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1 ROLE of TGF SIGNALING IN THE PATHOGENESIS OF VASCULAR DISEASES By CHUL HAN 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 2011
2 2011 Chul Han
3 To my loving wife, parents, and brother
4 ACKNOWLEDGMENTS Most o f all, I thank God for giving the chance to have these great mentor s in my life T heir thoughtful guidance made me to enjoy my journey from a curious boy to a young scientist Dr s. Seongman K ang and Hyangshuk Rhim taught me the alphabet of scientific research beginning from gene cloning a nd cell culture. Most of all, they emphasiz ed keeping a consistent attitude not to make a big deal of the smallest things Even now it is one of my most important attitudes toward s cience. Truly, Dr. S. Paul Oh was a great m entor to me L ike a fathe r, he showed a big picture w hen I was perflexed, or uncertain about what I was doing. He encouraged me to get th rough difficult moments when I was depressed and humbled me when I am doing well. L ike a friend he gave me high five s whene ver I made even small success Like a big brother, he has been a good listener even for complains about trivial things. Most of all, he and I share the same passion and enthusiasm for the science. I am very proud to be his disciple and h e is my role model both as a person and as a scientist I also thank my thesis committee members, Drs. Mohan Raizada Naohiro Terada and Peter Sayeski for providing passionate assistance during my graduate career. Since I had a laboratory rotation in Dr. Terada s lab, he ha s been always open to me. He made me enjoy talk ing with him about any topics Even when I asked stupid questions, he g ave me wise answers. I appreciate Dr. Raizada s passion ate and sh arp suggestions for me Whenever I h ad a committee meeting I looked forw ard to his creative questions and comments. Furthermore, h e showed me how to live younger by keeping his humor. I thank Dr. Sayeski s thoughtful consideration. When ever I requested something, he always helped me with all his available resources He has been a good listener. I am also inde bted to past and current Oh lab members for productive discussions and for
5 comfortable environment Dr. K won Ho Hong taught me important concepts and techniques necessary for the pulmonary hypertension project I appreci ate his invaluable suggestions about be ing a great scient ist who can see the big picture. Halong is my good colleague and lab member. I appreciate her open mind edness and friendship Dr. Sung Ok P ark helped me a lot expand my knowledge and learn many tech nique s necessary for the HHT study. Her positive attitude help me appreciate for even the small est things. I like Yong H wan Kim s perfectionism as well as his careful cons ideration of me and others. I was able to enjoy the lab life much more because of his unlaughable jokes. I thank Dr. Peter Dickie for his wisdom, patience, and mentorship. Whenever I shared trouble s and difficulties, h e always pointed ou t what I missed and did not forget to give me critical suggestions for future experiment s He edited m y writings for my poor English and encouraged me to focus on the contents I am very grateful to Korean students in the interdisciplinary program As Korean students, we not only shared the common troubles as foreign students but also intensively discussed a bout contemporary scientific issues and our own experimental data. The s e active meeting s were very helpful to improve my critical thinking skills Specially, I appreciate Sewoon Choi. He helped me with my research in imaging analysis. Furthermore, he is on e of the people I can talk openly without any hesitation Thanks to the gathering s I had wi th him, I was able to reduce half of my stress by sharing my joy and sadness spare her suggestions and encouragement as a PhD to fill my deficiencies as a PhD
6 s tudent. I would like to give love and thank s to my mother father, and younger brother w ho have been supporting throughout
7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 SMAD1 DEFICIENCY IN EITHER ENDOTHELIAR OR SMOOTH MUSCLE CELLS RESULTS IN PULMONARY ARTERIAL HYPERTENSION ....................... 14 Introduction ................................ ................................ ................................ ............. 14 Pulmonary Arterial Hypertension (PAH) ................................ ........................... 14 Pathology of Pulmonary Arterial Hypertension ................................ ................. 14 Current Treatment Options for PAH ................................ ................................ 16 Prostacyclin (prostaglandin I 2 ) ................................ ................................ ... 16 Endothelin 1 receptor antagonists ................................ ............................. 17 Nitric oxide (NO) and cyclic GMP (cGMP) ................................ ................. 18 Calcium channel blocker ................................ ................................ ............ 18 Pathologic Mechanism of PAH ................................ ................................ ......... 19 Involvement of BMP signaling based on genetic studies ........................... 19 TGF ................................ ................................ ...... 20 Endothelial dysfunction in PAH ................................ ................................ .. 21 Compromised immune response and inflammation ................................ ... 22 Role of TGF .................. 22 Is SMAD Important Signaling Mediator for PAH Caused by BMPR2 Deficiency? ................................ ................................ ................................ .... 23 Endothelial BMPR2 Deficient PAH Mouse Model ................................ ............ 24 Deletion of Smad1 Gene in ECs or SMCs ................................ ........................ 24 Results ................................ ................................ ................................ .................... 25 SMAD1/5/8 Phosphorylation was Impaired in the Lungs of Pulmonary Hypertensive L1cre(+); Bmpr2 2f/2f Mice. ................................ ......................... 25 Smad1 Deletion in Pulmonary ECs or SMCs by L1cre or Tagln cre ................ 26 Some Mice with the Smad1 Deletion in Pulmonary ECs or SMCs Exhibited Elevated Pulmonary Pressure and Right Ventricul ar Hypertrophy. ............... 26 Positive Distal Arteries and Medial Wall Thickness Were Increased in the Smad1 Mutant Mice. ................................ ................. 27 Isolation of Pulmonary Endothelial Cells Carrying R26 creER/+ ; Bmpr2 2f/2f Allele .. 27 Deletion of Bmpr2 Gene and Impaired BMP Signaling ................................ ..... 28 Enhanced TGF Deficient pECs ................................ .... 29 Opposing Balance Between TGF and BMP S ignaling s in pECs ................... 29
8 Discussion ................................ ................................ ................................ .............. 30 SMAD1 is a Downstream Mediator of BMP Signaling in Pathogenesis. ........... 30 SMAD1 Involvement in the Pathogenesis of PAH ................................ ............ 31 SMAD1 Deficiency in ECs has a Greater Impact on PAH Than That in SMCs ................................ ................................ ................................ ............ 32 Incomplete Penetrance of PAH in Smad1 Conditional KO Mice ....................... 33 Imbalance in TGF ...................... 33 T hickening of the Smooth Muscle Layer Might not be Sufficient to Lead to High Pulmonary Pressure ................................ ................................ ............ 35 Smad1 cKO Mice and Inducible Bmpr2 KO pECs are Useful Resources fo r Future Mechanism Research, Drug Screening and Preclinical Study. .......... 36 2 THERAPEUTIC EFFECT OF VEGF BLOCKADE ON ARTERIOVENOUS MALFO RMATIONS (AVMS) IN AN ANIMAL MODEL FOR HEREDITARY HEMORRHAGIC TELANGIECTASIA ................................ ................................ ..... 46 Introduction ................................ ................................ ................................ ............. 46 Hereditary Hemorrhagic Telangiectasia (HHT) ................................ ................. 46 Limitation of Current Therapeutic Interventions in HHT ................................ .... 47 Genetic Studies in HHT ................................ ................................ .................... 47 Genetic Mouse Models for HHT ................................ ................................ ....... 47 Secondary Hits are Required for AVM Development. ................................ ...... 48 Results ................................ ................................ ................................ .................... 49 Vascular Endothelial Growth Factor (VEGF) or Lipopolysaccharide (LPS) can Induce De Novo AVMs in Alk1 Deficient Subdermal Vessels. ................ 49 VEGF and LPS Stimulated Angiogenesis. ................................ ........................ 50 VEGF Blockade Suppressed Wound Induced AVM in Alk1 Deficient Subdermal Vessels. ................................ ................................ ...................... 51 VEGF Blockade Alleviated Internal Bl eeding and Low Hemoglobin Occurring in Alk1 Deficient Mice. ................................ ................................ .. 51 VEGF Blockade Suppressed LPS Induced AVMs in Alk1 Deficient Subdermal Vessels. ................................ ................................ ...................... 52 TNF ...................... 53 Discussion ................................ ................................ ................................ .............. 53 Therapeutic Potential of Anti Angiog enic Drugs in HHT ................................ ... 53 Therapeutic Potential of Anti Inflammatory Intervention ................................ ... 54 Inflammatory Stimulation Associated with Angiogenesis is Important for Inducing AVM Formation. ................................ ................................ .............. 55 3 MA TERIALS AND METHODS ................................ ................................ ................ 65 Smad1 Conditional Knockout Mice ................................ ................................ ......... 65 Hemodynamic Analysis ................................ ................................ ........................... 65 Right Ve ntricular Hypertrophy (RVH) ................................ ................................ ...... 66 Pulmonary Vessel Morphometry ................................ ................................ ............. 66 Establishment of Immortalized Pulmonary Endothelial Cells ................................ .. 67 Semi Quantitative RT PCR ................................ ................................ ..................... 68
9 West ern Blotting ................................ ................................ ................................ ..... 68 Alk1 Conditional Knockout Mice ................................ ................................ ............. 69 Preparation of PLGA Microparticles ................................ ................................ ........ 69 Injection of PLGA Particles into Subdermal Area ................................ .................... 70 Skin Wound Generation and VEGF Antibody Treatment ................................ ........ 71 Latex Dye Injection and Image Processing ................................ ............................. 71 Hemoglobin Concentration and Hemorrhage Index ................................ ................ 72 Hypertrophy of the heart ................................ ................................ ......................... 72 Stat istical Analysis ................................ ................................ ................................ .. 73 4 CONCLUDING REMARKS AND FUTURE DIRECTIONS ................................ ...... 74 LIST OF REFERENCES ................................ ................................ ............................... 77 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 97
10 LIST OF TABLES Table page 1 1 Primers for genomic and semi quantitative PCR reaction ................................ .. 37 2 1 The number of mice with hemorrhage in saline and VEGF antibody treated groups ................................ ................................ ................................ ................ 57
11 LIST OF FIGURES Figure page 1 1 BMP response was severely impaired in the lungs of PH mice [ L1cre(+); Bmpr2 f/f ] ................................ ................................ .............................. 38 1 2 Smad1 de letion in L1cre(+); Smad1 f/f and Tagln cre(+); Smad1 f/f mice. ............... 39 1 3 Smad1 deletion in SMCs and ECs resulted in elevation of RVSP and RV hypertrophy.. ................................ ................................ ................................ ....... 40 1 4 Elevated pulmonary pressure is associated with pulmonary vascular remodeling. ................................ ................................ ................................ ......... 41 1 5 Deletion of Bmpr2 gene in pulmonary endothelial cells (pECs) .......................... 42 1 6 Bmpr2 deleted pECs display ed a reduce d response to BMP4 but not BMP7 .... 43 1 7 Reduced BMP response induced overactivation of TGF ................ 44 1 8 BMP and TGF form an opposing balance in pECs. .......................... 45 2 1 Angiogenic or inflammatory stimulation induces AVMs in Alk1 deficient adult subdermal vessels but not Alk1 wild type vessels ................................ .............. 58 2 2 Angiogenesis blockade (VEGF neutralizing antibody) suppressed wound induced AVM formation in Alk1 defi cient adult subdermal vessels. .................... 59 2 3 Angiogenesis blockade improved hemoglobin levels and visceral hemorrhage in Alk1 deficint adult mice.. ................................ ............................. 60 2 4 Angiogenesis blockade suppressed LPS induced AVMs in Alk1 deficient mice ................................ ................................ ................................ .................... 61 2 5 TNF Alk1 deficient mice. ................................ 62 2 6 Quantification of AVM formatio n by various stimuli in Alk1 deficient subdermal vessels. ................................ ................................ ............................. 63 2 7 Lung (top) and GI bleeding (bottom) was categorized into three g roups (weak, moderate, and severe) depending on the severity. ................................ 64
12 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 ROLE of TGF SIGNALING IN THE PATHOGENESIS OF VASCULAR DISEASES By Chul Han August 2011 Chair: S. Paul Oh Major: Medical Sciences Physio logy and Pharmacology Gene tic mutations causing diverse genetic vascular diseases ha ve been found in the genes that encode components of the transforming growth factor ( TGF ) signaling pathway suggesting that TGF essential for hom e ostasis o f the vascular system T o explore the role of TGF signaling in the pathogenesis of a couple of vascular diseases, our laboratory developed genetic mouse models for two major vascular diseases pulmonary arterial hypertension (PAH) and hereditary hemorrhagic telangiectasia (HHT) PAH is a rare but fatal disease that increases pulmonary pressure due to thickening of pulmonary arterial walls Although d eficiency of bone morphogenetic protein type II receptor (BMPR2) in TGF signaling is known as a g enetic contributor, whether dysfunction of SMAD1 one of the canonical transducers of BMPR2 is critical for PAH development remains unknown. We hypothesized that d eficiency of SMAD1 would lead to PAH and tested the hypothesis using Smad1 con ditional knocko ut (cKO) mice. The Smad1 gene was de leted in endothelial cells or smooth muscle ce lls using L1cre and Tagln cre lines respectively. We discovered that these Smad1 c KO mice develop PAH, suggesting that SM AD1 could b e a critical downstream mediator of BMPR2 in PAH. HHT2 is a vascular disease with arteriovenous
13 malformation s (AVMs) and visceral hemorrhages due to ALK1 deficiency. Our previous studies showed that wounding play s an essential ro le in AVM development in the A lk 1 deficient context. We investigated the involvement of tw o major wound healing responses: inflammat ion and angiogenesis. We found that either LPS (for inflammation) or VEG F (for angiogenesis) could recapitulate the wound induced AVM formation in A lk 1 deficient skin. The inhib it ion of angiogenesis with VEGF neutralizing antibo dy (Ab) significantly inhibited both LPS and wound induced AVMs and ameliorated internal bleeding s in Alk1 deficient mice s uggesting a critical role for angiogenic st imulation in AVM development. These dat a provide a better scientific basis for the therapeutic effect of a VEGF blockade (Bevacizumab) for epistaxis, GI bleeding, and liver AVMs in HHT patients. Our data also demonstrated that the skin AVM model by Alk1 conditional knockout mice is reliable for preclinical screening of drug candidates for epistaxis and GI bleedings.
