Role Of Connective Tissue Growth Factor In Vascular Remodeling Of Rat Pulmonary Hypertension

BackgroundPulmonary arterial hypertension (PAH) is characterized pathologicallyby vascular remodeling, which includes abnormal proliferation of vascularendothelial and smooth muscle cells, and deposition of extracellular matrixprotein, resulting in occlusion of the lumen of small pulmonary arteries andincreased pulmonary vascular resistance and right-sided heart failure.Vascular smooth muscle cell (VSMC) proliferation has been known to bepredominant in vascular remodeling of hypertensive pulmonary vasculardisease. Connective tissue growth factor (CTGF) was first identified as a38-kDa cysteine-rich secreted protein that regulates cell proliferation/apoptosis, angiogenesis, migration, adhesion, and fibrosis. CTGF wasrecently found to be expressed abundantly in pulmonary arterial smoothmuscle cell (PASMC) in pulmonary hypertensive rat injected withmonocrotaline(MCT), but only marginally in normal vascular tissues. It washypothesized that CTGF is one of the factors involved in the development ofpulmonary vascular remodeling of PAH. CTGF expression is strongly upregulated by mechanical stress or static pressure and by several otherfactors involved in vascular damage, including elevated glucoseconcentrations, TGF-β, VEGF, angiotensin (Ang)â…¡, and ET-1, but not byfactors such as epidermal growth factor and PDGF, and is downregulated bycAMP and TNF-α. On the basis of the benefits of compounds withantiproliferative effects on vascular endothelial and smooth muscle cells,such as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductaseinhibitors, statins, in decreasing neointimal vascular occlusion and improvingblood flow in the systemic arterial circulation, we hypothesized that statinsmight show benefits in experimental hypertensive pulmonary vasculardisease. Here, we characterized the effects of simvastatin on expression of,CTGF, development of neointimal formation, pulmonary arterialhypertension, and fight ventricular hypertrophy in pneumonectomized,monocrotaline (MCT)-treated rats.Objective1. To explore differentiation of the pattern of pulmonary vascularremodeling in response to injury by comparing 4 approaches in establishingpulmonary hypertension model in rat.2. To investigate the expression of CTGF gene in pneumonectimized,MCT-treated rats and explore the effect of regulation of CTGF expression bysimvastatin on pulmonary vascular remodeling.3. To determine the effect of simvastatin on development of pulmonaryvascular remodeling of PAH animal model.4. To investigate the modulation of proliferation, migration, cell cycleand mRNA expression of collagenâ… -α1, collagenâ…£-α1 and fibronectin-1 in PASMC stimulated by reconstructed human CTGF in vitro.Methods1.4 approaches in establishing pulmonary hypertension model in rat:Abdominal aortocaval fistula shunting Group (A-VF, n=10); Left lungpneumonectomy Group (PE, n=10), MCT injection (subcutaneous 60mg/kg)Group (MCT, n=10); and MCT injection Day 7 after pneumonectomyGroup(PM, n=22), which includes Group PM_1-21(n=10) and PM_1-35 (n=12)received neither simvastatin nor vehicle, and were sacrificed on Day 21 orDay 35 to provide reference point for disease progression in this model.Additional 48 pneumonectomized rats were randomized to receivesimvastatin or vehicle by daily gavage: Rats in Group PMV_1-35 (n=12)received a vehicle from Day 1 to Day 35; Group PMV_21-35(n=12) received avehicle from Day 21 to Day 35; Group PMS_1-35 (n=12) received simvastatin(2mg/kg, per day) from Day 1 to Day 35; Group PMS_21-35 (n=12) receivedsimvastatin (2 mg/kg per day) from Day 21 to Day 35. Ten additional ratswere observed as a control Group without any intervention. On Day 35 afterpneumonectomy, hemodynamic measurements was performed before ratswere sacrificed, and organs harvested for the further analysis.2. Morphological parameters relevant to pulmonary vascular remodelingpattern was performed in each group of rats, including percent medialthickness (WT%), muscularization of nonmuscular arteries in the region ofthe alveolar duct, vascular oclussion scores (VOS), proliferating cell nuclearantigen (PCNA) and TUNEL staining.3. CTGF expression was analysised by hybridization in situ, real timefluorescent quantitation