, targeting specific CV regulatory area such as PVN in the brain to stimulate sympathetic nervous system BM innervations pathway would be interesting Lastly, the involvement of re nin angiotensin system including AT 1 R, Ang 1 7, ACE and ACE2 in the regulation of brain BM regulatory mechanism cannot be ruled out. This study provides evidence in support of neural microglial BM regulation of cardiovascular system. It raises important issues regarding neuroinflammatory signals in other CV regulatory regions including NTS and RVLM, and BM neural retrograde signal transduction with the involvement of microglia. Additional examination would be necessary to provide further proof of concept for this novel neural peripheral axis.
88 Figure 5 1 AngII AT 1 R medi ated ROS production in neurons and glia cells NADPH oxidase is activated by AT 1 R, producing cellular ROS. In addition, mitochondrial ROS Ca 2+ accumulation is triggered by AT 1 R and NADHP oxidase, which produce superoxide. Increased expression of Ndufa10 results in mitochondrial ROS production, leading to increase in Ang II independent cellular ROS production. Phospholipase C (PLC), Phosphokinase C (PKC), cellular superoxide dis mutase (SOD 1 ), SOD mimetic (TEMPOL), mitochondrial suporoxide dismutase (SOD2), mitoTEMPO (mito chondrial targeting antioxidant ).
89 Figure 5 2 Proposed hypothesis of activated microglia neuron astrocyte interaction in the CNS of hypertension AT 1 R s are p resent in mic roglia, neuron and astrocyte Microglia are activated by Ang II signal, oxidative stress and microglia activator factors released from nearby damaged neuron such as MMP3 and neuromelanin Cytokines are released from activated microglia and they stimulate microglia as autocrine signaling Neuronal activity is increased by cytokines from microglia and astro cyte and produce ROS, resulting in increase in sympathetic drive.
90 Figure 5 3 Proposed regulatory mechanism of ne ural microglial BM connection in neurogenic hypertension AT 1 Rin circumventricular organs (SFO, OVLT) are activated by increased circulating Ang II, and signals are transmitted to the PVN neuron and microglia. Increased neuronal mitochondrial ROS mediates microglial activation and neuronal activity, resulting in increase in sympathetic drive from brain to BM. An imbalance in EPCs/ICs is due to the d ecrease in EPCs and increase in ICs associated with blood pressure and ca rdiovascular pathophysiology. Peripheral Ang II also directly affect s BM EPCs and ICs. ( Modified from Zubcevic et al 2011 )
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104 BIOGRAPHICAL SKETCH Joo Yun Jun was born in Sangju, South Korea in 1981. She is the first childwith two younger sisters to In Hee Hwang and Byung Gon Jun Joo Yun graduated from Sangju Elementary School in 1993, Sung Shin Girls Junior High School in 1997, and Sangju Girls High School in 2000. After that, she was accepted to Chung Ang University in Seo ul where she majored in biology in department of Life Science. She earned a Bachelor of Science in 2005 During her college years, she had studied abr oad at the University of Oregon, US, for 6 months and enrolled at the American English Institute. After graduation, she started her Master of Scie nce degree at Korea University and Korea Institute of Science and Technology in 2005. Joo Yun o btained her masters degree in F ebruary 2007 and join ed the interdisciplinary program in biomedical sciences at t he University of Florida, College of Med icine in Gainesville, Florida in fall 2007.She did her graduate work in Dr. Mohan K. Raizadas laboratory of the Department of Physiology and Pharmacology and completed h er Ph.D. dissertation in Spring 20 12