14 CHAPTER 1 SMAD1 DEFICIENCY IN EITHER ENDOTHELI AR OR SMOOTH MUSCLE CELLS RESULTS IN PULMONARY ARTERIAL H YPERTENSION Introduction Pulmona ry Arterial Hypertension (PAH) Pulmonary Arterial Hypertension (PAH) (formerly primary pulmonary hypertension PPH ) is rare but fatal lung vascular disease and is characterized by sustained elevation of mean pulmonary arterial pressure (PAP) and increased pulmonary vascular resistance leading to right heart failure. PAH is one of 5 types of pulmonary hypertension (PH) which is caused by chronic thrombosis, embo lization, elevated left ventricular end diastolic pressure, lu ng disease/hypoxemia or valve disease. While PH is diagnosed by the sole criterion being a resting mean PAP > 25 mmHg, diagnosis of PAH is required two additional criteria : more than 3 Wood unit s of pulmonary vascular resistance (PVR), and less than 15 mmHg of pulmonary capillary wedge pressure without other causes of PH. PAH is subclassified into idiopathic PAH (IPAH), heritable PAH (HPAH), and PAH associated with other diseases (APAH). Other di seases include congenital heart defect, portal hypertension, HIV infection, connective tissue disease, appetite suppressant drug use 1 Pathology of Pulmo nary Arterial Hypertension The basic pathological phenotype of PAH is a narrowing and thickening of small pulmonary vessels. All PAH patients exhibit pulmonary vascular remodeling of all layers of the vessel: intimal thickening, smooth muscle cell hypertrophy, adventitial fibrosis a nd occluded vessels by in situ thrombosis 2 The intimal layer is a single layered lining of endothelial cells (ECs) between the internal elastic lamina and lumen. Normally, ECs are q uiescent but in PAH lungs, they are activated, rapidly proliferat ing and fo rm
15 neointima layers Myofibroblast is one of major cell types in the neointima and expresses sm MA) and vimentin instead of EC makers such as CD31 and the von Willebrand factor (vWF) 3 Although the origin of the myofibroblasts remains unknown, in vitro data s uggest that myofibroblasts are likely originated from vascular SMC or transdifferentiated from EC. 4 5 Plexiform lesions are commonly observed in the severe form of PAH showing multiple capillary like channels in a pulmonary artery 6 They contain several cell types including ECs, myofibroblast, and connective tissues 7 ECs are the most responsible cell types for initiation of plexiform lesions through monoclonal proliferation of tumorlet like clusters of ECs A study showed that ECs with BMPR2 mutation are more susceptible to apoptotic stimulus and after repeated apoptosis, the surviving ECs are apoptos is resistant and undergo considerable proliferation and develop plexiform lesions 8 Vascular SMCs are a predominant cell type of the medial layer and normally unresponsive to mitogen s 9 Hyperplastic SMCs are a common phenotype in all the different forms of PAH. In precapillary vessels, cells inside internal elastic lamina appear to be involved in differentiation into SMCs 10 and in distal vessels lacking elastic lamina, pericyte and interstitial fibroblast surrounding lung parenchyma seem to contribute to muscularization 11 Remodeling of a dventitia layer by increase of fibroblasts is associated with induction of many proinflammatory cytokines including monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 2, interleukin (IL) 6 12 Endothelial level of prostacyclin ( prostaglandin I 2 ), an endogenous vasodilator, an inhibitor of platelet aggregation and a suppresso r of vascular SMCs proliferation, was shown to be decreased in patients with PAH 13 The mechanism
16 involved in this observation could be that lowered expression of prostacyclin synt hase impaires balance between prosta cyclin and thromboxane A2, a vasoconstrictor and a potent stimulator of platelet aggregation 14 Thromb otic lesions in PAH are associated with imbalanced ratio of prostacyclin and thromboxane A2 and potential outcome of the serotonin pathway 15 16 Current T reatment O ptions for PAH The incidence of PAH is 2 3 cases per million people every year 17 There is age variability for the initial onset of PAH 18 19 Without proper treatments, mortality is predicted within 3 years after diagnosis. Up to now, the following tre atments are available and have been used widely Prosta cyclin (prostaglandin I 2 ) One of the most extensively and succ essfully used therapies for PAH patients is to increase the level of prostacyclin with exogenous prostanoids. Fatty acid cyclooxygenase metabolizes arachidonic acid to prostaglandin H 2 a substrate for both prostacyclin (prostaglandin I 2 ) synthase and thromboxane synthase. Prostacyclin is expressed by prostacyclin synthase in endothelial cells and works as a vasodilator through stimulation of cyclic AMP (cAMP) and inhibits proliferation of SMCs 20 On the other hand, thromboxane A2 ( TxA2 ) is produced by throboxane synthase in platelet and endothelial cells and stimulates vasoconstric tion and platelet aggregation PAH patients showed decreased prostacyclin metabo lites and increase of TxA2 production 14 indicating that endothelial dysfunction and platelet activation in PAH might impair the balance between vasodilator s and vasoconstrictor s Continuous intravenous infusi on of epoprostenol ( Floran ) started in the early 1980s, has shown to decr ease pulmonary vascular resistance (PVR), increased cardiac output, and improve d exercise capacity
17 and overall survival rates 21 23 Due to poor stability, high expense, and side effects from intravenous treatment of epoprostenol 24 more stab le analogs and alternative delivery of prostacyclin were developed: subcutaneous (treprostinil), oral (beraprost), or inhaled (iloprost) delivery of prot acyclin analog s 25 27 Recent combination therapy of prostanoid and a PDE5 inhibitor was effective in improving pulmonary hemodynamic change and exercise toleran ce in PAH 28 2 9 Endothelin 1 receptor a ntagonists Endothelin 1 (ET 1) works as a vasoconstrictor via 2 type s of receptors ( ETA and ETB ) and has a mitogenic effect on SMC s 30 31 Both receptors are expressed in SMCs and mediate vasoconstriction while ETB in ECs promotes vasodilation through NO and prostacyclin production 32 33 PAH patients exhibit ed high levels of lung and circula ting ET 1 34 35 Endothelin receptor antagonist such as Bosentan ( a nonselective ET receptor antagonist ), sitaxsentan and ambrisentan (selective ETA receptor blocker ) were FDA approved and showed significant but moderate improvement in pulmonary hemodynamics and 6 minute walk distance 36 39 However, there is no clear evidence showing advantage of selective ETA antagonism over combined antagonism for both ETA and ETA. Liver toxicity and teratogenicity are class ic side effects. Despite a re port that bosentan monotherapy increased survival 40 more robust dat a for survival are necessary for clinical trials. Since the endothelins are produced from pro endothelin by endothelin converting enzyme, inhibitors for endothelin converting enzyme could be an alternative approach to blocking overproduction of endothelin in PAH. Studies with this inhibitor (eg, daglutri l) have been conducted in systemic hypertension and heart failure 41 and application for PAH patients are being investigated.
18 Nitric o xide (NO) and cyclic GMP (cGMP) NO produced by endothelial NO synthase (eNOS) dilates blood vessel in the pulmonary circulation. Endothelium derived NO s timulates soluble guanylate cyclase (sGC) to produce intracellular cGMP in SMCs 42 I ncreased cGMP activates cGMP dependent protein kinase (cGKI) and decrease s the sensitivity of myosin to calcium induced contraction and lowers concentration of calcium released from the sarcoplasm ic reticulum 42 P hosphodiesterase type 5 (PDE5) the target of sildenafil opposes NO dependent vessel dilation by suppressing the rise of NO induced cGMP 43 NOS expression and NO bioavailability were shown to be reduced in the lungs of PAH patients. 44 46 Furthermore, PDE5 is upregulated in the hypoxia mediated animal model for PAH which worsens cGMP availability 47 48 Endogenous NOS inhibitors, asymmetrical and symmetrical dimethylarginines (ADMA and SDMA) appeared to be more abundant in PAH 49 50 Thus, Inhaled NO gas dilates pul monary arteries, which lowers vascular re sistance, PA press ure, and RV afterload 51 52 T he oral delivery of s i l denafil citrate an inhibitor of PDE5, improved exerc ise capacity and hemodynamics in PAH patients 53 Calcium channel blocker I ntracellular calcium and calcium mediated signals play a crucial role in smooth muscle contraction 54 55 A study has shown that a high dose of calcium channel blockers have a beneficial effect on the survival of some PAH patients 56 However, i t should be used very carefully because calcium channel blockers are only convincingly effective in the 5% of patients who showed an acute vasoactive response to vasoreactivity testing 56 57
19 In summary, c urrent t reatments for this disease alleviate symptoms and improve heart functions but are not a cure of the disease. B etter understand ing on the pathogenetic mechanism s of disease would allow development of drugs targeting the cure. Patholog ic M echanism of PAH Involvement of BMP signaling based on genetic studies Genetic studies have found the linkage of a locus for the gene, named PPH1 to chromosome 2q31 32 in 1997 58 59 and later showed that Bone Morphogenetic Prote in type 2 Receptor ( BMPR2 ), one of the receptors in sup erfamily signaling is responsible for heritable PAH in an autosomal dominant manner 60 63 A heterozygous BMPR2 mutation w as found in nearly 70% of HPAH patients and also in 25% of sporadic IPAH patients. 63 65 67 The levels of B MPR 2 mRNA and proteins are markedly reduced in the lung s of PAH patients with heterozygous B MPR 2 mutation s 64 indicating that BMPR2 mutation s are associated with hap loinsufficiency P enetrance of disease is low and disease express ivity varies even within members of a fami ly Estimates showed that only a bout 20% of individuals wit h BMPR2 mutation develops PAH during their entire life This low penetrance in PAH suggests that a dditional factors such a s inflammation may be neces sary for clinical manifestations of PAH in addition to the genetic predisposition. While Bmpr2 +/ mice displayed mild PAH phenotype, a denovirus mediated pulmonary overexpression of 5 lipoxygenase (5 LO), a mediator of inflammation, or a chronic infusion of serotonin (5HT) develop ed full blown PAH in Bmpr2 +/ mice 68 69 Interestingly, BMPR2 is downregulated in the lung tissues and cells from idiopathic PAH patients without Bmpr2 mutation 64 70 implying a wide range of influence of BMPR2 deficiency to other forms of
20 PAH. Furthermore, B MPR 2 signaling plays an important role in the proliferation of local endothelial cells and the migration and local proliferation of smooth muscle cells 8 71 Taken together, impaired BMP signaling due to BMPR2 deficiency would be a considerable contribut or for PAH development. TGF perfamily signaling TGF including cell proliferation, migration, ap optosis, pattern formation, and immunosupression. TGF TGF proteins (BMPs), growth and dif ferentiation factors (GDFs), activins/inhibins and m lle rian inhibiting substance (MIS) / anti Mllerian hormone (AMH ) 72 74 TGF transduction is initiated by binding of ligands to heteromeric complex of transm embrane serine/threonine type 2 and type 1 receptors Once ligands bind to a type 2 receptors, the ligand type 2 receptor complex recruits and trans phosphorylates type 1 receptor, which, in turn, activates receptor regulated SMADs (R SMADs): SMAD2/3 for TGF and SMAD1/5/8 for BMPs. R SMADs then form a complex with a common partner, SM AD4 (Co SMAD) and enter the nucleus and initiate transcription of target genes. On the other hand, there is mounting evidence demonstrating that independent ly of SMADs, TGF signaling can be transduced through mediators other than SMADs such as the mitoge n activated protein kinases (MAPKs), including p38MAPK, p42/44MAPK (ERK1/2), and c Jun N terminal kinase/stress acti vated protein kinase (JNK/SAPK) 75 76 For instance, exogenous BMP ligands stimulate phosphorylation of p3 8MAPK and p42/44MAPK, and affect prolifera tion of SMCs 70 77
21 Endothelial dysfunction in PAH The endothelium is the cells lining the interior surface of blood vessels in the entire circulatory syst em. Endothelial c ells function in many aspect s of vascular biology : vasoconstriction and vasodilation and hence the control of blood pressure blood clotting ( thrombosis & fibrinolysis ), atherosclerosis formation of new blood vessels ( angiogenesis ), inflammation and swelling ( oed ema ) and also control of the passag es of materials and blood cells (permeability). Thus, endothelial dysfunction may result in increased coagulation proliferation, and vasoconstriction 78 81 Endothelial dysfunction seems to play an integral role in mediating the structural changes in the pulmonary vasculature. Disordered endothelial cell prolifera tion along w ith concurrent neoangiogenesis results in the formation of glomeruloid structures known as the plexiform lesions, which were found in the pulmonary vessels of patients with severe PAH 82 In vitro studies with endothelial cells suggest a plausible mechanism of angioproliferative change of ECs in PAH Loss of BMPR2 function cannot protect ECs from apoptosis representing a possible initiating step and increased apoptosis of ECs promoted appearance of the apoptosis resistant cells 8 83 In add ition, an altered prod uction of various endothelial vasoactive mediators such as Nitric oxide (NO), prostacylin, endothelin 1 (ET 1), serotonin, and thromboxane, has been increasingly recognized in patients with PAH 14 34 84 86 Since change of these mediators can affect the growt h of the smooth muscle cells, alteration in their production may facilitate the development of pulmonary vascular hypertrophy. Thus, it is conceivable that the beneficial effects of currently available treatments for PAH, such as prostacyclin, NO, and ET ant agonists, result in part from restoring the balance betwee n these mediators. Furthermore, e n dothelial dysfunction may bring about a change in the EC permeability
22 and allow direct contact of serum proliferative mediators with the subendothelium, leading to cell proliferation in the medial and advential layers 87 89 C ompromised i m mune r esponse and i nflammation Pulmonary hypertension has been associated with connective tissue disease 90 human immunodeficiency virus (HIV) 9 1 and auto antibodies 92 Mononuclear cell infiltration was often observed in the PAH vascular lesions 64 Inflammatory cells including macrophag es and lymphocytes are highly accumulated in the plexiform lesions of hypertensive pulmonary vessels 7 Induction of many proinfl a mmatory cytokines and chemoki nes, such as monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1 IL 6, RANTES, fractalkine has been implicated in PAH 12 93 97 In the classical PH animal model, Monocrotalline (MCT a pyrrolizidine alkaloid plant toxin ) injected rats elevated pulmonary pressure with apoptosis of ECs and SMCs and massive mononuclear infiltration into the perivascular region s of pulmonary arterioles 98 99 Therefore, the observations f rom a variety of h uman and animal studies suggest a compelling conclusion that intact immune system is required to maintain homeostasis of pulmonary circulation. In other words, dysregulated immunity could be an environmental second hit for clinical mani festation of PAH in individuals with Bmpr2 mutation. Role of TGF / BMP B alance in P ulmonary V ascular H omeostasis Only 20% disease penetrance in individuals with Bmpr2 mutation implies that the Bmpr2 mutation is necessary but not sufficient alone for clinical manifestation of PAH. This fact suggests that other genetic or environmental modifiers are necessary for initiation and progression of PAH. TGF could be a possible candidate of modifiers in PAH.
