Simvastatin reduces venous stenosis formation in a murine hemodialysis vascular access model

Venous neointimal hyperplasia (VNH) is responsible for hemodialysis vascular access malfunction. Here we tested whether VNH formation occurs, in part, due to vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP)-9 gene expression causing adventitial fibroblast transdifferentiation to myofibroblasts (α-SMA-positive cells). These cells have increased proliferative and migratory capacity leading to VNH formation. Simvastatin was used to decrease VEGF-A and MMP-9 gene expression in our murine arteriovenous fistula model created by connecting the right carotid artery to the ipsilateral jugular vein. Compared to fistulae of vehicle-treated mice, the fistulae of simvastatin-treated mice had the expected decrease in VEGF-A and MMP-9 but also showed a significant reduction in MMP-2 expression with a significant decrease in VNH and a significant increase in the mean lumen vessel area. There was an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and decreases in α-SMA density, cell proliferation, and HIF-1α and hypoxyprobe staining. This latter result prompted us to determine the effect of simvastatin on fibroblasts subjected to hypoxia in vitro. Simvastatin-treated fibroblasts had a significant decrease in myofibroblast production along with decreased cellular proliferation, migration, and MMP-9 activity but increased caspase 3 activity suggesting increased apoptosis. Thus, simvastatin results in a significant reduction in VNH, with increase in mean lumen vessel area by decreasing VEGF-A/MMP-9 pathway activity. Venous neointimal hyperplasia (VNH) is responsible for hemodialysis vascular access malfunction. Here we tested whether VNH formation occurs, in part, due to vascular endothelial growth factor-A (VEGF-A) and matrix metalloproteinase (MMP)-9 gene expression causing adventitial fibroblast transdifferentiation to myofibroblasts (α-SMA-positive cells). These cells have increased proliferative and migratory capacity leading to VNH formation. Simvastatin was used to decrease VEGF-A and MMP-9 gene expression in our murine arteriovenous fistula model created by connecting the right carotid artery to the ipsilateral jugular vein. Compared to fistulae of vehicle-treated mice, the fistulae of simvastatin-treated mice had the expected decrease in VEGF-A and MMP-9 but also showed a significant reduction in MMP-2 expression with a significant decrease in VNH and a significant increase in the mean lumen vessel area. There was an increase in terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, and decreases in α-SMA density, cell proliferation, and HIF-1α and hypoxyprobe staining. This latter result prompted us to determine the effect of simvastatin on fibroblasts subjected to hypoxia in vitro. Simvastatin-treated fibroblasts had a significant decrease in myofibroblast production along with decreased cellular proliferation, migration, and MMP-9 activity but increased caspase 3 activity suggesting increased apoptosis. Thus, simvastatin results in a significant reduction in VNH, with increase in mean lumen vessel area by decreasing VEGF-A/MMP-9 pathway activity. In the United States, approximately 600,000 patients have end-stage renal disease, with the vast majority of patients requiring chronic hemodialysis for long-term survival, and this population of patients will double in the coming decades.1.Collins A.J. Kasiske B. Herzog C. et al.Excerpts from the United States Renal Data System 2003 Annual Data Report: Atlas of end-stage renal disease in the United States.Am J Kidney Dis. 2003; 42: A5-A7Abstract Full Text Full Text PDF PubMed Google Scholar Arteriovenous fistula (AVF) is the preferred vascular access; however, venous stenosis formation and lack of maturation are major problems, and thus 1-year patency rates are estimated to be 62%.2.Rooijens P.P.G.M. Tordoir J.H.M. Stijnen T. et al.Radiocephalic wrist arteriovenous fistula for hemodialysis: meta-analysis indicates a high primary failure rate.Eur J Vasc Endovasc Surg. 2004; 28: 583-589Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar Venous stenosis occurs in AVFs because of neointimal hyperplasia.3.Rekhter M. Nicholls S. Ferguson M. et al.Cell proliferation in human arteriovenous fistulas used for hemodialysis.Arterioscler Thromb. 1993; 13: 609-617Crossref PubMed Scopus (129) Google Scholar, 4.Swedberg S.H. Brown B.G. 