Muscle Cell Proliferation Research Articles

Role of CGRP modified mesenchymal stem cells in restenosis in rat model with carotid angioplasty

To explore the role of calcitonin gene-related peptide (CGRP) in the proliferation of vascular smooth muscle cells (VSMCs) and restenosis. The high-expression CGRP lentivirus [Lenti-green fluorescent protein (GFP)-CGRP] was constructed. And mesenchymal stem cells (MSCs) were transfected with Lenti-GFP-CGRP, Lenti-GFP and phosphate buffer saline (PBS). Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the CGRP gene and protein expression level of smooth muscle cells in each group respectively. Then MSCs were co-cultured with VSMCs. Experimental groups were MSCs(CGRP+/+)+VSMCs, MSCs(CGRP-/-)+VSMCs and MSCs(PBS)+VSMCs groups. The method of methyl thiazolyl tetrazolium (MTT) was employed to detect the proliferation of smooth muscle cells. Sacculus damaged atherosclerotic carotid was prepared according to the previous study. MSCs were transfected with Lv-CGRP-EGFP and Lv-CGRP and then transplanted into rat model. At Day 7 post-transplantation, injured carotid artery was harvested to detect the C expression of GRP by Western blot and the expression of CD31 by immunofluorescence. At Day 28 post-transplantation, injured carotid artery was harvested to assess organization morphology by hematoxylin and eosin stain and detect the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical stain. As compared with control and Lenti-GFP groups, the expressions of CGRP protein and CGRP mRNA increased at 72 h after transfecting with Lenti-GFP-CGRP (P < 0.05). After 72-hour co-culturing with VSMCs, the proliferation ability of VSMCs was the lowest in MSCs(CGRP+/+)+VSMCs group versus other three groups (P < 0.05). At Day 7 post-transplantation, as compared with control and Lenti-GFP groups, the expression of CGRP proteins increased significantly in MSCs-CGRP group (P < 0.05). A continuous expression of CD31 was found in damaged carotid intima in MSCs-CGRP group, but not in control group. At Day 28 post-transplantation, the area of intimal hyperplasia was smaller in MSCs-CGRP group than that in control and Lenti-GFP groups. Also the expression of PCNA decreased more in MSCs-CGRP group than control and Lenti-GFP groups (P < 0.05). CGRP protein and CGRP mRNA are expressed after CGRP transfection. And CGRP can suppress the proliferation of VSMCs and reduce intimal hyperplasia so as to facilitate a recovery of damaged endothelium.

Role of transmembrane protein 16A in vascular remodeling in portal hypertension induced by bile duct ligation of SD rats

Objective To investigate the role of transmembrane protein 16A(TMEM16A)in murine portal vein remodeling in cirrhotic portal hypertension induced by bile duct ligation(BDL). Methods Sixty male SD rats were randomly divided into the model group(n= 40)and the control group(n= 20).Rats in the model group were subjected to BDL.Animals were sacrificed at 4th week.Hematoxylin and eosin(HE)staining was used to observe the liver cirrhosis in rats, and the portal vein pressure was measured to determine portal hypertension in rats.The change of histological structure of the portal vein was observed by HE staining.In addition, immunohistochemistry and Western blotting were applied to detect the expression of TMEM16A, phosphorylated extracellular signal- regulated kinase 1/2(p- ERK1/2)and extracellular signal - regulated kinase 1/2(ERK1/2) proteins. Results Compared with sham control group, the liver of rats in model group had false lobules and the portal vein pressure significantly increased[(16.79±2.65) mm Hg (1 mm Hg= 0.133 kPa)vs.(8.86±0.53)mm Hg,P<0.05].And the intima- media thickness of portal vein in model group was increased as compared with sham control group[(43.27±9.62)μm vs.(21.75±5.56) μm,P<0.05].The protein expression of TMEM16A was significantly decreased, and that of p- ERK1/2 was significantly increased in model group as compared with sham control group.The protein expression of ERK1/2 almost had no changes Conclusion TMEM16A may regulate the proliferation of portal vein smooth muscle cells by influencing the activity of mitogen- activated protein kinase(MAPK)/ERK signal pathway. Downregulation of TMEM16A may play an important role in the occurrence of portal vein remodeling induced by portal hypertension. Key words: Transmembrane protein 16A; Portal hypertension; Vascular remodeling; Cell proliferation; Mitogen-activated protein kinase/extracellular signal-regulated kinase signal pathway

P21 deficiency delays regeneration of skeletal muscular tissue.