23 BMP and TGF ling form an opposing balance in pulmonary vascular homeostasis 100 Thus, BMPR2 deficiency may result in predominant TGF which favors fibrosis and smooth muscle cel l growth. R ecent studies suggest that an imbalance between TGF s to PAH development Activation of BMP signaling suppresses proliferation of pulmonary vascular smooth muscle cells 64 Furthermore, TGF early onset of PAH and increased penetrance of heritable PAH 101 Zaiman et al reported that increased TGF monocrotaline induced PH 102 Long et al showed that suppression of TGF via activin receptor like kinase 5 inhibitor prevents development and progression of PAH in the monocrotaline model 103 Impaired BMP signaling from BMPR2 deficiency would not be sufficient to develop PAH and when in conjunction with predominance of TGF finally would elevate pulmonary pres sure. Is SMAD I mportant S ignaling M ediator for PAH C aused by BMPR2 D eficiency? Human genetic studies have identified various BMPR2 mutations throughout the exons coding for the BMPR2 protein 104 These data imply that d eficiency of BMPR2 is a crucial genetic factor in the PAH development However, which downstream signaling molecules of BMPR2 such as MAPKs or SMADs contribute to PAH is still unknown. Mutations in the kinase domain ( about 50% of total mutations) suggested that downstream SMAD signaling play a role for PAH development because of its reduced SMAD dependent transcriptional activity 105 However, mutation s in cytoplamic tail domain ( about 20 % of total mutations) such as R899X leave SMAD signaling intact, indicating that the cytoplas mic tail of BMPR 2 may not be essential for transduction of BMP signals through S MAD s 106 107 In other words, S MAD deficiency may not be
24 associated with the pathogenesis of PAH. James West et al addressed this question using transgenic mice expressing BMPR2 R899X transgene in smooth muscle 108 In these mice, truncated BMPR2 proteins with a tail domain mutation were overexpressed in smooth muscle cells by doxycycline treatment from 4 weeks of age. They elevated right ventricular systolic pressure, associated with extensive pruning, musculariz ation of pulmonary arterioles and perivascular infiltration of immune cells, not affecting SMAD activity. These in vivo results suggest that the SMAD deficiency may not be asso ciated with PAH caused by BMPR2 deficiency. Endothelial BMPR2 D eficient PAH M ou se M odel Previously we produced Bmpr2 conditional knockout (cKO) mice [L1cre(+); Bmpr2 f/f or +/f ] by deleting the Bmpr2 gene in the pulmonary endothelium using the novel L1cre line 109 A subset of mice exhibited elevated right ventricula r systolic pressure (RVSP) and right ventricular hypertrophy (RVH) which are the representative phenotypes of pulmonary hypertensive (PH) mice 110 and these PH mice showed increased muscularization of pulmonary arterioles and thickening of vessel wall compared to non PH mice and controls. Deletion of S mad1 G ene in ECs or SMCs SMAD1 is a canonical signal transducer of BMPR2 and its reduced activity has been associated with PAH Yang et al showed tha t phosphorylation of SMAD1 was reduced in the pulmonary arterial SMCs of PAH patients with BMPR2 mutation 70 Thus, w e hypothesized that SMAD1 is a contributing downstream mediator of BMPR2 in the pathogenesis of PAH. To test this hypothesis, we produced Smad1 cKO mice by deleting Smad1 gene in the pulmonary endothelial cells (pECs) or smooth muscle ce lls (SMCs) using L1 cre and Tagln cre lines, respectively and found that Smad1 deletion in
25 pECs or SMCs can develop PAH in mice with muscularization of pulmonary vessels suggesting that SMAD1 may be a critical downstream molecule in PAH. Recent studies reported that enhanced TGF impaired BMP signaling 103 111 113 In Bmpr2 deleted pulmonary endothelial cel ls we found that a reduced BMP response resulted in a predominant TGF supporting the role of a balance between TGF of pulmonary blood pressure 100 Results SMAD1/5/8 Phosphorylation was Impaired in the Lungs of Pulmonary H ypertensive L1cre(+); Bmpr2 2f/2f M ice We have previously shown that about 40% of mice in which Bmpr2 gene was specifically deleted in endothelial cells by L1cre exhibited pulmonary hypertensive phenotype 110 To assess the extent to which the conditional Bmpr2 deletion affects on its downstream SMAD1/5/8 signaling, lungs of cre negative controls, Non PH L1cre(+); Bmpr2 2 f/2f PH L1cre(+); Bmpr2 2 f/2f mice were immunostained with anti phospho SMAD1/5/8 antibodies (Fig. 1 1 A through 1 1 F). While pSMAD1/5/8 positive cells were readily detected in the cre negative controls (Fig. 1 1 A through 1 1 B) and Non PH Bmpr2 mutants (Fig. 1 1 C), they appeared to be much less in the PH Bmpr2 mutants (Fig. 1 1 D through 1 1 F). Consistent with this immunostaining result, the levels of SMAD1/5/8 phosphorylation in the whole lung samples were significantly reduced in the PH Bmpr2 mutants compared to the cre negative controls and Non PH Bmpr2 mutants (Fig. 1 1 G H). These data s uggested to us that impaired SMAD1 signaling may play a pivotal role in the pathogenesis of pulmonary arterial hypertension associated with Bmpr2 deficiency.
26 Smad1 Deletion in P ulmonary ECs or SMCs by L1cre or Tagln cre To investigate the role of SMAD1 in the pathogenesis of PAH, we exploited conditional knockout approaches for deleting the Smad1 gene in endothelial cells or smooth muscle cells by L1cre or Tagln cre lines, respectively, because Smad1 null mice were embryo nic lethal 114 Both L1cre(+); Smad1 f/f and Tagln cre(+); Smad1 2f/2f mice we re viable and normal compared to their cre negative littermates. We analyzed the Cre activity by detecting the Smad1 null allele in several organs of 2 month old mice, including the lung, heart, liver, kidney, and spleen. Consistent with our expectations b ased on our previous reports 109 110 the Cre mediated Smad1 deletion was detected primarily in the lungs of L1cre(+); Smad1 f/f mice, and found in most organs of Tagln cre(+); Smad1 f/f mice ( Fig 1 2 ). Some M ice with the Smad1 Deletion in Pulmonary ECs or SMCs Exhibited Elevated Pulmonary Press ure and Right Ventricular H ypertrophy. To assess the pulmonary pressure, we measured the right ventricular systolic pressure (RVSP) of L1cre(+); Smad1 f/f Tagln cre(+); Smad1 f/f and their age matched cre( ) control mice. While RVSPs of controls were cluster ed in a 20 27 mmHg range, those of L1cre(+); Smad1 f/f and Tagln cre(+); Smad1 f/f mice were scattered in a wide range (Fig. 2A) from 22 to 45 mmHg. About 40% (14/35) of L1cre (+); Smad1 f/f mice and 12% (4/33) of Tagln cre(+); Smad1 f/f mice had their RVSPs great er than 30 mmHg, and we designated them as pulmonary hypertensive (PH) group (Fig. 1 3 A). The mean RVSPs of L1cre(+); Smad1 f/f (28.1 mmHg) was significantly higher than that of cre( ); Smad1 2f/2f (23.9 mmHg). Fulton index, the ratio of RV free wall weight ov er septum plus left ventricular free wall weight, was used to estimate RV hypertrophy. The Fulton index of L1cre(+); Smad1 f/f and Tagln cre(+); Smad1 f/f mice was significantly greater
27 than that of cre( ); Smad1 f/f controls (Fig. 1 3 B). It was greater in the PH mice than that in N PH mice, indicating that sustained elevation of pulmonary pressure might have resulted in RV hypertrophy in the PH groups (Fig. 1 3 C). There was no difference in systemic blood pressure among three groups (F ig. 1 3 D). Positive Distal Arteries and Medial Wall Thickness Were I ncreased in the Smad1 Mutant M ice. To examine whether the elevated RVSP and RV hypertrophy in the Smad1 mutants is associated with pulmonary vascular remodeling, anti actin positive pulmonary arteries ranging from 30 and the wall thickness was measured. The PH group L1cre(+); Smad1 f/f mice showed positive pulmonary arterioles and thicker arterial walls compared to the N PH group and the cre( ); Smad1 f/f (Fig. 1 4 B, E, F). In Tagln cre(+); Smad1 f/f mice, however, both PH and N PH groups showed a higher number of muscularized vessels and thicker walls compared to the cre( ); Smad1 f/f controls. Isolati on of Pulmonary Endothelial Cells C arrying R26 creER/+ ; Bmpr2 2f/2f A llele It has been hypothesized that an opposing balance between TGF signalings is critical for homeostasis of p ulmonary vasculature and that imbalance of TGF contribute to the pathogenesis of PAH 100 In order to investigate this hypothesis and to examine the extent to which Bmpr2 deficiency impact on this balance, we established a n in vitro model as follows. We isolated pulmonary ECs (pECs) from the lung of R26 creER/+ ; Bmpr2 2f/2f recombinase function. Three days of culture of the pECs with medium co 4 hydroxy tamoxifen (OH TM) efficiently deleted exons 4 and 5 of the Bmpr2 gene (Fig.