RT-PCR, immunohistochemical staining and western blot, and endothelin-1 mRNA was detected by real-time fluorescentquantitation RT-PCR in lung tissue of pulmonary hypertensive rats andsimvastatin-treated rats.4. Survival analysis was performed among pneumonectomized,MCT-treated rats receiving vehicle or simvastatin from Day 1 or Day 21 toDay 35.5. Collagen synthesis was evaluated by Masson staining and content ofhydroxyproline (HYP) by hydrolization.6. Proliferation and cell cycle of PASMC stimulated by rhCTGF wasinvestigated by Cytometry, PCNA staining, and flow cytometry, respectively.7. PASMC migration was measured using a scrap injury of confluentcell layers treated with rhCTGF in vitro.8. Expression of extracellular matrix genes such as fibronection-1,collagenâ… -α1 and collagenâ…£-α1 mRNA in PASMC stimulated by rhCTGFwas assessed by real-time fluorescent quantitation RT-PCR in vitro.9. Cytoskeleton in PASMC treated with rhCTGF was observed usingCoomassie brilliant blue R₂₅₀ stainging.Results1. Neointima proliferation was observed in 88.5% of right lungintra-acinar arteries on Day 35 in pneumonectomy, MCT-treated rats, and hadsignificantly increased mean pulmonary arterial pressure (mPAP), RV/(LV+S)ratio, vascular occlusion scores (VOS), percent medial thickness and distalarteriole muscularization on Day 35, compared with A-VF, PE, MCT rats andControl animals. Neointima formation did not develop in Groups A-VF, PE,MCT rats and Control animals. 2. CTGF mRNA and protein were found to be expressed abundantly inVSMC of the arteries and arterioles, and infiltrated macrophages inpneumonectomized, MCT-treated rats by in situ hybridization andimmunohistoehemical staining respectively. Real-time fluorescentquantitation RT-PCR and Western Blot assay of the lung tissue of thesedisease rats at Day 35 indicated significantly increased expression of CTGFas compared with normal rats. The level of ET-1 mRNA was alsosignificantly increased in these disease rats.3. The expression of ET-1 and CTGF was significantly down-regulatedin pneumonectomy and MCT-treated rats after receiving simvastatin. Thesesimvastatin-treated rats also had significantly decreased mPAP, RV/(LV+S)ratio, VOS, percent medial thickness and distal arteriole muscularization,compared with rats treated with vehicle. IOD of PCNA immunohistochemicalstaining in pneumoneetomized, MCT-treated rats also reduced remarkably inthese simvastatin-treated rats, while cell apoptosis rate using TUNEL stainingwas significantly increased as compared with. vehicle-treated rats. Survivalanalysis indicated that there was 100% or 83.33% survival inpneumoneetomized, MCT-treated rats receiving simvastatin from Day 1 orDay 21 to Day 35.4. The lung sections stained with Masson' triehrome indicated increasedcollagen deposition in neointima, vascular wall and lung interstitium, andcontent of HYP also significantly increased in pneumonectomy, MCT-treatedrats, compared with normal rats. Collagen deposition and content of HYPreduced remarkably in simvastatin-treated rats compared with rats treatedwith vehicle, respectively. 5. In this study, we investigated the function of CTGF protein inregulating proliferation and migration of PASMC and found that proliferationand migration in PASMC stimulated by rhCTGF were significantly increasedcompared with control. In addition, exogenous CTGF also promoted theexpression of extracellular matrix such as collagenâ… -α1, collagenâ…£-α1 andfibronectin-1 mRNA, and modulated transiently cytoskeleton of PASMC invitro.Conclusion1. The neointimal model of pulmonary hypertension can be successfullyestablished by introduction of hemodynamic stress to the injury of MCTinjection rats and should be utilized as an preferable model for the researchon human severe obstructive pulmonary hypertension.2. Our results indicate that the development and progression ofpulmonary hypertension is prevented by simvastatin via a diversity ofpathways such as anti-proliferation and induced apoptosis of PASMC, anddirect or ET-1 inhibition related down-regulation of CTGF expression in lungtissue.3. Effect of rhCTGF on proliferation, migration, change of cytoskeletonand ECM deposition in PASMC was demonstrated in vitro, suggesting thatCTGF may play a predominant role in vascular remodeling of PAH.