The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

Abstract 500: Vitamin D Supplementation Attenuates ADAM-12-mediated Proliferation of Carotid Artery Smooth Muscle Cells of Hypercholesterolemic Swine

Introduction: Risk of hypertension, peripheral artery disease, myocardial infarction and development of human atherosclerosis has been linked to vitamin D deficiency. Atheromatous cytokines, including IL-6, TNF-α and epidermal growth factor receptor (EGFR) family growth factors are released at the site of atherosclerosis and boost the activity of proteolytic enzymes such as ADAMs (a disintegrin and metalloproteinases). ADAM-12 cleaves proHB-EGF (Heparin-binding EGF-like growth factor) activating EGFR, resulting in increased proliferation of smooth muscle cells (SMCs). The aim of this study was to examine the effect of vitamin D on IL-6 and TNF-α-induced ADAM-12, pEGFR expression, HB-EGF release and SMC proliferation. Methods: Micro-swine were fed with vitamin D-deficient high cholesterol diet, high cholesterol diet containing 900 IU of vitamin D, and high cholesterol diet containing 3000 IU of vitamin D for total of 12 months. After six months, serum cholesterol levels of 500-600 mg/dL were achieved in all the three groups. The protein expression of ADAM-12 &amp; pEGFR, and HB-EGF release, in presence or absence of IL-6, TNF-α and Calcitriol, in SMCs was quantified by western Blot. HB-EGF release was measured by ELISA. The proliferation was assayed by [3H]-Thymidine incorporation and cell counting method. Results: The protein expression of ADAM-12 &amp; pEGFR, HB-EGF release were significantly reduced in carotid artery SMCs isolated from Vitamin D-supplemented swine. IL-6 and TNF-α treatment increased the protein expression of ADAM-12 &amp; pEGFR and HB-EGF release in carotid artery SMCs. Proliferation capacity was higher in SMCs isolated from Vitamin D-deficient swine carotid artery, potentiated by IL-6 and TNF-α. Calcitriol inhibited the ADAM-12, pEGFR expression and HB-EGF released in SMCs of hypercholesterolemic swine. Calcitriol also inhibited the proliferation of carotid artery SMCs isolated from Vit D-deficient, D-sufficient and D-supplemented swine. Conclusion: Together, these results suggest that vitamin D deficiency enhances proliferation of SMCs, which is potentiated by atheromatous cytokines. Whereas, vitamin D supplementation regulates ADAM-12-mediated cleavage of proHB-EGF and activation of EGFR inhibiting SMC proliferation.

Abstract 440: Increased Migration and Proliferation of Human Venous Valve Wall Smooth Muscle Cells are Mediated by FGF2

Introduction: Lesions causing coronary and peripheral vein graft failure often occur at valves. To test the hypothesis that intrinsic differences in cells of the valve wall contribute to graft failure we measured cell migration from tissue explants and migration and proliferation of cultured cells from valve and non-valve sections of human saphenous vein (HSV). Methods: After removal of endothelium, HSVs were dissected into an outer wall with adventitia and some media and an inner wall with intima and some media. Cell migration from explants and migration (microchemotaxis chambers) and proliferation of cultured cells at passage 6 were measured. Results: Cells migrated faster from valve compared to non-valve tissue of the inner wall (Figure A; P&lt;0.01, N=24 veins), but not the outer wall (not shown). PDGF-BB- or serum-mediated migration of valve inner wall smooth muscle cells (VSMC) was greater than with non-valve SMCs (NVSMC; P&lt;0.05, N=6 pairs, 6.5 ± 1.2 vs. 4.2 ± 0.8 fold of control for PDGF-BB and 4.7 ± 0.6 vs. 3.4 ± 0.4 for serum). VSMC also proliferated faster than NVSMC after stimulation with PDGF-BB plus 2% serum (P&lt;0.01, N=6 pairs, 3.3 ± 0.4 vs. 2.2 ± 0.2 fold of control). Finally, a blocking antibody to FGF2 had no effect on PDGF-mediated migration or proliferation of NVSMC (migration: IgG 2.2 ± 0.3 vs anti-FGF2 2.0 ± 0.4, N=6; proliferation: IgG 2.6 ± 0.1 vs anti-FGF2 2.5 ± 0.2, N=7), but largely blocked the enhanced migration and proliferation of VSMC (migration: Figure B; P&lt;0.01, N=6; proliferation: control IgG 3.4 ± 0.3 vs. Anti-FGF2 Ab 2.7 ± 0.3 fold; P&lt;0.01, N=7). Conclusion: The increased migration and proliferation of VSMC compared to NVSMC is largely mediated by FGF2.