28 1 5 A). When the Bmpr2 deleted cells were subsequently cultured with OH TM free media for 10 days, no overgrowth of undeleted cell p opulations were observed (Fig. 1 5 B). Henceforth, Bmpr2 intact and deleted cells will be designated as Bmpr2 2f/2f and Bmpr2 1f/1f cells, respectively. To examine whether OH TM treatment and Bmpr2 deletion affected the expression of other genes involved in TGF characteristics, semi quantitative RT PCR analyses were performed. Expression of EC specific markers including Nos3, Tie2, Eng, and Flk1 were maintained in Bmpr2 1f/1f pECs (Fig. 1 5 D ). Bmpr2 transcript level was undetectable while transcripts for other TGF Bmpr2 1f/1f pECs (Fig. 1 5 C ). Deletion of Bmpr2 Gene and Impaired BMP S ignaling In order to assess the extent to which Bmpr2 deletion affects BMP signaling, BMP4 or BMP7 was added to Bmpr2 2f/2f and Bmpr2 1f/1f cells at various doses (0, 5, 25, 50 ng/ml) after 15 hr serum starvation. First, we examined the protein level of BMPR2 in these cells. While BMPR2 was readily detected i n Bmpr2 2f/2f cells, BMPR2 protein was undetectable in Bmpr2 1f/1f cells (Fig 1 6 A, B). Level of phosphorylation of SMAD1/5/8 was augmented in a dose dependent manner in Bmpr2 2f/2f cells whereas it was suppressed at all BMP4 doses in Bmpr2 1f/1f cells (Fig. 1 6 A, C), indicating that the BMP4 signaling is impaired in Bmpr2 1f/1f pECs. BMP7 signaling can be compensated by ACVR2A in BMPR2 depleted pulmonary artery SMCs (PASMCs) 77 As shown in Fig. 1 6 D, phosphorylation of SMAD1/5/8 was elevated in a dose dependent manner in both Bmpr2 2f/2f and Bmpr2 1f/1f cells, suggesting that BMP7 signaling can be compensated in Bmpr2 1f/1f pECs.
29 Enhanced TGF D eficient pECs To investigate whether i mpaired BMP signaling affects TGF examined the basal level of SMAD2 phosphorylation in Bmpr2 2f/2f and Bmpr2 1f/1f cells cultured in medium containing 10% fetal bovine serum (F ig 1 7 A). The level of SMAD2 phosphorylated in Bmpr2 1f/1f c ells was much higher than that in Bmpr2 2f/2f cells. To test if Bmpr2 deficient cells are more sensitive to TGF phosphorylation as a response to TGF (F ig 1 7B ) While both Bmpr2 2f/2f and Bmpr2 1f/1f cells showed a dose dependent augmentation of pSMAD2, the level of SMAD2 phosphorylation was significantly higher in Bmpr2 1f/1f cells at 1 and 2 ng/ml TGF 1 compared to Bmpr2 2f/2f cells, su ggesting that impaired BMP signaling may potentiate the TGF Opposing Balance Between TGF and BMP S ignaling s in pECs To investigate whether BMP and TGF signaling form an opposing balance in pECs we examined whether TGF induced SMAD2 phosphorylation is suppressed by BMP treatment in pECs. TGF 1 ( 0, 0. 1, 1, and 2 ng/ml) and BMP4 (25 ng/ml) or BMP7 (25 ng/ml) were treated for 30 min after serum starvation in Bmpr2 2f/2f and Bmpr2 1f/1f cells T he level of SMAD2 phosphoryla tion by 1 and 2 ng/ml of TGF 1 treatment in Bmpr2 2 f/ 2 f cells was decreased by either BMP4 or BMP7 implying that BMP and TGF have a competitive relationship by opposing each other (Fig. 1 8). However, TGF mediated SMAD2 phosphorylation in Bmpr2 1f/1f cells was not affected by BMP4 treatment, suggesting that BMP4 signaling is mediated mainly through BMPR2 (Fig. 1 8A) Interestingly, BMP7 could suppress overactivated TGF signaling in Bmpr2 deficient pECs as well as Bmpr2 intact pECs, suggesting that BMP7 signaling is not mediated
30 mainly through BMPR2 and also mediated by other type 2 r eceptors such as ACVR2a (Fig. 1 8B) Discussion In this study, we showed that genetic ablation of Smad1 in pECs or SMCs predisposes mice to PAH, suggesting that impaired SMAD1 signaling is relevant to the pathogenesis of PAH. Furthermore, the findings of reduced SM AD1/5/8 activation and the over activation of TGF deficient pECs suggests that a predominance of TGF genetically disturbed balance between TGF and BMP signaling, may be an important facto r triggering PAH development. SMAD1 i s a Downstream M ediator of BM P S ignaling in P athogenesis. SMAD1 is a canonical downstream mediator of the BMP pathway that functions in a variety of cellular and developmental events. Therefore, reduced BMP activity i n association with the reduced expression of its receptors is accompanied by decreased phosphorylation of SMAD1/5/8 in the development of diseases. Liu et al found that pSMAD1/5/8 and BMPR 1 b were decreased in malignant glioma tissues compared with normal brain tissues 115 Yang et al reported that the activated form of SMAD1 is deficient in the pulmonary vasculature of patients with a BMPR2 mutation suggesting that inactivation of SMAD1 plays a role in the PAH pathogenesis 70 Our data demonstrate that Smad1 deletion in either ECs or SMCs ca n induce PH in mice, implying that SMAD1 may be an important downstream signaling molecule of BMPR2 in PAH development.
31 SMAD1 I nvolvement in the P athogenesis of PAH Our data showing the role of SMAD 1 in PAH may appear to be con flict with the previous data from James West s group demonstrated that overexpression of tail domain BMPR2 mutant (R899X) not affect ing the SMAD1 acti vation resulted in PAH 108 However, f rame shift and nonsense mutations consist of 70% of total mutations of BMPR2 gene and interestingly, approximately one quarter of this type mutations are collected in the cytoplamic tail domain. Nonsense mutation decay (NMD) is a cellular mechanism to destroy defective RNA transcripts with nonsense mutation to block the production of truncated proteins 116 Thus, most of BMPR2 mRNAs with the cytopla smic tail mutation are destroyed through NMD mediated processing, leading to BMPR2 deficiency 104 117 In James West s studies, truncated BMPR2 was not destroyed by NMD and showed normal SMAD1 activation. Thus, their mutant mice may not represent human PAH patients carrying a cytoplasmic tail muation of BMPR2 Otherwise, t his discrepancy suggests that some BMPR2 associat ed PAH might be SMAD dependent (mutation in the kinase domain) and some are not (mutation in the tail domain). M utations in the tail domain have been shown to interrupt interactions between BMPR2 and the dynein light chain Tctex 1 as well as LIMK1, a key r egulator of actin dynamics 118 119 Thus, the cytoplamic tail mutation may contribute to PAH development by disrupting these interactions with potentially critical signaling molecules in SMAD independent manner. As an alterna tive interpretation, SMAD1 signaling is critical in ECs, not in SMCs for PAH development. Hansmann et al presented a novel anti proliferative axis in PAH 120 spontaneously developed PAH with elevated RVSP, RVH, and increased muscularization of the distal pulmonary arteries. This was independent of SMAD1/5/8
32 phosphorylation. Li et al suggested a novel role of NOTCH3 in controlling proliferation of SMCs and in maintaining SMCs in an undifferentiated state 121 They found that the severity of disease in human PAH patients and rodent PH models correlated with the amount of NOTCH3 prote in in vascular SMCs of the lung, suggesting that NOTCH3 signaling pathway in SMCs is crucial for the development of PAH. Even though it still remains unknown whether NOTCH3 and PPAR signaling in ECs are also critical contributors to PAH pathogenesis, up to now, our data with Smad1 cKO mice demonstrated that SMAD1 deficiency in ECs is a important contributing factor for spontaneous PAH development. SMAD1 D eficiency in ECs has a G rea ter I mpact on PAH T han T hat in SMCs Mice with endothelial Smad1 deletion showed 40% penetrance while mice with Smad 1 deficiency in SMCs displayed 13% penetrance. Why did endothelial deletion make a greater influence on PAH pathogenesis compared to deletion in SMCs? Malfunctioning of SMCs usually increase their growth rate, leading to thickening of vessel wall, whereas endothelial dysfunction contributes to PAH pathogenesis in various ways. Disordered endothelial proliferat ion forms neoinitimal layers 82 and disrupted balance of vasoactive mediators exacerbate s vasoconstriction 14 34 84 86 and inc reased cell permeability allows serum growth factors to affect SMC pr oliferation and release of mitogen such as serotonin from ECs induces smooth muscle hyperplasia 122 and the number of occluded vessels are increased by in situ thrombosis 2 Therefore, because of these various effect s of ECs, Smad1 deletion in ECs may accelerate PAH development compared to Smad1 deletion in SMCs.
33 Incomplete P enetrance of PAH in Smad1 C onditional KO M ice All the mice with endothelial or smooth muscle Smad1 deletion did not develop PAH. O nly a subset of mice showed PH phenotypes including eleva tion of RVSP and thickening of smooth muscle layer. First SMAD1/5/8 could be functionally redundant and compensate for the absence of other molecules Cre mediated genetic ablation of either Smad1 or Smad5 in ovarian granulosa cells results in normal reproductive function but that combined loss of Smad1 and Sma d5 results in fertility defects and granulosa cell tumors 123 S MAD 1, S MAD 5, and S MAD 8 function redundantly in activating regression of the M llerian duct mesoepithelium 124 Second, additional environmental insult would be required for the manifestation of PAH w ith genetic predisposition. Inflammation has been associated with PAH as an influencing environmental factor I nflammatory cells including macrophages and lymphocytes were accumulated and many proinflammatory cytokines were induced in the PAH vascular lesions 7 12 64 93 97 Thus, if treated with inflammatory stimulants such as IL 6 125 and 5 lipoxygenase (5 LO) 68 Smad1 cKO mice may show increased penetrance. Conversely, blocking inflammatory activation could be therapeutically beneficial for PAH patients Studies with animal models support this. Platelet activating factor ( PAF ) antagonists an anti inflammat ory drug inhibit ed pulmonary vascular remodeling induced by hypobaric hypoxia in rats 126 Inhibition of 5 lipoxygenase activating protein (FLAP) suppressed hypoxia induced pulmonary vasoconstriction in vitro and the development of chronic hypoxic pulmon ary hypertens ion in rats 127 Imbalance in TGF alings in PAH P athogenesis Many studies have been focused on impaired BMP signaling to explain PAH pathogenesis. However, as dysfunction of BMP pathway alone is not sufficient to
34 develop PAH, r ecent studies suggest a role of imbalance in TGF / BMP signaling for PAH development because BMP and TGF pulmonary vascular homeostasis 128 BMPR2 deficient pulmonary vessels lose control of overactivation of TGF leading to predominan ce of TGF 101 103 129 Based on these reports, we hyp othesized that BMPR2 d eficiency results in overactivated TGF monstrate that BMPR2 d eficiency directly elevates TGF in vivo sys tem because even though our Bmpr2 or Smad1 cKO mice exhibit TGF pSMAD2/3, it could be a secondary effect of PAH. Hence, we isolated and immortalized pECs from a R26 creER+/ ; Bmpr 2 2f/2f m ous e, in which tamoxifen treatment induce s Bmpr2 deletion. This inducible system is advantageous because we can exclude strain and individual difference using the same parental cells Bmpr2 deleted pECs showed the low level of pSMAD1/5/8 by BMP4 treatment and enhanced phosphorylation of SM AD2 not only at the basal level but also by TGF 1 treatment compared with Bmpr2 intact pECs, supporting our hypothesis that BMPR2 d eficiency overactivates TGF in pECs. In addition, we found a marked reduction of total SMAD2 with an acc ompanyin g high level of pSMAD2. This may have arisen because activated SMAD2 is multi ubiquitinated and is degraded by the proteosome 130 132 These results from pECs present the possibility of the future therapeutic options to restore TGF /BMP imbalance. Recent reports support our results and suggest potential candidates for inhibitor of TGF pathway. Inhibition of TGF signaling by ALK5 inhibitor, IN 1233 prevented PAH in the monocrotalin treated rat which showed increased TGF activity 103 T he angiotensin II type 1 recepto r (AT1) blocker, losartan
35 averted aortic aneurysm in a mouse model of Marfan syndrome ( MFS ), which is associated with increased TGF signaling 133 In the aspect of BMP activation, Irrespective of BMPR2 deficiency pECs activated SMAD1/5/8 in response to BMP7. This intact BMP7 response was also shown in SMCs with Bmpr2 deletion 77 Therefore, restoration of pSMAD1/5/8 by BMP7 could b e beneficial to PAH patients with BMPR2 deficiency T hickening of the Smooth Muscle Layer Might not be Sufficient to Lead to High Pulmonary P ress ure The PH group in L1cre(+); Smad1 2f/2f mice showed thickened vessel walls, implying that high pulmonary pressure was associated with the muscularization of pulmonary arterioles. However, Tagln cre(+); Smad1 2f/2f mice including N on PH and PH groups showed thickening of the vascular smooth muscle layer, suggesting that SMAD1 dysfunction in SMCs directly affected proliferation of vascular smooth muscles. Yang et al reported that SMAD1 dysfunction promoted MAPK signaling leading to aggressive proliferation of PASMCs 70 More importantly, our result suggests that thickening of the smooth muscle layer might not be sufficient to lead to high pulmonary pressure in the absence of related endothelial dysfunction. We speculate that SMAD1 deficiency in pECs results in endothelia l dysfunction. Pulmonary endothelial dysfunction induces sustai ned constriction of blood vessels due to an increase of vasoconstrictors such as endothelin or decrease of vasodilators such as eNOS or prostacyclin. These alterations of vasoactive molecules w ere readily observed in PAH patients 14 44 134 135 and PH animal models 136 138 C ontinued vascular constriction leads to high pulmonary pressure and results in thickening of the v ascular smooth muscle layer. However, although Smad1 deletion in SMCs induces thickening of smooth muscle layer, unaffected
36 endothelial cells may enable vessels to dilate more in response to the high blood pressure gained from thickened blood vessels. Ther efore, thickening of the smooth muscle layer might not be sufficient to sustain high pulmonary blood pressure. Smad1 cKO M ice and I nducible Bmpr2 KO pECs are U seful R esource s for F uture M echanism R esearch, D rug S creening and P reclinical S tudy. In this study, we developed two useful resources for PAH studies; Smad1 cKO mice and inducible Bmpr2 KO pECs. I n the future, these mice and pECs will be usefully utilized for mechanistic research to find downstream targets of BMPR2/SMAD1 pathway for novel PAH ther apy and when candidate drugs are developed or preexisting medicines with potential effect are not tried to PAH patients, we can get valuable information about efficacy and effectiveness of candidate drugs for PAH using t hese materials.