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Sigley R. et al.Intimal fibromuscular hyperplasia at the venous anastomosis of PTFE grafts in hemodialysis patients. Clinical, immunocytochemical, light and electron microscopic assessment.Circulation. 1989; 80: 1726-1736Crossref PubMed Scopus (260) Google Scholar, 5.Roy-Chaudhury P. Kelly B.S. Miller M.A. et al.Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts.Kidney Int. 2001; 59: 2325-2334Abstract Full Text Full Text PDF PubMed Google Scholar Recent experimental studies have demonstrated a pivotal role for adventitial and medial fibroblasts that convert to myofibroblasts (α-SMA-positive cells) and can subsequently contribute to the formation of venous neointimal hyperplasia.6.Misra S. Doherty M.G. Woodrum D. et al.Adventitial remodeling with increased matrix metalloproteinase-2 activity in a porcine arteriovenous polytetrafluoroethylene grafts.Kidney Int. 2005; 68: 2890-2900Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 7.Li L. Terry C.M. Blumenthal D.K. et al.Cellular and morphological changes during neointimal hyperplasia development in a porcine arteriovenous graft model.Nephrol Dial Transplant. 2007; 22: 3139-3146Crossref PubMed Scopus (36) Google Scholar, 8.Wang Y. Krishnamoorthy M. Banerjee R. et al.Venous stenosis in a pig arteriovenous fistula model anatomy, mechanisms and cellular phenotypes.Nephrol Dial Transplant. 2007; 22: 3139-3146Crossref PubMed Scopus (56) Google Scholar As a consequence, over a billion dollars are spent annually to maintain the functioning of hemodialysis AVFs and grafts.1.Collins A.J. Kasiske B. Herzog C. et al.Excerpts from the United States Renal Data System 2003 Annual Data Report: Atlas of end-stage renal disease in the United States.Am J Kidney Dis. 2003; 42: A5-A7Abstract Full Text Full Text PDF PubMed Google Scholar Effective, noninvasive treatments, which would prevent and/or reduce AVF stenosis, could greatly benefit patients with end-stage renal disease. Vascular endothelial growth factor-A (VEGF-A) has been shown to be involved in the pathogenesis of arterial stenosis, vein bypass grafts, and venous neointimal hyperplasia associated with hemodialysis vascular access.5.Roy-Chaudhury P. Kelly B.S. Miller M.A. et al.Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts.Kidney Int. 2001; 59: 2325-2334Abstract Full Text Full Text PDF PubMed Google Scholar, 9.Bhardwaj S. Roy H. Heikura T. et al.VEGF-A, VEGF-D and VEGF-D(DeltaNDeltaC) induced intimal hyperplasia in carotid arteries.Eur J Clin Invest. 2005; 35: 669-676Crossref PubMed Scopus (71) Google Scholar, 10.Di Marco G.S. Reuter S. Hillebrand U. et al.The soluble VEGF receptor sFlt1 contributes to endothelial dysfunction in CKD.J Am Soc Nephrol. 2009; 20: 2235-2245Crossref PubMed Scopus (144) Google Scholar, 11.Hutter R. Carrick F.E. 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Hiasa K. et al.Essential role of vascular endothelial growth factor and Flt-1 signals in neointimal formation after periadventitial injury.Arterioscler Thromb Vasc Biol. 2004; 24: 2284-2289Crossref PubMed Scopus (71) Google Scholar, 16.Ohtani K. Egashira K. Hiasa K.-i. et al.Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells.Circulation. 2004; 110: 2444-2452Crossref PubMed Scopus (123) Google Scholar, 17.Misra S. Fu A.A. Puggioni A. et al.Increased shear stress with up regulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts.Am J Physiol Heart Circ Physiol. 2008; 294: H2219-H2230Crossref PubMed Scopus (56) Google Scholar, 18.Misra S. Shergill U. Yang B. et al.Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency.J Vasc Interv Radiol. 2010; 21: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Previous work from our laboratory and other laboratories has demonstrated increased expression of VEGF-A and other profibrotic genes, including matrix metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9), at the site of venous stenosis in murine and porcine models of hemodialysis AVF and AV graft failure. However, the mechanism(s) through which VEGF-A and MMPs have a role in venous neointimal hyperplasia formation has not been carefully investigated.17.Misra S. Fu A.A. Puggioni A. et al.Increased shear stress with up regulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts.Am J Physiol Heart Circ Physiol. 2008; 294: H2219-H2230Crossref PubMed Scopus (56) Google Scholar,19.Yang B. Shergill U. Fu A.A. et al.The mouse arteriovenous fistula model.J Vasc Interv Radiol. 