Abstract 645: Heparin’s Effects on Vascular Cells Require Transmembrane Receptor 184A

Introduction: Though heparin has been used in the clinic for decades, molecular mechanism(s) underlying heparin’s functions independent of anti-coagulation are still unclear. Transmembrane protein 184A (TMEM184A) is conserved, yet its physiological and molecular functions remain unknown. There are few studies reporting its expression and potential role in membrane trafficking in exocrine cells, and involvement in signaling during male germ cell differentiation. Sequence analysis reveals that the C terminal domain includes a putative binding site for heparin. We have previously shown that heparin decreases proliferation in vascular smooth muscle cells (VSMCs) by inducing expression of MKP-1 that decreases Elk-1 and ERK activation. Hypothesis: We hypothesized that TMEM184A plays a role in mediating heparin effects in vascular cells. Methods and Results: We observed TMEM184A expression in vascular cells through immunofluorescence and western blotting using three primary antibodies against different regions in TMEM184A, and visualized cells with confocal microscopy. To investigate whether heparin effects were dependent on TMEM184A, VSMCs were electroporated with 20 μg/ml control or TMEM184A shRNA. Control and knockdown VSMCs were treated with 200 μg/ml heparin for 20 min followed by 2 μg/ml platelet-derived growth factor (PDGF). Activated ERK or Elk-1 in the nucleus was compared to untreated controls or cells treated with PDGF alone. Quantitative immunofluorescence of over 100 cells for each treatment from at least three independent experiments showed that heparin treatment prior to 20 min PDGF stimulation significantly decreased active ERK by nearly 50% in control shRNA cells compared to cells treated with PDGF alone (20 min PDGF = 100.0% ± 3.76%, heparin pre-treatment = 55.5% ± 2.20%; p&lt;0.001). In TMEM184A knockdown cells, heparin pre-treatment did not decrease ERK activation (20 min PDGF = 100.0% ± 3.27%, heparin pre-treatment = 109.8% ± 3.06%). Similar results were observed for Elk-1. Heparin also did not decrease proliferation in response to PDGF in knockdown VSMCs as shown with BrdU incorporation assays. Conclusions: Our results provide functional evidence that heparin signaling in VSMCs is mediated at least in part by TMEM184A.

Abstract 102: Cd98 Modulates Atherosclerotic Plaque Morphology by Regulating Smooth Muscle Cell Proliferation via the P-38 Map Kinase Pathway

Smooth muscle cells (SMC) are known to migrate and proliferate to form a stabilizing fibrous cap that encapsulates atherosclerotic plaques. It has been shown that CD98hc, a transmembrane protein with a known role in amino acid transport and integrin signaling, is involved in proliferation and survival of various cell types including SMC. Based on these data, we hypothesized that CD98hc deficiency selectively in SMC would have pathogenic effects on atherosclerosis development and plaque composition. To test this, we utilized mice with SMC-specific deletion of the CD98hc ( CD98hc fl/fl SM22Cre + ) to determine the effects of CD98hc deficiency on SMC function in the context of atherosclerosis. We performed in vitro proliferation and survival/apoptosis assays to investigate the role of CD98hc in the proliferation and survival of primary mouse aortic vascular smooth muscle cells. We found that VSMC isolated from whole aortas of CD98hc -/- animals displayed approximately 60% reduced cell counts compared to control (41 ± 8.2% of control) after 5 days in culture. EdU assays in vivo showed a defect in the ability of CD98hc -/- SMC to proliferate, with 25% reduction in EdU-positive VSMC compared to controls (2.3 ± 0.2% vs 3 ± 0.2%). In addition, caspase-3 staining of SMC in vitro displayed a 41% increase in propensity of CD98hc -/- SMC to undergo apoptosis compared to controls (7.9 ± 0.6% vs 5.6 ± 0.5%). Furthermore, the absence of CD98hc in SMC caused a sharp increase in phosphorylated p-38, which was partially abrogated towards control levels when the cells were treated with PDGF-BB to induce proliferation. Long-term atherosclerosis study using SMC-CD98hc -/- /LDLR -/- mice showed that atherosclerotic plaque morphology was altered with increased necrotic core area (25.8 ± 1.9% vs 10.9 ± 1.6% necrotic core area per plaque area) due to a reduction in infiltration of SMC within the plaque (2.1 ± 0.4% vs 4.3 ± 0.4% SM22α positive area per plaque area) compared to control LDLR -/- mice. These data support an important role for CD98hc and its regulation of p-38 MAP kinase signaling in aortic vascular smooth muscle cell proliferation and survival. We conclude that CD98hc is critical for the formation of fibrous cap that is important in maintaining the stability of atherosclerotic plaque.