37 Table 1 1 Primers for genomic and semi quantitative PCR reaction Genomic PCR Smad1 A CACCTGTGCCCCCTCCAAGT Smad1 B GAGCTCTGCTCCGCCACTCA Alk1 F CAGCACCTACATCTTGGGTGGAGA Alk1 R ACTGTTCTTCCTCGGAGCCTTGTC Bmpr2 2A CACACCAGCCTTATACTCTAGATAC Bmpr2 6R CACATATCTGTTATGAAACTTGAG Bmpr2 2C TTATTGTAAGTACACTGTTGCTGTC L1cre F GTTTTCCTTTGAAAAACACGATGA L1cre R ATCAGGTTCTTGCGAACCTCATCA Tagln cre F CTCCTTCCAGTCCACAAACGAGC Tagln cre R GGGCGATCCCTGAACATGTCC R26R F GTCGTTTTACAACGTCGTGACT R26R R GATGGGCGCATCGTAACCGTGC RT PCR Gapdh F CAATGCATCCTGCACCACCAA Gapdh R GTCATTGAGAGCAATGCCAGC Bmpr2 F GTTGACAGGAGACCGGAAACAG Bmpr2 R GGAGACTCAGATATTTGCACAG Tgfbr2 F TTGCCTGTGTGACTTCGGGCT Tgfbr2 R CTATTTGGTAGTGTTCAGCGA Acvr2a F CGTTCGCCGTCTTTCTTATC Acvr2a R AGGATTTGAAGTGGGCTGTG Alk1 F TCATGGTGCACAGTGGTGCTG Alk1 R CAAATCCCGCTGCTTCTCCTG Alk2 F AGTCATGGTTCAGGGAGACG Alk2 R TGCAGCACTGTCCATTCTTC Alk3 F TAAAGGCCGCTATGGAGAAG Alk3 R CCAGGTCAGCAATAAGCAA Alk6 F CACTCCCATTCCTCATCAAA Alk6 R TTCCAATCTGCTTCACCATC F CGGAAGCGCATCGAAGCCATCC R GCAAGCGCAGCTCTGCACGG Nos3 F TTCCGGCTGCCACCTGATCCTAA Nos3 R AACATATGTCCTTGCTCAAGGCA Tie2 F CTCATCTGTGGACGCTGGATG Tie2 R GGCACTGAGTGGATGAAGGAG Eng F TGCACTCTGGTACATCTATTC Eng R TGGATTGGGCAGTTCTGTAAA Flk1 F AGAACACCAAAAGAGAGGAACG Flk1 R GCACACAGGCAGAAACCAGTAG
38 Figure 1 1 BMP response was severely impaired in the lungs of PH mice [ L1cre (+); Bmpr2 f/f ] (A F) The levels of pSMAD1/5/8 and SMAD1 were examined to compare BMP response among lungs of cre negative controls (A,B) N PH (C) and PH mice (D F) (G H) The level of pSMAD1/5/8 was significantly decreased in the PH lungs compared to controls and N PH lungs.
39 Figure 1 2 Smad1 deletion in L1cre(+); Smad1 f/f and Tagln cre(+); Smad1 f/f mice. Deleted Smad1 allele was examined in various organs of Smad1 conditional KO (cKO) mice and cre negative control mice by genomic PCR analysis. Null Smad1 was detected at 300 bp. A primer set amplifying the Alk1 locus (190 bp) was used as a control for the PCR amplification. Genomic segment between 2 loxp sites including exon2 was deleted by cre recombinase activated by tamoxifen leading to production of PCR product by a primer set (Smad1 A and Smad1 B).
40 Figure 1 3 Smad1 deletion in SMCs and ECs resulted in elevation of RVSP and RV hypertrophy. ( A ) closed circles indicate RVSP of each mouse. More than 30 mmHg of RVSP was designated as pulmonary hypertensive pressure. 40% of L1cre(+);Smad1f/f mice and about 12% of Tagln cre(+);Smad1f/f mice were pulmonary hypertensive. RVSP of L1cre(+);Smad1f/f was signifi cantly higher compared to either that of controls or that of Tagln cre(+);Smad1f/f ( B ) Systemic blood pressure was not significantly different by genotypes of mice. ( C ) Both Smad1 cKO mouse lines showed significant RV hypertrophy compared to cre negative controls. ( D ) W hen mice were divided into non pulmonary hypertensive (N PH) and pulmonary hypertensive (PH) groups, RV hypertrophy of PH mice was significantly higher than cre negative controls and N PH mice of each cKO group. Statistical differences (p<0 .05) were indicated by asterisks above each bar
41 Figure 1 4 Elevated pulmonary pressure is associated with pulmonary vascular remodeling. ( A D ) pulmonary arterioles were visualized by staining with anti arrowheads) is shown at higher magnification at the left bottom corner of each mice of each Smad1 cKO mice showed muscularized vessels (E) and thickened vessel walls (F) compared with cre negative controls. In terestingly, N PH group of Tagln cre(+); Smad1 f/f also exhibited muscularized vessels with thickened vessels walls. indicate s significant statistical difference (p<0.05) compared to cre negative controls.
42 Figure 1 5 Deletion of Bmpr2 gene in pulmonary en dothelial cells (pECs) ( A ) pECs were treated with tamoxifen (TM) for 3 days. As shown in the diagram, TM enables cre recom binase to loop out the genomic segment containing 6R primer binding sequence and exon 4 5 which is flanked by two loxp sites (triangles). This deleted allele can produce a PCR product with the 2A 2C primer pair. Deletion of Bmpr2 gene was examined using the 2A 6R primer pair. ( B ) Following growth in TM free media, reappearance of an intact Bmpr2 gene in TM treated pECs was examined using prime r pairs for the deleted allele [ 2A 2C ] and the intact allele [ 2A 6R ] The mRNA levels of TGF signaling molecules (C) and endothelial markers (D) were examined by semi quantitative RT PCR to determine whether tamoxifen treatment for Bmpr2 deletion affected expression of other molecules in TGF characteristic.
4 3 Figure 1 6 B mpr2 deleted pECs displayed a reduced response to BMP4 but not BMP7. ( A ) BMP4 (0 50 ng/ml) was added to Bmpr2 intact ( Bmpr2 2f/2f ) and Bmpr2 deleted ( Bmpr2 1f/1f ) cells for 30 minutes after serum s tarvation. The levels of pSMAD1 / 5 / 8 were examined to evaluate the BMP4 response. ( B ) The protein levels of Bmpr2 were determined for Bmpr2 2f/2f and Bmpr2 1f/1f cells. ( C ) BMP4 actin in Bmpr2 2f/2f and Bmpr2 1f/1f cells. ( D ) BMP7 (0 50 ng/ml) was added to Bmpr2 intact ( Bmpr2 2f/2f ) and Bmpr2 deleted ( Bmpr2 1f/1f ) cells for 30 minutes after serum s tarvation. The levels of pSMAD1 / 5 / 8 were determined to evaluate differences in response to BMP7. The BMP7 response was quantified by the ratio actin in Bmpr2 2f/2f and Bmpr2 1f/1f cells.
44 Figure 1 7 Reduced BMP response induced overactivation of TGF ( A ) The levels of pSMAD2 were examined to compare basal TGF Bmpr2 2f/2f and Bmpr2 1f/1f cells under normal media condition without additional actin in Bmpr2 2f/2f and Bmpr2 1f/1f cells. ( B ) In order to compare TGF Bmpr2 2f/2f and Bmpr2 1f/1f cells, TG F 2 ng/ml) was added for 30 minutes after serum starvation. TGF actin in Bmpr2 2f/2f and Bmpr2 1f/1f cells.
45 Figure 1 8 BMP and TGF form an opposing balance in pECs (A) Activation of TGF was supp ressed by either BMP4 or BMP7 in Bmpr2 intact pECs. 1 (0 2 ng/ml) was treated on Bmpr2 2f/2f and Bmpr2 1f/1f cells for 30 minutes after serum starvation. TGF r actin However, (B) this sup pression was induced only by BMP7, not by BMP4 in Bmpr2 deficient pECs
46 CHAPTER 2 THERAPEUTIC EFFECT OF VEGF BLOCKADE ON ARTERIOVENOUS MALFORMATIONS (AVMS) IN AN ANIMAL MODEL FOR HEREDITARY HEMORRHAGIC TELANGIECTASIA Introduction Hereditary Hemorrhagic Telangiectasia (HHT) Hereditary Hemorrhagic Telangiectasia (HHT) is an inherited autosomal dominant vascular disease affecting 1 in 5,000~8,000 individuals worldwide 139 141 The disease is characterized by epistaxis (spontaneous and recurrent nosebleeds), mucocutaneous telangiectases as well as arteriovenous malformations (AVMs) in the brain, lungs, and visceral organs including the liv er and GI tract 142 145 An AVM is a direct connection between arteries and veins without intervening capillaries 142 and is an underlying defect associated with recurrent nose bleeding, telangiectases, and visceral hemorrhaging. In AVMs, high velo city arterial blood invades venous vascular beds creating turbulent and swirling flows leading to tortuous and irregular vascular remodeling. When these blood vessels rupture, it results in chronic and severe anemia due to massive blood loss. Brain and lun g hemorrhages are life threatening and the major cause of death in young patients 146 147 To compensate for the lost blood from GI and nose bleedi ng, patients receive regular blood transfusions and/or iron supplementation therapy. More than 90% of HHT patients experience recurrent nosebleeds by their 60s 148 and this affliction impacts profoundly on the patients quality of life 149 150 All the pati procedure for completely blocking nostrils said that the quality of their lives was improved following the surgery 151
47 Limitation of Current Therapeutic I nterventions in HHT Therapeutic treatments are available to alleviate telangiectasia and visceral lesions. Embolization can prevent blood flow to AVM lesions by occluding blood vessels with metal coils and is effective for pulmonary and cereb ral AVMs 152 153 For acute nosebleeds, packing is a common method and in chronic and recurrent nose bleeding, procedure are applied depending on the unique condition of the patient 154 Systemic hormonal treatments or prothrombotic strategies using antifibronolytic agents have been useful in limiting excessive hemorrhag ing 155 156 However, the mechanism of hormonal efficacy remains unknown and the negative impacts of prothrombogenic agents must be considered before treatment 15 7 Thus, the development of therapies targeting the pathogenic mechanisms underlying AVM formation remains an urgent issue. Genetic S tudies in HHT H eterozygous mutations in HHT causing genes have been identified: ENG for HHT1 158 160 ALK1 for HHT2 161 162 and SMAD4 for Juve nile Polyposis and HHT (JP HHT). 163 Over 80% of HHT patients possess heterozygous mutations in one of these genes, while mutations at additional loci are responsible for the remaining 20%. These loci include the HHT3 locus (chromosome 5) 164 and the HHT4 locus (chromosome 7) 165 even though causative genes remain unidentified. Genetic Mouse M odels for HHT The role of ALK1 in HHT pathogenesis has been explored using genetically modified mouse models 166 167 Heterozygous Alk1 null mice displayed HHT like phenotypes such as age dependent subcutaneous and mucocutaneous vascular lesions as well as hemorrhages in the lungs, GI tract, liver, brain and spleen.
