Venous Smooth Muscle Cells Research Articles

MicroRNA-29a Involvement in Phenotypic Transformation of Venous Smooth Muscle Cells Via Ten-Eleven Translocation Methylcytosinedioxygenase 1 in Response to Mechanical Cyclic Stretch.

Mechanical stimuli play an important role in vein graft restenosis and the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) are pathological processes contributing to this disorder. Here, based on previous high-throughput sequencing data from vein grafts, miR-29a-3p and its target, the role of Ten-eleven translocation methylcytosinedioxygenase 1 (TET1) in phenotypic transformation of VSMCs induced by mechanical stretch was investigated. Vein grafts were generated by using the "cuff" technique in rats. Deep transcriptome sequencing revealed that the expression of TET1 was significantly decreased, a process confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. MicroRNA-seq showed that miR-29a-3p was significantly up-regulated, targeting TET1 as predicted by Targetscan. Bioinformatics analysis indicated that the co-expressed genes with TET1 might modulate VSMC contraction. Venous VSMCs exposed to 10%-1.25 Hz cyclic stretch by using the Flexcell system were used to simulate arterial mechanical conditions in vitro. RT-qPCR revealed that mechanical stretch increased the expression of miR-29a-3p at 3 h. Western blot analysis showed that TET1 was significantly decreased, switching contractile VSMCs to cells with a synthetic phenotype. miR-29a-3p mimics (MI) and inhibitor (IN) transfection confirmed the negative impact of miR-29a-3p on TET1. Taken together, results from this investigation demonstrate that mechanical stretch modulates venous VSMC phenotypic transformation via the mediation of the miR-29a-3p/TET1 signaling pathway. miR-29a-3p may have potential clinical implications in the pathogenesis of remodeling of vein graft restenosis.

A novel function of calcium sensing receptor in chronic hypoxia-induced pulmonary venous smooth muscle cells proliferation.

Chronic hypoxia (CH) causes remodeling not only in pulmonary arteries but also in pulmonary veins. Pulmonary vascular remodeling stems from increased pulmonary vascular myocyte proliferation. However, the pathogenesis of CH-induced proliferation of pulmonary venous smooth muscle cells (PVSMCs) remains unknown. The present study aimed to explore the mechanisms by which CH affects PVSMCs proliferation. PVSMCs were isolated from rat distal pulmonary veins and exposed to CH (4% O2 for 60 h). The expression of calcium sensing receptor (CaSR) was determined by immunofluorescence, real-time quantitative PCR and Western blotting. Cell proliferation was assessed by cell counting, CCK-8 assay, and BrdU incorporation. Apoptosis analysis was examined by flow cytometry. In rat distal PVSMCs, CH increased the cell number and cell viability and enhanced DNA synthesis, which is accompanied by upregulated mRNA and protein expression levels of CaSR. Two negative CaSR modulators (NPS2143, NPS2390) not only attenuated CH-induced CaSR upregulation but also inhibited CH-induced increases in cell number, cell viability and the proliferation index of PVSMCs, whereas two positive modulators (spermine, R568) not only amplified CH-induced CaSR upregulation but also intensified CH-induced increases in cell number, cell viability and the proliferation index of PVSMCs. Silencing CaSR with siRNA similarly attenuated the CH-induced enhancement of cell number, cell viability and DNA synthesis in PVSMCs. Neither CH nor downregulation of CaSR with siRNA had an effect on apoptosis in PVSMCs. These results suggest that CaSR mediating excessive proliferation is a new pathogenic mechanism involved in the initiation and progression of distal PVSMCs proliferation under CH conditions.

Issue Highlights

IUBMB LifeVolume 71, Issue 7 p. 770-770 Issue HighlightsFree Access Issue Highlights First published: 20 June 2019 https://doi.org/10.1002/iub.2106AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 845 LncRNA MIAT Facilitates Osteosarcoma Progression by Regulating miR-128-3p/VEGFC Axis Chunyan Zhang, Linsen Xie, Huiling Liang and Yuanbo Cui In this study, the results display that lncRNA MIAT is highly expressed in osteosarcoma (OS) tissues and cell lines. High expression of MIAT is closely associated with large tumor size and distant metastasis in OS patients. Knockdown of MIAT inhibits cell proliferation and metastasis, and promotes apoptosis in OS cells in vitro. MIAT promotes OS progression by functioning as a ceRNA via sponging miR-128-3p to up-regulate VEGFC in vitro. These findings may provide new insight into the roles of MIAT in the tumorigenesis of OS. 908 MicroRNA-365 Promotes the Contractile Phenotype of Venous Smooth Muscle Cells and Inhibits Neointimal Formation in Rat Vein Grafts Bo-Jun Cao, Lei Zhu, Xiao-Wen Wang, Rong-Jiang Zou and Zhi-Qian Lu MicroRNA-365 (miR-365) levels are substantially reduced in rat vein grafts at 7, 14, and 28 days post-grafting. Overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced graftinginduced neointimal formation and effectively improved the hemodynamics of the vein grafts, suggesting its potential therapeutic application for prevention of vein graft failure. REFERENCES 1Zhang C., Xie L., Liang H. and Cui Y. LncRNA MIAT Facilitates Osteosarcoma Progression by Regulating miR-128-3p/VEGFC Axis. IUBMB Life. 2019, 71, 845– 853. 2Cao B.-J., Zhu L., Wang X.-W., Zou R.-J. and Z.-Q. Lu. MicroRNA-365 Promotes the Contractile Phenotype of Venous Smooth Muscle Cells and Inhibits Neointimal Formation in Rat Vein Grafts. IUBMB Life. 2019, 71, 908– 916. Volume71, Issue7July 2019Pages 770-770 ReferencesRelatedInformation