Abstract 446: Ebp50 Regulates Oxidative Stress Through Skp2 and Foxo1

Inflammation and oxidative stress play important roles in cardiovascular disease. Reactive oxygen- and nitric oxide (NO)-derived species affect proliferation and migration of endothelial and vascular smooth muscle cells (VSMC), and amplify vascular inflammation. In this work we explore the role of Ezrin-Radixin-Moesin (ERM) binding protein of 50kDa (EBP50) as a novel mediator of oxidative stress. EBP50 plays a critical role in vascular remodeling. We reported that EBP50 mediates migration and proliferation of VSMC. In addition, EBP50 potentiates inflammatory responses (including iNOS expression) in macrophages and vascular cells. EBP50 stabilizes S-phase kinase associated protein 2 (Skp2) - a substrate recruiting component of the E3 ubiquitin ligase that targets FoxO1 for degradation. FoxO1 is a transcription factor that controls the expression of two key antioxidant enzymes - manganese superoxide dismutase (MnSOD) and catalase. Therefore, we hypothesize that EBP50 regulates oxidative stress through Skp2-FoxO1 axis. Our findings show a significantly greater nuclear localization of FoxO1 in EBP50 depleted VSMC compared to WT VSMC. Moreover, EBP50 knockdown increased FoxO1 stability following cycloheximide treatment. Consistent with these observations, expression of MnSOD and catalase were higher in EBP50 knockout primary macrophages and VSMC. To begin addressing the role of EBP50 on oxidative stress in vivo we used bone marrow transplants in LDLR-/- mice. Following reconstitution, mice were place on a high cholesterol diet for 12 weeks. We observed a significant 25% reduction in plaque area in EBP50-deficient marrow recipients compared to WT marrow recipients. In these mice there was a significant reduction in 4-hydroxynonanal (a marker of oxidative stress) and of 3-nitro-tyrosine (a marker of oxygen- and NO-derived reactive species) staining in the atherosclerotic plaques. Collectively, these observations suggest that EBP50, by regulating the localization and Skp2-mediated degradation of FoxO1, increases oxidative stress and inflammation during atherosclerosis. These studies further highlight the actions of EBP50 in controlling multiple signaling pathways that play a critical role in the pathogenesis of vascular disease.

Abstract 140: Nuclear Targeting Apelin Induces Phenotypic Transition of Vascular Smooth Muscle Cells

Apelin, and its receptor APJ, are a peptidic system playing a crucial role in vascular diseases. However, the role of apelin in atherogenesis and smooth muscle cell (SMC) proliferation remains unclear. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as being a marker of the R-SMCs in vitro and of intimal SMCs, both in pig and human. The aim of our study was to investigate the effects of apelin on SMC phenotypic transition and S100A4 expression. Apelin was highly expressed in R-SMCs including in their nucleus. P-SORT software analysis of preproapelin sequence suggested that N-terminal truncated apelin may target the nucleus, and we confirmed this in SMCs by overexpression of mutated preproapelin-His-tag. Transfection of mutated preproapelin-His-tag encoding plasmid in differentiated S-SMCs induced a transition towards a R-phenotype associated with increased proliferative activity, downregulation of SMC differentiation markers, and increased nuclear expression and release of S100A4. In contrast, transfection of S-SMCs with wild type preproapelin-His-tag encoding plasmid did not induce nuclear targeting of apelin or S100A4, and did not change the S-phenotype. Stimulation of S-SMCs with PDGF-BB, known to induce a transition to the R-phenotype, yielded nuclear targeting of both apelin and S100A4. In vivo, Apelin was expressed in SMC nuclei of stent-induced intimal thickening while its expression in the media was mainly cytoplasmic. Our results suggest that nuclear targeting of apelin in SMCs acts on S100A4 expression and release, cell proliferation and differentiation. The pathophysiological consequences of this retargeting could be instrumental in the understanding of artherosclerosis.

Abstract 654: XBP1 Splicing is Involved in Endothelial and Smooth Muscle Cells Interaction