48 Penetra nce, however, was not complete and symptoms when observed were not as severe as observed in humans. Homozygous Alk1 knockout (KO) mice died in utero displaying vascular defects such as hyper dilated blood vessels and AVMs 166 168 Targeted Alk1 deletion in endothelial cells of transgenic mice resulted in AVMs and hemorrhages in the vascular beds of brain, lung and GI tract 109 Recently, our lab demonstrated that the conditional deletion of Alk1 in ad ult mice using the tamoxifen inducible RosacreER driver recapitulated HHT phenotypes common in human patients, such as severe hemorrh aging and low hemoglobin levels. 169 Secondary Hits are Required for AVM D evelopment. AVM lesions in HHT patients and Alk1 muta nt mice appear in selective organs such as the lung, GI tract, liver and brain despite the global presence of ALK1 or ENG mutations. Even in the same organ, some vascular beds form AVMs and hemorrhage while other vessels remain normal in appearance. This s uggests that genetic predisposition is not sufficient to cause disease and other genetic or environmental factors play a role. When excisional wounds were performed on the dorsal skin and ear of Alk1 de novo AV to the wounded area 169 Vessels removed from the wounded area were unaffected. This was evidence to suggest that ALK1 deficiency and injury combined to induce the vascular lesion. Angiogenesis is variably regulated by processes associated with wound healing. These include the actions of infiltrating inflammatory cells, the turnover of extracellular matrix via matrix metalloproteinase (MMP) secretion, and the local activity of growth factors and cytokines produced by endogenous and recruited cells 170 Which event is most critical for AVM formation in Alk1 deficient blood vessels is a question we addressed in this study. We focused on LPS induced inflammation and
49 growth factor induced angiogenesis. Mucocutaneous telangiectasia occur s frequently on the lips, tongue, and nose which are constantly exposed to infection. Increased levels of inflammatory markers factors such as MMP9 and interleukin 6 (IL 6) have been demonstrated in brain AVM tissue 171 172 Inflammatory cells including neutrophils and macrophages/microglia were frequently identified in the vascular walls of AVM tissue 173 In ad dition, the tissues and plasma of HHT patients contain high levels of vascular endothelial growth factor (VEGF). Vessel density was also increased 174 175 Brain AVMs usually develop congenitally during vascularization. Some case reports showed that anti angiogenesis therapy using bevazicumab and thalidomide worked well to prevent epistaxis and liver AVMs 176 177 In this report, we demonstrate that inflammation or direct angiogenic stimulation can induce AVMs in Alk1 deficient subdermal ves sels. Angiogenic stimulation is a necessary sequella to inflammation in the induction of AVM formation. Then, angiogenesis bl ockade using a VEGF neutralizing antibody is effective in alleviating wound induced AVMs and internal bleeding in GI tract and lungs in ALK1 deficient adult mice. Taken together, our findings suggest that angiogenesis blockades could be promising and funda mental medication in future clinical trials for HHT patients. Results Vascular Endothelial Growth Factor (VEGF) or Lipopolysaccharide (LPS) can I nduce De N ovo AVMs in Alk1 Deficient Subdermal V essels. In order to examine whether inflammation or angiogenesi s can induce AVM formation in Alk1 deficient mice, VEGF or LPS was administered to mice in the form of PLGA particles implanted subdermally under the dorsal skin. LPS PLGA and VEGF PLGA particles were injected into cre negative mice as a control to assess whether
50 either agent alone could induce vascular lesions in the absence of an Alk1 deficiency. After 7 days, blue latex dye was infused through the left heart to visualize the blood vessels and to locate arterial venous (AV) shunts. The dye should be prohi bited from traversing capillary beds unless an AV shunt is present. The particles formed an orange round aggregation on the subdermal area because of rhodamine. After removing particle aggregates, the skin was cleared to reveal the vascular network. In Alk 1 wild type mice, a higher vascular density was evident around LPS and VEGF particle injected areas compared to neighboring regions, but vascular malformation such as AV shunts were not observed (Fig. 2 1A, B). PLGA particles themselves occasionally induce d higher vascular densities around implantation sites in Alk1 deficient mice but did not induce AV shunts. This was demonstrated in tamoxifen treated R26 creER/+ ; Alk1 2f/2f mice in which PBS PLGA was implanted into the dorsal skin. PBS PLGA had no affect on subdermal vessels (Fig. 2 1C) or, on rare occasions, induced a moderate level of abnormal vasculature (Fig. 2 1D). When VEGF PLGA was injected into Alk1 deficient skin, tortuous, irregular, and excessive vasculature developed around the particles and surrounding veins were dilated and contained latex dye, indicating the presence of AV shunts (Fig. 2 1E). LPS PLGA particles also induced a comparable level of tortuous vessels and AV shunting (Fig. 2 1F). VEGF and LPS Stimulated A ngiogenesis. VEGF is a strong angiogenic stimulator in vascularization 178 179 and tumors 180 and LPS has been to stimulate angiogenesis 181 182 Therefore, we questioned whether VEGF or LPS released from PLGA particles elicited an angiogenic response. We observed the vascular morphology around VEGF or LPS PLGA injected area 4 days following PLGA particle injection. Sprouting blood vessels were seen to be drawn
51 towards the VEGF and LPS particles (Fig. 2 1G, H). This result was consistent with angiogenesis being a common, and necessary, element to both VEGF and LPS induced AVM formation. It could also be a critical factor in wo und induced AVMs. VEGF Blockade Suppressed Wound Induced AVM in Alk1 Deficient Subdermal V essels. To test the participation of VEGF in the development of inflammation induced or wound induced AVM formation, experiments were repeated in the presence of VEG F neutralizing antibody to block angiogenesis. Alk1 deficient and wounded mice were simultaneously treated with VEGF antibody or saline alone. The local vasculature was evaluated 9 days following the application of the wound. Saline treated control mice exhibited a typical AV shunts with excessive, tortuous, and dilated vessels surrounding the wound ed area (Fig. 2 2A top panels ). In contrast, AV shunts were only occasionally observed in VEGF antibody treated animals and there were fewer tortuous and irr egular vessels near the wound (Fig. 2 2A, bottom panels). Blood vessels positive for latex dye were quantified to measure the severity of AVMs in saline treated and VEGF antibody treated groups. The VEGF antibody treated group (mean: 17.38 % vessel area) d isplayed a reduction in vessel content of approximately 40% in mean vessel area relative to saline treated group (17.38 % versus 29.04 %, respectively). Thus VEGF antibody was capable of suppressing AVM formation in Alk1 deficient subdermal vessels respon ding to a wound (Fig. 2 2B). VEGF Blockade Alleviated Internal Bleeding and Low Hemoglobin O ccurring in Alk1 Deficient M ice. Alk1 deleted mice (tamoxifen treated R26 creER/+ ; Alk1 2f/2f mice) die within 2 3 weeks of tamoxifen treatment displaying severe GI, lung, and uterine hemorrhage 169 Since AVMs are a major cause of bleeding in HHT, we questioned whether
52 angiogenesis blockade would suppress visceral bleeding in Alk1 deficient mice. VEG F antibody (or saline, as a control) was injected into R26 creER/+ ; Alk1 2f/2f mice following tamoxifen treatment. After 9 days, the concentration of blood hemoglobin was determined as an indirect measure of internal hemorrhaging. Normal mice had hemoglobin l evels at 15 17 g/dl (data not shown). Alk1 deficient mice treated with saline exhibited significantly lower hemoglobin levels (mean: 8.6 g/dl) indicative of severe hemorrhaging. On the other hand, VEGF antibody treated mice displayed near normal levels of hemoglobin (13.8 g/dl), suggesting that internal bleeding was greatly reduced by VEGF antibody treatment (Fig. 2 3A). Thoracic and abdominal cavities were explored for direct evidence of hemorrhaging. In addition, heart size was compared as well because poorly oxygenated blood due to severe hemorrhage would force the heart to work harder to maintain oxygen levels, leading to enlargement of the heart. The relative severity of GI and lung bleeding and heart enlargement was categorized as weak, mo derate, or severe ( Fig. 2 7 ). The saline treated group showed moderate to severe bleeding in the lungs and GI tract (1 1/12 animals; T able 2 1, Fig. 2 3B). The majority of the VEGF antibody treated group displayed weak GI and lung hemorrhaging (9/12 mice; T able 2 1, Fig 2 3B ). The hearts of saline treated group were more enlarged compared to the VEGF antibody treated group (Fig. 2 3B) suggestive that severe hemorrhaging in the mock protected group adversely affected the size of the heart. VEGF Blockade Suppressed LPS I nduced AVMs in Alk1 Deficient Subdermal V essels. To determine whether inflammation alone, in the absence of angiogenesis, could induce AVMs in Alk1 deficient mice, LPS particles were implanted in subdermal sites and animals were simultaneously treated wit h VEGF antibody to block angiogenesis.
53 VEGF antibody suppressed AVM formation associated with LPS implantation and mock treatment with vehicle (saline; Fig. 2 4A through 2 4 D). This result indicates that LPS induced AVM formation is linked to angiogenic st imulation. TNF Inflammatory stimulation largely independent of an angiogenic response was evaluated with the implantation of TNF PLGA particles into Alk1 deficient mice. TNF is less actively involv ed in the monocyte induced angiogenesis compared to other inflammatory cytokines such as IL 8 183 A higher vascular density was observed around TNF ver, it did not provoke severe form of AVMs shown around LPS particles (Fig. 2 5D through 2 5 F) Discussion Although HHT is a genetic disease, vascular lesions appear only in limited vascular beds, indicating that localized secondary genetic or environmenta l insult is involved in development of AVMs Recently, we have demonstrated that wound could induce AVMs in the subdermal vessels of a mouse model of HHT2 169 In the present study, we showed that angiogenic stimulation by VEGF or LPS could mimic th e wound effect for de novo AVM formation in subdermal vessels of ALK1 deficient mice ( Fig. 2 6). We further demonstrated that treatment with VEGF neutralizing antibody could inhibit both wound and LPS induced AVMs (Fig. 2 6) and could alleviate the inter nal bleeding in the Alk1 conditional KO model These data attest to the use of angiogenesis blockade for treating epistaxis and GI bleeding in HHT patients. Therapeutic P otential of Anti A ngiogenic D rugs in HHT Recent anecdotal clinical evidence suggests that a nti angiogenic drugs can be an effective therapy for HHT The first ground breaking report came from an Australian
54 group showing VEGF neutralizing antibody, Bevacizumab (Avastin) treatment effectively regressed hepatic AVMs 177 More recently, Bevacizumab treatment was shown to be effective for pancreatic AVMs 184 GI bleeding 185 186 and epistaxis 187 190 in HHT patients. Lebrin et al showed that thalidomide known to have anti angiogenic and anti inflammatory functions, enhanced mural cell recruitment in Eng+/ mouse model, and alleviated the frequency and severity of nos ebleeds in HHT patients 176 V arious side effects of Bevacizumab including cardiac failure, GI bleeding, wound healing complication, and arterial thrombo embolic events have been reported 191 In order to reduce side effects thro ugh systemic administration of B evacizumab, topical application to na sal area using spray has been tried and shown to be effective 192 193 Therapeutic Potential of Anti Inflammatory I ntervention More than 90% of patients experience nosebleeds that stem from nasal Telangiectasia, 194 indicating that the nasal muc osa is very susceptible to AVM formation. We speculate that chronic inflammation infection and immune activation in the nose might be related to the high susceptibility. Inflammatory cytokines gene expression was implicated in AVM formation as the promoter polymorphisms in IL IL 6, TNF presentation of intracranial hemorrhage 171 195 197 More recently, Torsney et al showed that inflammation in the ears and eyelids almost invariably caused bleeding from thin walled dilated vessels in the E ng +/ mouse 198 Our skin wound model demonstrated that AVMs form only in the wound areas in Alk1 deficient mice 169 and here we showed that LPS could mimic the wound effect.