2009; 20: 946-950Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Our investigations were performed in a murine model of chronic kidney disease in animals with an AVF. Simvastatin has been shown to decrease VEGF-A and MMP expression.20.Porter K.E. Naik J. Turner N.A. et al.Simvastatin inhibits human saphenous vein neointima formation via inhibition of smooth muscle cell proliferation and migration.J Vasc Surg. 2002; 36: 150-157Abstract Full Text PDF PubMed Scopus (132) Google Scholar, 21.Turner N.A. Midgley L. O'Regan D.J. et al.Comparison of the efficacies of five different statins on inhibition of human saphenous vein smooth muscle cell proliferation and invasion.J Cardiovasc Pharmacol. 2007; 50: 458-461Crossref PubMed Scopus (51) Google Scholar, 22.Turner N.A. O'Regan D.J. Ball S.G. et al.Simvastatin inhibits MMP-9 secretion from human saphenous vein smooth muscle cells by inhibiting the RhoA/ROCK pathway and reducing MMP-9 mRNA levels.FASEB J. 2005; 19: 804-806Crossref PubMed Scopus (131) Google Scholar We tested the hypothesis that the reduction of VEGF-A and MMP-9 gene expression via systemic delivery of simvastatin before the placement of an AVF leads to a reduction in venous neointimal hyperplasia with positive vascular remodeling. Gene and protein expression studies, as well as histomorphometric analyses, were performed at the outflow vein removed from animals treated with either simvastatin or controls. We ascertained whether simvastatin reduces fibroblast to myofibroblast (α-smooth muscle actin–positive cells) differentiation induced by hypoxia, and we determined its effect on several important cellular functions including proliferation and migration, with increased caspase 3 activity and decreased MMP-9 activity. Sixty-nine male C57BL/6 mice weighing 25–30g were used for the study. Five mice died after nephrectomy, and two mice (control) had thickened arteries at the time of AVF placement and were excluded. Therefore, 62 mice comprise this study18.Misra S. Shergill U. Yang B. et al.Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency.J Vasc Interv Radiol. 2010; 21: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,19.Yang B. Shergill U. Fu A.A. et al.The mouse arteriovenous fistula model.J Vasc Interv Radiol. 2009; 20: 946-950Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar (Figure 1a and b). Either 40mg/g of simvastatin (SV, n=35) or phosphate-buffered saline only (control, C, n=27) was given intraperitoneally every other day starting 1 week before fistula placement until the time of killing (Figure 1c). The serum blood urea nitrogen (BUN) and creatinine was used to assess the kidney function. After nephrectomy, the average BUN was significantly higher for the simvastatin and the control group at all time points (P<0.001) when compared with baseline (Figure 1d). At 5 to 8 weeks after nephrectomy, the average BUN was significantly higher in the control group when compared with the simvastatin group (Figure 1e). At 8 weeks after nephrectomy only, the average serum BUN was significantly higher in the control group when compared with the simvastatin group (P<0.001). At 8 weeks after nephrectomy, the average serum creatinine was significantly increased in the control group when compared with the simvastatin group (P<0.001). VEGF-A expression is increased in failed hemodialysis vascular accesses (AV fistulas or AV grafts) and in experimental animal models.5.Roy-Chaudhury P. Kelly B.S. Miller M.A. et al.Venous neointimal hyperplasia in polytetrafluoroethylene dialysis grafts.Kidney Int. 2001; 59: 2325-2334Abstract Full Text Full Text PDF PubMed Google Scholar, 6.Misra S. Doherty M.G. Woodrum D. et al.Adventitial remodeling with increased matrix metalloproteinase-2 activity in a porcine arteriovenous polytetrafluoroethylene grafts.Kidney Int. 2005; 68: 2890-2900Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17.Misra S. Fu A.A. Puggioni A. et al.Increased shear stress with up regulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts.Am J Physiol Heart Circ Physiol. 2008; 294: H2219-H2230Crossref PubMed Scopus (56) Google Scholar, 18.Misra S. Shergill U. Yang B. et al.Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency.J Vasc Interv Radiol. 