Background: The X-box binding protein 1 (XBP1) is a stress inducible transcription factors, essential for cell survival under stress conditions. Our previous studies have revealed that XBP1 participates in endothelial cell (EC) proliferation, autophagic response and survival and in smooth muscle cell (SMC) proliferation. Whether XBP1 is involved in EC/SMC interaction remains unclear. Methods and Results: In this study, we first performed hindlimb ischemia model in XBP1loxP/loxP and SM22-Cre+/XBP1loxP/loxP mice. The XBP1 deficiency in SMCs significantly attenuated the recovery of foot blood perfusion in ischemic mice. Immunofluorescence staining revealed that ECs lost contact to each other in XBP1 deficient mice, suggesting that XBP1 in SMCs may contribute to EC/SMC interaction. The conditioned medium from Ad-XBP1s-infected SMCs increased EC migration and proliferation. The conditioned medium from Ad-XBP1s-infected ECs also increased SMC migration but suppressed SMC proliferation. The migration effect could be attenuated by depletion with anti-col4A1 antibody. Proteomics analysis and Western blot assays demonstrated that over-expression of the spliced XBP1 in SMCs increased the secretion of a 50KD band of collagen IVα1. RT-PCR analysis indicated that this 50KD band might be derived from an unconventional splicing, in which exon 4 and exon 42 directly joins together, resulting in a 533 amino acid peptide with the internal part shortened. Further experiments with immunohistological staining revealed that a functional XBP1 was essential to collagen IVα1 expression during embryonic development. Very little amount of collagen IV could be observed in XBP1-/- embryos. Conclusion: XBP1 in SMCs may modulate EC-SMC interaction via regulation of collagen IV expression and splicing, which may contribute to vasculature remodelling.

Abstract 648: Reduced Expression of Phosphoprotein Enriched in Astrocytes-15 (PEA-15) Increases Neointimal Hyperplasia

Neointimal hyperplasia from increased vascular smooth muscle cell (VSMC) proliferation remains a significant clinical problem. Phosphoprotein enriched in astrocytes-15 (PEA-15) is an extracellular signal-regulated kinases 1/2 (ERK1/2) binding protein. We have previously demonstrated in vitro that PEA-15 acts as a cytoplasmic “anchor” in VSMCs and prevents ERK1/2 translocation to the nucleus, decreasing proliferation. We now assess the hypothesis that expression of PEA-15 regulates the development of neointimal hyperplasia. Arterial injury was induced by wire denudation with ligation of the carotid artery in C57BL/6 mice and examined 3, 7 and 14 days after surgery. Neointima formation was significantly increased in injured arteries from day 7 compared to control arteries. PEA-15 protein expression was significantly decreased in injured arteries in comparison to control vessels 3 days after arterial injury (ratio to GAPDH - 0.29±0.10 vs. 0.91±0.1, n=3), preceding significant neointima formation at day 7. A similar decrease in PEA-15 protein and mRNA levels was observed in human saphenous vein rings incubated ex vivo in 30% serum for 14 days to induce neointimal hyperplasia (protein ratio to GAPDH - 0.36±0.05 vs. freshly isolated veins 1.34±0.20, n=5). This coincided with increases in proliferative markers. To determine a causal relationship between decreased PEA-15 expression and neointimal formation, we induced arterial injury in mice deficient in PEA-15 (PEA-15-/-). There was a significantly exaggerated neointima formation in PEA-15-/- mice 7 days after surgery compared to C57BL/6 mice at the same time point (intima-to-media ratio - 0.95±0.26 vs. 0.35±0.09, n=3-5). The mechanisms involved were investigated. Immunolocalization of ERK1/2 was assessed in primary cultured VSMCs isolated from aortae of C57BL/6 and PEA-15-/- mice. In unstimulated VSMCs from PEA-15-/- mice, there was a significant increase in nuclear staining of ERK1/2 compared with cells from C57BL/6 mice. In conclusion, decreased PEA-15 expression occurs in VSMCs during the development of neointima hyperplasia which may increase proliferation. Targeting this pathway could be beneficial in the treatment of restenosis.

Abstract 191: Immobilized ApoA-I and High Density Lipoproteins are Anti-thrombotic and Inhibit Smooth Muscle Cell Attachment and Proliferation

Aim: The clinical efficacy of current endovascular stents is limited by thrombosis and restenosis. Plasma activated coated stainless steel (PAC) is able to covalently bind proteins to stainless steel (SS) surfaces in their bioactive state. High density lipoproteins (HDL) and its main apolipoprotein constituent apoA-I regulate key biological processes involved in restenosis and thrombosis, highlighting their potential for immobilization on PAC stent surfaces. We hypothesized that covalently bound apoA-I or reconstituted HDL (rHDL) to PAC will retain its biological properties and improve stent patency. Methods and Results: Covalent binding of 125I labelled apoA-I and rHDL was increased on PAC compared to SS (p&lt;0.001). Thrombosis formation under static conditions was assessed by scanning electron microscopy. Thrombi were strikingly reduced on PAC and completely absent on PAC+apoA-I and PAC+rHDL, while extensive thrombi formed on all SS samples at all time points. Thrombus formation under flow conditions using a Chandler Loop (0.5U/ml heparin) showed PAC, PAC+apoA-I and PAC+rHDL had 98%, 97% and 94% lower thrombus weights, compared to SS samples, respectively (p&lt;0.05). Lower heparin concentrations (0.3U/ml) revealed that PAC+apoA-I and PAC+rHDL were superior to PAC alone in reducing thrombus weights (98% and 95%, respectively, p&lt;0.01). P-selectin protein expression was measured by ELISA in the plasma from the Chandler loop assay (0.3U/ml heparin). Plasma exposed to PAC+apoA-I had reduced p-selectin levels (32%) compared to the PAC control (p&lt;0.05). Immobilized apoA-I and rHDL on PAC reduced smooth muscle cell (SMC) attachment by 70% and 80%, respectively, p&lt;0.05. Conversely, endothelial cell (EC) attachment was increased by 36% on PAC+apoA-I (p&lt;0.05). Immobilized PAC+apoA-I and PAC+rHDL also reduced TNF-α-induced SMC proliferation (26% and 20% respectively), compared to the PAC control (p&lt;0.05). Conclusion: ApoA-I and rHDL covalently bind to PAC surfaces. Immobilized apoA-I and rHDL retain their anti-thrombotic properties, inhibit SMC attachment and proliferation, and increase EC attachment. This may represent a novel site-directed approach to increase stent patency.