55 Inflammatory Stimulation Assoc iated with A ng iogenesis is Important for Inducing AVM F ormation. LPS is an endotoxin that provokes an acute inflammatory response by releasing proinflammatory cytokines including tumor necrosis factor ( TNF ) IL 6, IL 8. 199 TNF flammatory response by recruiting immune cells, induces additional proinflammatory cytokines such as IL 1 and IL 6 and inhibits apoptosis of inflammatory cells 200 201 We show that LPS can induce angiogenic stimulation in the context of an inflammatory response. Neovascularization and recruitment of sprouting blo od vessels occurred around LPS injected sites. Mattsby Baltzer et al have suggested that endotoxin mediated neovascularization is a component of inflammation and wound healing 181 LPS is associated with increased angiogenesis, vascul ar permeability, tumor cell invasion, and VEGF expression in macrophages 182 202 TNF 203 Activation of the Eck RPTK by its inducible ligand B61 i s involved in TNF induced angiogenesis 204 We showed that the AVMs formed by LPS were more severe than those by TNF Perhaps it happened because LPS might have provoke d stronger inflammatory stimulat ion by releasing various proinflammatory cytokines (including TNF induced inflammation, leading to more intensive angiogenic stimulation. For instance, it was shown that production of IL 8 which may be play a role in a more lasting my eloid cell driven angiogenesis could be induced by LPS, but not TNF 183 Taken together, our data suggest that blocking angiogenesis or inflammation could be an effective therapy for epistaxis and GI bleeding in HHT patients. Number of FDA approved drugs inhibiting angiogenesis or inflamm ation is rapidly increasing. In addition
56 to angiogenesis blockade and inflammatory inhibitors, drugs targeting some other aspect of AVM formation such as antifibrinolytic agent (Tranexamic acid) 156 205 antioxidants (N Acetyl Cystein) 206 estrogen analogs (Raloxifene) 207 have been suggested for treating HHT. The preclinical model presented here would be an invaluable resource with which these potential drugs can be screen ed for effectiveness in preventing skin de novo AVMs and GI bleeding. Thus, inflammation prevention could be therapeutic for HHT patients suffering nosebleeds.
57 T able 2 1. The number of mice with hemorrhage in saline and VEGF antibody treated group s Saline (n=12) VEGF Ab (n=12) GI bleeding Weak 1 7 Moderate 6 3 Severe 5 2 Lung bleeding Weak 2 9 Moderate 5 2 Severe 5 1 Liver AVM Weak 11 10 Moderate 0 1 Severe 1 1 Lung, GI bleeding and liver AVM of each mouse were categorized into three groups (weak, moderate, severe) and the number of groups was counted in saline treated (n=12) and VEGF treated (n=12) Alk1 deficient mice.
58 Figure 2 1. Angiogenic or inflammatory stimulation induces AVMs in Alk1 deficient adult subdermal vessels but not Alk1 wild type vessels. VEGF PLGA and LPS PLGA were implanted under dorsal skin as an angiogenic or inflammatory stimulus, respectively. Tamoxifen (TM) was intraperitoneally injected to effect Alk1 deletion. A fter 7 days, blue latex dye was infused through the heart to visualize blood vessels in the dorsal skin and particle injected skins were cleared (see methods). (A B) VEGF (A) and LPS (B) particles did not induce AVMs in Cre negative controls ( R26 +/+ ; Alk1 2f /2f + TM) bearing wild type Alk1 alleles. (C D) PBS PLGA was implanted to investigate whether PLGA itself can induce AVM formation in Alkl deficient mice ( R26 creER/+ ; Alk1 2f/2f + TM). PBS PLGA did not induce AVMs (C) or rarely formed abnormal vasculature ( D). (E F) VEGF (E) and LPS (F) particles induced severe AVMs and tortuous vessels in ALK1 depleted vessels ( R26 creER/+ ; Alk1 2f/2f +TM). VEGF (G) and LPS (H) particles induced angiogenesis by recruiting sprouting vessels toward particles as seen 4 days post implantation of loaded particles. At left bottom are higher magnifications of recruited sprouting vessels. Scale bars in each panel indicate 1 mm.
59 Figure 2 2. Angiogenesis blockade (VEGF neutralizing antibody) suppressed wound induced AVM formation in Al k1 deficient adult subdermal vessels. (A) Saline or VEGF antibody (Ab) were injected into Alk1 deficient adult mice (R26creER/+;Alk12f/2f ) to block angiogenesis. AVMs were induced by incisional wound of dorsal skin. VEGF Ab suppressed wound induced AVMs c ompared to saline treated group. Tamoxifen (TM) was treated to effect Alk1 deletion. VEGF Ab (bottom panels) suppressed wound induced AVMs compared to saline treated animals (top panels). (B) The severity of AVMs formed surrounding wound sites was calculat ed as the percentage of vessel area containing latex dye (MATLAB program). Panels on the left are representative of one experimental comparison. The graph on the right is a compilation of all data showing a significant reduction in the percentage of vessel area in VEGF treated group compared to the saline treated group. Scale bars in each panel indicate 1 mm.
60 Figure 2 3. Angiogenesis blockade improved hemoglobin levels and visceral hemorrhage in Alk1 deficint adult mice. (A) Hemoglobin concentration was m easured in saline treated (n=12) and VEGF treated (n=12) Alk1 deficient mice 9 days after tamoxifen treatment to effect Alk1 deletion. VEGF blockade improved hemoglobin levels in Alk1 deficient mice relative to the saline treated group. (B) The severity of GI and lung bleeding (top) was expressed as a severity index based on direct observation and categorization. H ypertrophy of the heart (bottom) was expressed as a ratio of heart volume and tibia length ( l/cm) VEGF blockade significantly suppressed related hemorrhaging and heart enlargement in Alk1 deficient mice.
61 Figure 2 4. Angiogenesis blockade suppressed LPS induced AVMs in Alk1 deficient mice. LPS particles formed aggregates on subdermal surfaces in both saline treated (A) and VEGF antibody treated (B) Alk1 deficient mice. After skins were cleared to enhance the visualization of blood vessels, AVMs were appar ent in saline treate d mice (C) but not mice treated with VEGF antibody (D). Scale bars in each panel indicate 1 mm.
62 Figure 2 5. TNF Alk1 deficient mice. TNF particles were implanted under dorsal skin in tamoxifen treated Alk1 deficient mice ( R26 creER/+ ; Alk1 2f/2f ). Vessels were observed after 7 days with the latex dye. TNF treated mice (A, B and D, E) are compared with an LPS treated mouse (C and F) before (A C) and after (D F) the clearing of aggregated particles. TNF However, AVMs formed by TNF induced by LPS (F). Scale bars in e ach panel indicate 1 mm.
63 Figure 2 6. Quantification of AVM formation by various stimuli in Alk1 deficient subdermal vessels. VEGF and LPS treated groups significantly promoted AVM formation compared to PBS treated group. TNF gnificantly induce AVM formation in Alk1 deficient mice. LPS induced AVM formation was suppressed by VEGF neutralizing antibody (V Ab).
64 Figure 2 7 Lung (top) and GI bleeding (bottom) was categorized into three groups (weak, moderate, and severe) depending on the severity. Partial or focal hemorrhaging belonged to the weak level (left). Vast areas of hemorrhaging were categorized as severe (right). Intermediate levels of hemorrhaging were grouped as moderate (middle).
65 CHAPTER 3 MATERIALS AND METHODS Smad1 Conditional Knockout M ice Generation of a conditional Smad1 allele ( Smad1 f ) was previously described 208 Generation of the Tg( Alk1 cre) L1 (L1cre) line has also been described 109 Tg( Tagln cre) and R26R mice were purchased from the Jackson Laboratory. Smad1 f/f mice were intercrossed with L1cre or Tagln cre lines. The L1cre(+); Smad1 f/+ or L1cre(+); Smad1 +/2f ;R26R males were further intercrossed with Smad1 f/f ;R26R females to produce L1cre( ); S mad1 f/f and L1cre(+); Smad1 f/f Tagln cre( ); Smad1 f/f and Tagln cre(+); Smad1 f/f were produced by the same breeding scheme. More than half of the control and experimental mice contained the R26R allele to monitor the Cre activity. PCR primer sets and conditi ons for detecting the conditional as well as null alleles of Smad1 were previously described 208 Primer sets for the genotype of L1cre, Tagln cre, or R26R are shown in Table 1 1 Hemodynamic A nalysis To evaluate pulmonary artery pressure, right ventricula r systolic pressure (RVSP) was measured by right heart catheterization through the right jugular vein. Each mouse was anesthetized by isoflurane (1 2 %) and placed in the supine position. A 1 2 cm incision was made in the neck to expose the right jugular v ein. The Mikro Tip pressure transducer (SPR 835, Millar Instrument) was inserted into right external jugular vein and advanced into the right ventricle. Systemic blood pressure was recorded non invasively using the tail cuff method. A pneumatic pulse senso r was placed on the tail distal to an occlusion cuff controlled by a Programmed Electro Sphygmomanometer (PE 300, Narco Bio Systems), which is connected to the Powerlab system (ADinstrument). All
66 electrical outputs from the tail cuff, the pulse sensor and transducer were recorded and analyzed by the Powerlab 8/30 data acquisition system and associated Chart software (ADinstrument). Right Ventricular Hypertrophy (RVH) After hemodynamic analysis, mice were euthanized and using syringe generated flow, the pulmonary circulation was perfused with PBS containing heparin (3 units/ml). The hearts were isolated and outflow tracts and atria were removed. The right ventricle was cut out from the heart by the spring scissor and the right ventricle and the remaining left ventricle (LV) plus septum (S) were weighed. Right ventricular hypertrophy was determined by the ratio of RV/LV+S. Pulmonary Vessel M orphometry After the hemodynamic analysis, the left lung was inflated with PBS for 20 minutes followed by formalin at a constant inflation pressure of 23cmH2O, fixed with 4% paraformaldehyde overnight, and paraffin embedded. Each lung sample was transversely sliced i aldrich, mouse monoclonal, 1:800) using M.O.M kit (Vector laboratories) to visualize the vascular smooth muscle layer. To determine muscularizati on of pulmonary vessels, peripheral blood vessels ranging from 30 Zeiss Axioplan 2 optical microscope. The counted vessels were categorized as as fully muscularized (75 100% of medial layer covered by anti muscularized (1 74% of medial layer is covered by anti nonmuscularized vessels at the level of alveolar ducts. The percentage of pulmonary vessels in each category was calculated by dividing the number of vessels by the total
67 number of counted vessels in the same field. To calculate the percentage of wall thickness (WT), circular and fully muscularized vessels were selected. WT1 (the thickness between the outer boundary and the in layer) was measured at one point of the vessel wall and WT2 at the point which was diametrically opposite, guided by Openlab 5.03 Beta software (Improvision Inc.). External diameter (ED) was also measured at the same ve ssel. The percentage of medial wall thickness was calculated as (WT1 + WT2) x100/ED. Establishment of Immortalized Pulmonary Endothelial C ells Whole lungs were removed from an eight week old R26 CreER/+ ; Bmpr2 2f/2f mouse and washed in HEPES followed by addi tional washing in DMEM. Lung tissues were finely minced using a sterile scalpel. The chopped tissues were subjected to serial digestion using 2 ml of a 1X trypsin solution [0.25% trypsin, 0.5 M EDTA (pH 8.0) in DMEM] at 37C, with frequent shaking, for thr ee times at 8 minutes each. Trypsin digestion was inactivated by adding 6 ml of normal endothelial cell media (ECM) [10% fetal bovine serum, 1 mg/ml heparine 0.1 mg/ml endothelial mitogen (Biomedical technologies), 1 mM non essential aminoacids (Cellgro), 1 mM sodium pyruvate, 50 units/ml penicillin/streptomycin] After the large debris sinking down, the supernatant was carefully collected and plated into 2 wells of a 6 well culture plate. For immortalization, when the culture reached 50% confluency post i solation, they were transfected with 4.0 g of SV40 DNA: ATCC (VRMC 3), pUCSV40 B2E 209 210 using After a couple of passages, the endothelial cells were sorted out by Fluorescence Activated Cell Sorting (FACS) using Dio Ac LDL (Biomedical technologies) and lectin (Sigma aldrich). For deleting the Bmpr2 gene, the immortalized pulmonary ECs were plated on 6 well plates