2010; 21: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar By day 7, the mean gene expression of VEGF-A at the simvastatin-treated vessels was significantly lower than the control vessels (average reduction: 44%, P<0.01 (Figure 2a)), and also at day 14 (average reduction: 49%, P<0.01). Taken collectively, these results indicate that the average gene expression of VEGF-A is reduced at the outflow vein in simvastatin-treated vessels when compared with control vessels. Studies have shown increased expression of MMP-9 in failed hemodialysis vascular accesses (AV fistulas or AV grafts) and in experimental animal models.6.Misra S. Doherty M.G. Woodrum D. et al.Adventitial remodeling with increased matrix metalloproteinase-2 activity in a porcine arteriovenous polytetrafluoroethylene grafts.Kidney Int. 2005; 68: 2890-2900Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 17.Misra S. Fu A.A. Puggioni A. et al.Increased shear stress with up regulation of VEGF-A and its receptors and MMP-2, MMP-9, and TIMP-1 in venous stenosis of hemodialysis grafts.Am J Physiol Heart Circ Physiol. 2008; 294: H2219-H2230Crossref PubMed Scopus (56) Google Scholar, 23.Misra S. Fu A.A. Rajan D.K. et al.Expression of hypoxia inducible factor-1 alpha, macrophage migration inhibition factor, matrix metalloproteinase-2 and -9, and their inhibitors in hemodialysis grafts and arteriovenous fistulas.J Vasc Interv Radiol. 2008; 19: 252-259Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar By day 7, the average gene expression of MMP-9 was significantly lower in the simvastatin-treated vessels when compared with controls (average reduction: 69%, P<0.0001 (Figure 2b)), and also at day 14 (average reduction: 41%, P<0.0001). Overall, these results indicate that simvastatin-treated vessels have a significant reduction in MMP-9 when compared with control vessels. Because the expression of the protein can lag behind the gene expression, we performed zymography at day 14 to assess MMP-2 and MMP-9 activity in simvastatin-treated vessels as compared with controls (Figure 2c). There was significant reduction in both the MMP-9 and MMP-2 activity in the simvastatin-treated vessels when compared with controls (MMP-9—average reduction: 18%, P<0.01 (Figure 2d); pro-MMP-2—average reduction: 33%, P<0.0001; and active MMP-2—average reduction: 18%, P<0.0001 (Figure 2e)). Because we observed a decrease in average serum BUN and creatinine in the simvastatin-treated animals when compared with controls at 8 weeks, we determined whether the improvement in kidney function was due to a decrease in genes implicated in causing chronic kidney disease, such as VEGF-A (Figure 2f), MMP-2 (Figure 2g), and MMP-9 (Figure 2h). The average gene expression of VEGF-A, MMP-2, and MMP-9 was significantly reduced at day 28 (VEGF-A—average reduction: 24%, P<0.01; MMP-2—average reduction: 42%, P<0.001; and MMP-9—average reduction: 57%, P<0.01) in the simvastatin-treated kidneys when compared with controls. On hematoxylin- and eosin-stained sections, we were able to differentiate between the neointima and media/adventitia (Figure 3a). Semiquantitative histomorphometric analysis was performed on sections removed from the outflow veins of simvastatin-treated vessels and control vessels for the following: the area of the neointima (Figure 3b), media/adventitia (Figure 3c), and lumen vessel (Figure 3d). There was a significant reduction in the average area of the neointima of the simvastatin-treated vessels when compared with the controls by days 14 to 28 (average reduction: 56%, P<0.0001; day 28—average reduction: 45%, P<0.001). By day 14, the average area of the media/adventitia was significantly lower in the simvastatin-treated vessels when compared with the control group (average reduction: 43%, P=0.0028). As the simvastatin-treated vessels had reduced average wall area when compared with controls, we wanted to determine whether the simvastatin-treated vessels had a larger average lumen vessel area (Figure 3d). By days 14 to 28, the average lumen vessel area was significantly higher in the simvastatin-treated vessels when compared with controls (average increase: 150%, P<0.001; day 28—average increase: 343%, P<0.001). Because VEGF-A is necessary for cellular homeostasis, we determined the cell density in the neointima (Figure 3e) and media/adventitia (Figure 3f). By days 14 and 28, the average cell density of the neointima and media/adventitia in the simvastatin-treated vessels was significantly lower than the control vessels (neointima—day 14: average reduction: 65%, P<0.0001; day 28: average reduction: 70%, P<0.001; media/adventitia—day 14: average reduction: 37%, P<0.001; day 28: average reduction: 35%, P<0.01). VEGF-A and MMPs are needed for cells to proliferate, and cellular proliferation was assessed using Ki-67 (brown-stained nuclei (Figure 4a)). By days 14 and 28, in the simvastatin-treated vessels, when compared with control vessels, the average Ki-67 density was significantly reduced (day 14: average