Abstract 190: Continuous Vascular Remodeling Related To Development Of Arteriosclerosis Or Atherosclerosis In Kawasaki Disease ~ Immunohistochemical Study using an Animal Model ~

Background: Atherosclerotic coronary heart disease has recently emerged as a clinical issue among young individuals with a history of Kawasaki disease (KD), which is a systemic vasculitis unique to children. However, whether or not and how KD promotes atherosclerosis remains unclear. We hypothesized that, analogous to the pathogenesis of arteriosclerosis or atherosclerosis, endothelial injury and the resultant intimal thickening are induced in coronary arteries after attenuation of vasculitis. Methods: We used a rabbit model of KD developed by Onouchi et al. and performed histopathological analysis of the coronary arteries at acute (1, 3, 5, and 7 days) and chronic (3 months) phases of the disease. Results: In these rabbit models, a pan-arteritis with significant intimal cellular hypertrophy was histologically detected in the acute phase, and arterial intimal thickening was observed during the chronic phase. Immunohistochemical analysis of the coronary arteries revealed that the thickened intimal lesions observed during the chronic phase comprised abundant α-smooth muscle actin (α-SMA)-positive cells, most of which concomitantly expressed vascular cell adhesion molecule-1 and nuclear factor-κB. Although macrophages positive for RAM11 were barely detected, macrophage colony stimulating factor was strongly expressed in migrating smooth muscle cells in the intimal layer. In addition, the accumulation of proteoglycan as extracellular matrix was distinctly visible in the thickened intima, indicating progressive accumulation of lipids and proliferation of smooth muscle cells within the lesion. Conclusions: These findings suggest that, in KD-associated vasculitis, the migration of α-SMA-positive cells into the thickened intima might induce continuous vascular inflammation and remodeling, which might progress to coronary arteriosclerosis or atherosclerosis.

Rosiglitzone Suppresses Angiotensin II-Induced Production of KLF5 and Cell Proliferation in Rat Vascular Smooth Muscle Cells

Krüppel-like factor (KLF) 5, which initiates vascular smooth muscle cell (VSMC) proliferation, also participates in Angiotensin (Ang) II-induced vascular remodeling. The protective effect of rosiglitazone on vascular remodeling may be due to their impact on VSMC proliferation. However, the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiproliferation effects of rosiglitazone are mediated by regulating Ang II/KLF5 response. We found that, in aortas of Ang II-infused rats, vascular remodeling and KLF5 expression were markedly increased, and its target gene cyclin D1 was overexpressed. Co-treatment with rosiglitazone diminished these changes. In growth-arrested VSMCs, PPAR-γ agonists (rosiglitazone and 15d-PGJ2) dose-dependently inhibited Ang II-induced cell proliferation and expression of KLF5 and cyclin D1. Moreover, these effects were attenuated by the PPAR-γ antagonists GW9662, bisphenol A diglycidyl ether and PPAR-γ specific siRNA. Furthermore, rosiglitazone inhibited Ang II-induced phosphorylation of protein kinase C (PKC) ζ and extracellular signal-regulated kinase (ERK) 1/2 and activation of early growth response protein (Egr). In conclusion, in Ang II-stimulated VSMCs, rosiglitazone might have an antiproliferative effect through mechanisms that include reducing KLF5 expression, and a crosstalk between PPAR-γ and PKCζ/ERK1/2/Egr may be involved in. These findings not only provide a previously unrecognized mechanism by which PPAR-γ agonists inhibit VSMC proliferation, but also document a novel evidence for the beneficial vascular effect of PPAR-γ activation.