68 (2x10 5 hydroxy tamoxifen (Sigma aldrich) for 3 days. Thereafter the cells were cultured with 4 hydroxy tamoxifen depleted growth medium. Primers (table 1) detecting null allele of Bmpr2 ( Bmpr2 1f/1f ) were used to confirm the deletion of Bmpr2 by tamoxifen. Semi Q uantitative RT PCR To determine the levels of transcripts of endothelial cell specific markers and genes involved in TGF Bmpr2 intact ( Bmpr2 2f/2f ) an d Bmpr2 deleted ( Bmpr2 1f/1f ) pECs were harvested at 100% confluency from a 25 cm 2 culture flask. Total RNAs from the cells were extracted using the NucleoSpin RNA purification The cDNAs were synthesized using SuperScript III First of cDNA was used as a template for PCR amplification for 25 cycles: denaturation at 94C for 45 seconds, annealing at 60C for 45 seconds and extension at 72C fo r 1 minute. The transcription level of each gene was normalized to Gapdh expression. The primers used for RT PCR analysis are shown in T able 1 1 Western B lotting Pulmonary ECs were harvested with a Chemicon lysis buffer containing 50 mM Tris (pH 6.8), 1 mM EDTA and 2% SDS and sonicated. Lysates were spun down at 13000 rpm for 15 minutes. The concentration of protein was determined using the Bio PAGE and transferred to nitrocellulos e membranes (Bio rad). Membranes were incubated with the primary antibody followed by the horseradish peroxidase linked secondary antibody. A chemiluminescent detection reagent (ECL PlusTM, Amersham Pharmacia Biotech Inc.) was used to visualize proteins. T he antibodies used for Western blotting analysis are
69 the following: SMAD1 (rabbit polyclonal, 1:1000), SMAD2 (mouse monoclonal, 1:1000), pSMAD2 (rabbit polyclonal, 1:1000), pSMAD1/5/8 (rabbit polyclonal, 1:1000) from Cell actin (mouse monoclon al, 1:10,000) from Sigma aldrich, BMPR2 (mouse monoclonal, 1:500) from BD Transduction Laboratories, Secondary antibodies include: mouse (1:5000) and rabbit (1:5000) from Sigma aldrich. Alk1 C ond itional Knockout M ice Establishment of the Alk1 2f allele in l aboratory mice was described previously 109 R26 creER/+ allele carrying mice were purchased from The Jackson Laboratory By crossing R26 creER/+ line with Alk1 2f/2f mice, R26 creER/+ ; Alk1 2f/2f mice were produced and 2 4 month old mice were used in all experiments. The inactivation of the Alk1 gene in adult mice was accomplished by treating animals as previously described 169 All in vivo procedures were conducted in accordance with animal use guidelines established by the University of Florida Institutional Animal Care and Use Committee. Preparation of PLGA M icroparticles Poly (d,l lactide co glycolide) (PLGA), 50:50 composition with inherent viscosity 0.55 0.75 dl/g in hexafluoroisopropanol, HFIP (Lactel) was used to generate the particles. The emulsion stabilizer, poly vinyl alcohol (PVA) (MW ~ 100,000 g/mol) was purchased from Fi sher Science. Phosphate buffered saline (PBS) solution (Hyclone) was used as the aqueous phase to form the emulsions while methylene chloride (Fisher Scientific) was used as an organic solvent to dissolve PLGA polymer. Microparticles were formed using a st andard water oil water solvent evaporation technique 211 212 Briefly, a 3% solution of PLGA polymer in methylene chloride was generated. 1 ml of 3% PLGA (Sigma Aldrich) in PBS, Fisher Scientific
70 systems) in PBS for TNF Al drich) in PBS for LPS PLGA at 26,500 rpm for 60 seconds using a tissue miser homogenizer (Fisher Scientific) to form the primary emulsion. The primary emulsion was added to 10 ml of 5% PVA solution in PBS and the homogenizing was continued at 19,500 rpm fo r 120 seconds to form the secondary emulsion. Then, the secondary emulsion was added to 100 ml of 0.5% PVA made in dIH 2 O. The particles thus formed were agitated using a magnetic stirrer for 16 hours to evaporate residual methylene chloride. The remaining solution was centrifuged at 10,000 rpm for 10 minutes to collect microparticles which were subsequently washed three times with PBS. The particle suspension in 5 ml of PBS was then flash frozen in liquid nitrogen and stored at 20 0 C until used. Injection of PLGA Particles into S ubdermal A rea The recipient mice ( R26 creER/+ ; Alk1 2f/2f and R26 +/+ ; Alk1 2f/2f ) were anesthetized by placing them within an induction chamber and introducing 4.0% isoflurane, and out of the chamber, anesthesia was maintained by 2.0% 3.0% isoflurane using a nose cone. area of the mid dorsal skin using 29 gauge 0.3 m l syringe (Monoject). Tamoxifen (TM, Sigma aldrich) was injected intraperitoneally at a dose of 2.5 mg/25 g of body weight for Alk1 gene deletion. Then, 0.6 ml of the blue latex dye (Connecticut Valley Biological Supply Co.) was injected via the left heart 7 days after TM injection to visualize the blood vessels.
71 Skin Wound Generation and VEGF Antibody T reatment The recipient mice ( R26 creER/+ ; Alk1 2f/2f ) were anesthetized as described above. The hairs on the back of the mice were shaved and one 2 mm diamete r full thickness excisional wounds were inflicted on the mid dorsum using a 2 mm dermal biopsy punch (Miltex). The wounds were left unsutured, and betadine was applied to the wounds. No analgesics or antibiotics were given in order not to affect the wound healing process. TM was injected intraperitoneally at a dose of 2.5 mg/25 g of body weight and VEGF neutralizing antibody (100 of saline was serially injected on the first, 4 th and 7 th day of post wounding. On the 9 th day of post wounding, 0.6 ml of the blue latex dye was injected via the left heart. Latex Dye I njection a nd Image P rocessing Mice were anesthetized with intraperitoneal injection of ketamine/xylazine (100 mg/15 mg/kg of body weight) a nd abdominal and thoracic cavities were opened. To perfuse all the blood out, heparin (200 unit/ml, Sigma aldrich) in 10 ml PBS was infused through the left ventricle of the heart at 120 ml/hour using a syringe pump (KD scientific) after a small cut was ma de on the left atria. Then, 10 ml of PBS dilator mixture containing heparin (10 unit/ml, Sigma Aldrich), papaverine (0.04 mg/ml, Sigma Aldrich), aldrich) was infused through the same hole made on the left ventricle t o maximize dilation of blood vessels. To fix the dilated vessels 10 ml of 10% formalin was infused through the same hole on the left ventricle. Then, 0.6 ml of the blue latex dye was slowly and steadily injected into the hole of left ventricle with a 26 g auge 1 ml syringe. Then, the hair removal cream (Veet) was put on the shaved back skin of dye injected mice and the hairs were completely removed after 10 min utes incubation. The mice were washed briefly in water and fixed with 10%
72 formalin overnight. Afte r fixation, the dorsal skin was peeled off, stretched, and flattened on the Styrofoam. The flattened skin was dehydrated by methanol series (20%, 50%, 75%, and 100%, each for 20 min utes ) and cleared with organic solvent (benzyl alcohol/benzyl benzoate, 1:1 ; Sigma aldrich). Blood vessels containing the latex dye in the cleared skin were imaged via a CCD camera (Leica) and processed by MATLAB (MathWorks) as described previously 169 Hemoglobin Concentration and Hemorrhage I ndex 2 3 drops of blood w ere collected from the tails of VEGF antibody treated (N=12) and saline treated mice (N=12) and soaked up by microcuvettes (STANBIO Laboratory). The concentration of hemoglobin was measured by the hemoglobin photometer (Hemopoint H2, STANBIO Laboratory) us ing the blood soaked microcuvettes. Then, after anesthesia with ketamine/xylazine, abdominal and thoracic cavities were opened and hemorrhages of lungs and gastrointestinal tract and the size of the heart were observed and categorized into three levels: we ak, moderate, and severe conditions according to the d egree of severity ( Fig ure 2 6 ). Hemorrhage levels were graded: 1.0 for weak, 2.0 for moderate, and 3.0 for severe level. The mean values were expressed as severity index. Hypertrophy of the heart After latex dye injection through the heart, hearts were taken and all the blood vessels connecting to the heart were removed. Hearts were submerged in 1.5 ml tube containing 500 l PBS and increased volume of PBS was measured as a volume of heart. Right le gs of mice were cut out in the middle of femur and were skinned off and were put in 1M NaOH overnight for digestion of tissues surrounding bones. After digestion, tissues were easily removed and the foot bone and broken femur were
73 separated from the tibia by slowly pulling them. The length of whole tibia was measured by the caliper. Statistical A nalysis T test was used to determine a statistical significance between two groups, and one way Anova was used for more than 3 groups, and multiple pairwise comp arisons were made by post hoc tests (Tukey) using the Sigmaplot.
74 CHAPTER 4 CONCLUDING REMARKS AND FUTURE DIRECTIONS Current therapeutic interventions for both PAH and HHT diseases are restricted to alleviate severe symptoms because they are not based on mechanistic pathology. There is a pressing need for more effective and pathology specific therapies. In PAH studies, I showed that l oss of SMAD1 function in ECs or SMCs predisposes mice to pulmonary hypertension and Impaired BMP signa ling makes TGF activated and leads to an imbalance of TGF pECs, which might be a critical factor for PAH development. From these results, I can suggest several therapeutic targets. First, reduced SMAD1 acti vity may be associated with PH development. Therefore, inhibiting phosphatase activity could be effective to maintain SMAD1 phosphorylation and its activity although it should be specific to SMAD1/5/8, not SMAD 2/3. Second, recovering from imbalance state o f TGF / BMP signaling would be therapeutic for PAH patients by suppressing over activated TGF TGF antagonists such as the angiotensin II type 1 receptor (AT1) blocker, Losartan 133 or the activin recetor like kinase 5 inhibitor, SB525334 213 may have therapeutic benefit. In addition to this, I found that BMP7 response was intact in Bmpr2 deleted pECs. Thus, treatment of BMP7 ligand could compensate for reduced level of pSMAD1/5/8 from BMPR2 deficiency. To determine whet her over activation of TGF response is a critical factor for PAH development in vivo we are planning to test whether inhibition of TGF models Smad1 or Bmpr2 cKO mice by additionally deleting genes involved in TGF
75 signaling such as Tgfbr2 and Smad4 or treatment with inhibitors blocking TGF signaling, such as Activin Receptor Like Kinase 5 (ALK5) inhibitor 103 Endothelial S mad 1 deletion did not result in 100% penetracne in PH, indicating that environmental or genetic second hits are required for PAH development. As shown in Figure 1 1 I observed the marked reduction of SMAD1/5/8 phos phorylation in the lungs of PH mice, suggesting that the complete loss of SMAD1 activity in the whole lung may be essential to manifest PAH phenotype. Thus, by mating L1cre(+); Smad1 f/f with Tagln cre(+); Smad1 f/f we can delete Smad 1 gene both in endothelial and smooth muscle cells. Although these double cre KO mice did not block SMAD 1 activity in the whole lung, we can expect synergistic effect of smooth muscle thickening and endothelial dysfunction and may expect increase of penetra nce. In HHT studies, I demonstrated that inflammation or angiogenesis are sufficient to induce AVMs as second hits for ALK1 deficiency. In terms of therapeutic aspect, antagonizing inflammatory or angiogenic pathway may be effective for HHT patients. I sh owed that VEGF neutralizing antibody can prevent AVM formation and internal bleeding in Alk1 deficient adult mice. Even though effective anti inflammatory reagents are not available, IL 9 neutralizing antibody could be a useful reagent to test because IL 9 is essential factor for inflammation induced angiogenesis Up to now, many studies have focused on assessing increased response to angiogenic stimulation in Alk1 deleted endothelial cells. However, what makes this increased sensitivity as a result of Alk1 deficiency remains unclear. To address this question, we have many useful resources: pulmonary endothelial cells (pECs) mice carrying Alk1 2f/2f and Alk1 1f/1 f allele respectively, and microarray data from pECs
76 ( Alk1 2f/2f and Alk1 1f/1 f ). I showed VEGF signaling is required for AVM formation with Alk1 deficiency. With above materials, examining whether Alk1 can suppress downstream of VEGF signaling or modulate interaction of VEGF and VEGF receptors and other possible hypotheses would be i nteresting and invaluable research to find novel therapeutic targets.
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97 BIOGRAPHICAL SKETCH Chul Han was born in Da ejon, South Korea in 19 75 He is the oldest of two sons born to Jeong gun Han and Duk ae Lee Chul attended Korea University from 1994 2003 where he studied molecular biology and biochemistry and earned a Bac helo r of Science in genetic engineering in 2001 and a Master of Science in molecular medical science in 2003 He served in the air force for two and half years (1998 2000). After graduation, Chul worked as a research scientist in the Samsung medical center (20 03 2006). Then he moved to Gainesville, Florida and joined the Interdisciplinary Program in Biomedical Sciences at the University of Fl orida College of Medicine. Chul did his graduate work in Dr. S. Paul Oh of Functional Geno mics and Physiology and complet ed his Ph.D. dissertation in August 20 11