reduction: 66%, P<0.001; day 28: average reduction: 76%, P<0.0001 (Figure 4b)). Current literature suggests that VEGF-A is needed for maintaining cellular homeostasis; therefore, we hypothesized that the decrease in cell density was due to an increase in apoptosis.24.Shay-Salit A. Shushy M. Wolfovitz E. et al.VEGF receptor 2 and the adherens junction as a mechanical transducer in vascular endothelial cells.Proc Natl Acad Sci USA. 2002; 99: 9462-9467Crossref PubMed Scopus (267) Google Scholar Apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (Figure 5a). By days 14 to 28, the average density of cells staining positive for TUNEL (brown) at the outflow vein of the simvastatin group was significantly higher than the control group (day 14: average increase: 366%, P<0.0001; day 28: average increase: 561%, P<0.0001 (Figure 5b)). Overall, these results indicate that simvastatin-treated vessels have increased TUNEL activity implying cellular apoptosis when compared with controls. The majority of cells that comprise the venous neointimal hyperplasia are α-SMA positive (brown-stained cells), and we determined whether the decrease in the cell density was due to a decrease in α-SMA-positive cells (Figure 6a). The average α-SMA density at the outflow vein of simvastatin-treated vessels was significantly lower than the control group by day 14 (average reduction: 46%, P<0.0001 (Figure 6b)). Smooth muscle myosin heavy chain (SMHC) and smoothelin are expressed by contractile smooth muscle cells. Qualitatively, by day 28, there was decreased staining for both smoothelin and SMHC in the simvastatin-treated vessels when compared with controls, which suggests a decrease in contractile smooth muscle cells along with myofibroblasts (Figure 6a). Several genes including connective tissue growth factor (CTGF) control the regulation of extracellular matrix. We assessed the gene expression of CTGF using real-time polymerase chain reaction (RT-PCR) analysis performed at different time points. The mean gene expression of CTGF (Figure 7a) at the simvastatin-treated vessels was significantly lower than the control vessels by day 14 (average reduction: 45%, P<0.001). Next, we assessed the changes in extracellular matrix using Sirus red staining, which allows for the evaluation of collagen 1 and 3. Sirus red staining was performed on outflow vein sections removed from simvastatin-treated and control vessels at day 14 and day 28, respectively (Figure 7b). Qualitatively, this demonstrated a reduction in the intensity of Sirus red staining in the simvastatin-treated vessels when compared with control vessels at both days 14 and 28. This implies that there is a decrease in constrictive remodeling in the simvastatin-treated vessels when compared with controls. Several studies have demonstrated increased hypoxia-inducible factor HIF-1α expression in animal models of hemodialysis graft failure and in clinical specimens from patients with hemodialysis vascular access failure.18.Misra S. Shergill U. Yang B. et al.Increased expression of HIF-1alpha, VEGF-A and its receptors, MMP-2, TIMP-1, and ADAMTS-1 at the venous stenosis of arteriovenous fistula in a mouse model with renal insufficiency.J Vasc Interv Radiol. 2010; 21: 1255-1261Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar,23.Misra S. Fu A.A. Rajan D.K. et al.Expression of hypoxia inducible factor-1 alpha, macrophage migration inhibition factor, matrix metalloproteinase-2 and -9, and their inhibitors in hemodialysis grafts and arteriovenous fistulas.J Vasc Interv Radiol. 2008; 19: 252-259Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The mean gene expression of HIF-1α (Figure 8a) at the simvastatin-treated vessels was significantly lower than the control vessels by day 7 (average reduction: 54%, P<0.001) and day 14 (average reduction: 54%, P<0.001). We assessed HIF-1α staining as well. Cells staining positive for HIF-1α are brown (Figure 8b). By days 14 to 28, there was a significant reduction in the average density of HIF-1α staining in the simvastatin-treated vessels when compared with controls (day 14: average reduction: 20%, P<0.001; day 28: average reduction: 36%, P<0.001 (Figure 8c)). We next performed hypoxyprobe staining in the outflow vein treated with either simvastatin or controls (Figure 8d). Cells staining positive for hypoxyprobe are brown. By days 14 to 28, there was a significant reduction in the average density of hypoxyprobe staining in the simvastatin-treated vessels when compared with controls (Day 14: average reduction: 40%, P<0.001; day 