Mo1761 Mesenteric Fat Cells Induce Proliferation of Intestinal Muscle Cells and Migrate Towards Muscle Cell-Derived Extracellular Matrix Products - A Feedback Loop Explaining the Association of Creeping Fat With Stricture Formation

Mo1761 Mesenteric Fat Cells Induce Proliferation of Intestinal Muscle Cells and Migrate Towards Muscle Cell-Derived Extracellular Matrix Products - A Feedback Loop Explaining the Association of Creeping Fat With Stricture Formation

Cyclopentenyl cytosine promotes endothelial protection by inducing smooth muscle re‐differentiation in vascular remodeling

Vascular remodeling due to endothelium denudation followed by pathological smooth muscle cell (SMC) proliferation is a major medical challenge in cardiovascular intervention. Our previous studies h...

EVALUATION OF LUNG GLUCOSE UPTAKE WITH FLUORINE-18 FLUORODEOXYGLUCOSE POSITRON EMISSION TOMOGRAPHY/CT IN PATIENTS WITH PULMONARY ARTERY HYPERTENSION AND PULMONARY HYPERTENSION DUE TO LEFT HEART DISEASE

Pulmonary arterial hypertension (PAH) is characterized by remodeling of the media with proliferation of smooth muscle cells in pulmonary arteries and arterioles. In contrast, pulmonary hypertension (PH) associated with left heart disease (group 2 PH) is caused by passive transmission of elevated

Role of NF-κB/survivin signal pathway on intima hyperplasia of rat carotid balloon injury restenosis model

To investigate the role of NF-κB/survivin signal pathway in the intima hyperplasia of rat carotid balloon injury restenosis model. NF-κB siRNA lentivirus vector (titer was 1 × 10⁸ TU/ml) was established. Carotid balloon injury restenosis model was made in 33 SD rats. The rats were divided into 4 groups according to different processing methods, including negative control (NC) group (n = 11), NF-κB siRNA group (n =11), NF-κB siRNA+YM155 (survivin inhibitor) (n = 11), the uninjured carotid artery served as the normal control group (n = 11). After 7 days, the carotid sample (n = 5 each group) were harvested to detect the NF-κB and survivin mRNA expression by RT-PCR.The carotid sample were harvested on 28 days (n = 6 each group) for HE staining and measuring intima hyperplasia. Immunohistochemical method was also used to detect the expression of intima proliferation cell nuclear antigen (PCNA) and media α-SM-actin. (1) After 7 days, NF-κB and survivin mRNA expression was significant higher in NC group than in normal control group (P<0.05), the NF-κB mRNA expression was significantly lower in NF-κB siRNA group than in NC group (P<0.05) and similar between NF-κB siRNA group and NF-κB siRNA+YM155 group. The survivin mRNA expression was significantly lower in NF-κB siRNA group compared to NC group (P<0.05) and significantly higher in NF-κB siRNA group than in NF-κB siRNA+YM155 group (P<0.05). (2) After 28 days, intima hyperplasia was observed in NC (0.13 ± 0.01), NF-κB siRNA (0.11 ± 0.01) and NF-κB siRNA+YM155 group (0.09 ± 0.01) mm² (P<0.05). Media area was similar among NC group, NF-κB siRNA group and NF-κB siRNA+YM155 group (P>0.05). I/M ratio was gradually reduced among NC group (1.55 ± 0.07), NF-κB siRNA group (0.92 ± 0.08), NF-κB siRNA+YM155 group (0.76 ± 0.06, all P<0.05). Similar results were found in the residual restenosis rate: NC group (58.71 ± 0.02) %, NF-κB siRNA group (32.13 ± 0.05) %, NF-κB siRNA+YM155 group (26.42 ± 0.03) % (all P<0.05) and expression of vascular smooth muscle cell PCNA: NC group (45.32 ± 7.21) %, NF-κB siRNA group (36.54 ± 6.42) %, NF-κB siRNA+YM155 group (28.57 ± 6.31) % (all P<0.05). On the contrary, the IOD of α-SM-actin in media increased gradually: NC group (0.055 ± 0.006), NF-κB siRNA group (0.072 ± 0.011), NF-κB siRNA+YM155 group (0.084 ± 0.008, all P<0.05). Inhibiting NF-κB expression can significant decrease intima hyperplasia in this model, and this effect may be mediated by inhibiting survivin and reducing the proliferation of vascular smooth muscle cells.

Involvement of interleukin-1 receptor (IL1R1) and myeloid differentiation primary response gene 88 (MyD88) signaling in pulmonary hypertension (PH)

Background Pulmonary hypertension (PH) results from complex vessel remodeling involving both pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammatory processes. Both toll-like receptors (TLR) and interleukine-1 receptor (IL1R1) are involved in innate immunity and they share common signaling pathways: their activation results in recruitment of the molecular adaptor Myd88, leading to the activation of NFKB and subsequently synthesis and release of many cytokines including IL-1s, IL-6 and TNFα. In this study we question whether reducing PH, it is efficient to target IL1R1 or Myd88. Methods We first examined expression and localization of IL1R1 and Myd88 in lungs from patient with iPAH or controls, as well as in mice with hypoxic PH. Secondly we evaluated the role for IL1R1 and Myd88 in PH by studying IL1R1-/-, MyD88-/− and WT mice treated daily by Anakinra (selective IL1R1 antagonist) exposed to chronic hypoxia, in comparison with WT mice. PA-SMCs from these mice were studied in vitro. Results Marked increase expression of IL1R1 and Myd88 were observed in patients with iPAH and in mice exposed to chronic hypoxia. In contrast to IL1R1, which was widely distributed in the lung, Myd88 was preferentially express in remodeled vessels and in particular in PA-SMC. IL1R1-/-, MyD88-/− and WT mice treated with anakinra were similarly protected against hypoxic PH development, as shown by decreases in right ventricular systolic pressure, Fulton index, muscularization of vessels and proliferative ki67-positive cells in arteries compared to untreated WT mice. The increase in lung perivascular macrophages, IL-1s and IL-6 associated with hypoxia exposure was abrogated in anakinra-treated WT mice and in IL1R1-/− and MyD88-/− mice. In vitro, the potent IL-1s-mediated growth-promoting activity on PA-SMCs was abolished by anakinra and by genetic deletion of IL1R1 or MyD88. Interestingly, the growth-promoting effect of IL-1s was stronger in cells from hypoxic WT mice than control. PA-SMC proliferation induced by conditioned media from alveolar macrophages was potentiated by previous macrophage treatment with IL-1s. Conclusion The IL-1s/IL1R1/Myd88 axis contributes to PH development, by stimulating PA-SMC growth through both direct and indirect macrophage-mediated effects. IL1R1 or Myd88 inhibition may represent a new option for treating PH.

Selective TSC1 deletion in smooth muscle results in mTOR signaling activation and development of pulmonary hypertension that can be reversed by rapamycin

Background Pulmonary artery (PA) smooth muscle cell (SMC) proliferation in pulmonary hypertension (PH) is associated with dysregulated mammalian target of rapamycin (mTOR) signaling. The mTOR pathway involves two independent complexes, mTORC1 and mTORC2, which phosphorylate S6 kinase (S6K) and serine/threonine kinase (Akt), respectively, and differ in their sensitivity to rapamycin. Here, we investigated whether selective mTOR activation in SMCs was sufficient to induce PH in mice. Methods To selectively activate the mTOR-S6K pathway in SMCs, we developed mice with selective deletion in SMCs of the tuberous sclerosis complex 1 gene (TSC1), which negatively controls mTOR, by crossing TSC1LoxP/LoxP mice with mice expressing the Cre recombinase under the control of the SM22alpha promoter (SM22/TSC1-/− mice). Results SM22/TSC1-/− mice had increased body and heart weights with normal survival at 1 year of age. Echocardiography showed normal left ventricle (LV) size and ejection fraction but increased anterior and posterior wall thicknesses. Right ventricle (RV) and LV weights were increased and the RV/LV + septum weight ratio markedly elevated. Marked activation of the mTOR-Akt signaling pathway was found in lungs from SM22/TSC1-/− mice compared to controls, with a marked PTEN decrease and activation of mTORC1 substrates S6K and 4EBP and of mTORC2 substrates P-AktSer473 and GSK3. In situ analysis of lung specimens revealed prominent P-Akt, P-S6K, and P-GSK3 staining in PA-SMCs. SM22/TSC1-/− mice spontaneously developed PH due to pulmonary vascular remodeling, as assessed by increased RV systolic pressure, muscularization of distal pulmonary arteries, and increased number of Ki67-positive dividing pulmonary vascular cells. Treatment with 5 mg/Kg rapamycin during 3 weeks reversed PH and reduced lung P-Akt, P-S6K, and P-GSK3 proteins to control levels. Cultured PA-SMCs from SM22/TSC1-/− mice exhibited an increased growth rate and sustained activation of mTORC1 and mTORC2 substrates compared to controls. PA-SMC exposure to rapamycin inhibited the serum-induced growth and diminished the difference in growth rate between cells from SM22/TSC1-/− and WT mice. Conclusion These results support a critical role in PH for mTOR signaling pathway activation, which can be suppressed by rapamycin treatment.