28: average reduction: 70%, P<0.01 (Figure 8e)). Overall, these results indicate that there is decreased expression of both HIF-1α and hypoxyprobe in simvastatin-treated vessels when compared with controls. Studies indicate that hypoxia can cause an increase in fibroblast to myofibroblast differentiation.25.Das M. Burns N. Wilson S.J. et al.Hypoxia exposure induces the emergence of fibroblasts lacking replication repressor signals of PKC{zeta} in the pulmonary artery adventitia.Cardiovasc Res. 2008; 78: 440-448Crossref PubMed Scopus (41) Google Scholar,26.Misra S. Fu A.A. Misra K.D. et al.Hypoxia-induced phenotypic switch of fibroblasts to myofibroblasts through a matrix metalloproteinase 2/tissue inhibitor of metalloproteinase-mediated pathway: implications for venous neointimal hyperplasia in hemodialysis access.J Vasc Interv Radiol. 2010; 21: 896-902Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar To determine whether simvastatin treatment could decrease the conversion of fibroblasts to α-SMA-positive cells under hypoxic stress, we used NIH 3T3 cells, which were treated with 1μmol/l of simvastatin (SV) or control (C) and subjected to incubation for 24h. We first determined the synthetic phenotype of the SMC using confocal imaging for phalloidin and α-SMA (Figure 9a). Cells staining red are positive for α-SMA, and cells staining green are positive for phalloidin, with the nuclei staining blue. As shown, this demonstrated a qualitative reduction in α-SMA plus phalloidin staining for the simvastatin-treated cells when compared with controls for both 24h of normoxia and hypoxia. We determined whether the proliferative capacity of simvastatin-treated NIH 3T3 cells is reduced under hypoxia when compared with controls. This demonstrated that there was significant reduction in the proliferative ability of fibroblasts treated with simvastatin as compared with controls for 24-h hypoxia (average reduction: 70%, P<0.0001 (Figure 9b)). We next determined whether the migratory capacity of simvastatin-treated NIH 3T3 cells is reduced under hypoxia when compared with controls using a Matrigel invasion assay (Figure 9c). This demonstrated that the migratory capacity of simvastatin-treated cells was significantly decreased for simvastatin when compared with controls for 24-h hypoxia (average reduction: 33%, P<0.0001 (Figure 9d)). We next determined the effect of simvastatin on caspase 3 activity on NIH 3T3 cells under hypoxia and normoxia. We observed a significant increase in caspase 3 activity in cells treated with simvastatin when exposed to hypoxia as compared with controls (average increase: 600%, P<0.0007 (Figure 9e)). Finally, we determined the effect of simvastatin on the activity of MMP-9 in NIH 3T3 cells under hypoxia and normoxia at 8 and 24 hours (Figure 9f). We observed a significant decrease in MMP-9 activity at 8 hours of hypoxia (average reduction: 33%, P<0.0001 (Figure 9g)) and at 24 hours of normoxia (average reduction: 33%, P<0.0001 (Figure 9g)). In this study, we demonstrated that the venous stenosis from the AVF of simvastatin-treated vessels have reduced gene expression of VEGF-A and MMP-9, with protein expression of pro- and active MMP-2 and MMP-9 and a significant reduction in the average area of the neointima and media/adventitia, as well as positive vascular remodeling. In simvastatin-treated vessels, we observed a significant decrease in cellular proliferation, cell density (α-SMA), smoothelin, and SMHC, and a significant increase in apoptosis. In addition, a significant decrease in the local vessel hypoxia was observed, which was confirmed using two different approaches: HIF-1α and hypoxyprobe staining with RT-PCR for HIF-1α. Simvastatin-treated vessels had a decrease in extracellular matrix, with a significant reduction in CTGF, a profibrotic gene responsible for regulating extracellular matrix. In vitro experiments showed that NIH 3T3 fibroblasts when exposed to hypoxia and treated with simvastatin had a decrease in α-smooth muscle cell expression, proliferation, migration, and MMP-9 activity with increased caspase 3 activity. These results, taken in aggregate, indicate pretreatment with simvastatin before the placement of AVF, and we hypothesize that this results because of a decrease in fibroblast to myofibroblast conversion mediated through a VEGF-A/MMP-9 pathway associated with a decrease in CTGF, resulting in positive vascular remodeling and decrease in venous neointimal hyperplasia. In patients with malfunctioning hemodialysis vascular access