MicroRNA‐365 promotes the contractile phenotype of venous smooth muscle cells and inhibits neointimal formation in rat vein grafts

The high rate of autologous vein graft failure caused by neointimal hyperplasia remains an unresolved issue in the field of cardiovascular surgery; therefore, it is important to explore new methods for protecting against neointimal hyperplasia. MicroRNA-365 has been reported to inhibit the proliferation of vascular smooth muscle cells (SMCs). This study aimed to test whether adenovirus-mediated miR-365 was able to attenuate neointimal formation in rat vein grafts. We found that miR-365 expression was substantially reduced in vein grafts following engraftment. In vitro, overexpression of miR-365 promoted smooth muscle-specific gene expression and inhibited venous SMC proliferation and migration. Consistent with this, overexpression of miR-365 in a rat vein graft model significantly reduced grafting-induced neointimal formation and effectively improved the hemodynamics of the vein grafts. Mechanistically, we identified that cyclin D1 as a potential downstream target of miR-365 in vein grafts. Specially, to increase the efficiency of miR-365 gene transfection, a 30% poloxamer F-127 gel containing 0.25% trypsin was mixed with adenovirus and spread around the vein grafts to increase the adenovirus contact time and penetration. We showed that adenovirus-mediated miR-365 attenuated venous SMC proliferation and migration in vitro and effectively inhibited neointimal formation in rat vein grafts. Restoring expression of miR-365 is a potential therapeutic approach for the treatment of vein graft failure. © 2019 IUBMB Life, 2019.

Dedicator of cytokinesis 2 silencing therapy inhibits neointima formation and improves blood flow in rat vein grafts

ObjectiveThe high rate of vein graft failure due to neointimal hyperplasia is a major challenge for cardiovascular surgery. Finding novel approaches to prevent neointimal hyperplasia is important. Thus, the purpose of this study was to investigate whether dedicator of cytokinesis 2 (DOCK2) plays a role in the development of neointima formation in the vein grafts. Methods and resultsWe found that DOCK2 levels were significantly elevated in the vein grafts following grafting surgery. In addition, overexpression of DOCK2 promoted venous smooth muscle cell (SMC) proliferation and migration. Conversely, knocking-down endogenous DOCK2 expression in venous SMCs inhibited SMC proliferation and migration. Consistent with this, knocking-down DOCK2 expression in the grafted veins significantly reduced neointimal formation compared with the controls 28 days after vein transplantation. Moreover, DOCK2 silencing treatment improved hemodynamics in the vein grafts. Mechanistically, knockdown of DOCK2 significantly alleviated the vein graft-induced down regulation of SMC contractile protein expression and impeded the vein graft-induction of both Cyclin D1 and PCNA expression. In particular, to ensure high efficiency when transferring the DOCK2 short hairpin RNA (shDOCK2) into the grafted veins, a 30% poloxamer F-127 gel incorporated with 0.25% trypsin was smeared around the vein grafts to increase the adenovirus contact time and penetration. ConclusionsDOCK2 silencing gene therapy effectively attenuates neointimal hyperplasia in vein grafts. Knock-down of DOCK2 would be a potential therapeutic approach for the treatment of vein graft failure.

Enhanced sensitivity to 4-hydroxynonenal and impaired redox signaling in human endothelial cells in gestational diabetes

Fetal exposure to gestational diabetes (GDM) in utero is strongly associated with a higher risk of cardiovascular disease and insulin resistance in later life, and accumulating evidence suggests this may be a consequence of fetal programming potentially involving epigenetic influences.1 Notably, offspring of mothers with GDM exhibit elevated blood pressure and reduced endothelium-dependent reactivity. We previously reported abnormal nitric oxide production, insulin resistance and reduced cell proliferation in fetal umbilical vein endothelial cells (HUVEC) from GDM pregnancies,2,3 and in parallel studies with endothelial cells from pre-eclamptic pregnancies, associated with in utero oxidative stress, identified abnormalities in NO production and regulation of [Ca2+]i.4,5 These phenotypic changes are maintained during culture, highlighting the involvement of fetal programming and epigenetics. As placental oxidative stress is propagated to the maternal and fetal vasculature via circulating lipid peroxides and H2O2 in GDM, we hypothesised that sustained oxidative stress in the fetal vasculature in utero may impair endogenous antioxidant defences. We established that activation of the redox sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2)6 and its downstream target antioxidant enzymes is markedly inhibited in HUVEC from GDM pregnancies. Fetal endothelial cells exhibited marked deficits in glutathione synthesis, increased basal mitochondrial superoxide production, reduced nuclear translocation of Nrf2, and diminished adaptive increases in the expression of the Nrf2 target genes heme oxygenase-1 (HO-1) and/or NAD(P)H quinone oxidoreductase 1 (NQO1) in response to the lipid peroxidation product 4-hydroxynonenal (4-HNE). A proteomic analysis of normal and GDM HUVEC confirmed the altered GDM phenotype, characterised by markers of increased oxidative stress, reduced antioxidant protection and reduced cell proliferation.7 More recently, we have confirmed increased 4-HNE induced damage and attenuated Nrf2-regulated gene expression in umbilical artery smooth muscle cells from GDM mothers. The altered phenotype of fetal venous endothelial and arterial smooth muscle cells derived from GDM pregnancies may underlie an increased risk of developing type 2 diabetes and cardiovascular disease in childhood and early adulthood.8-12

Glycyrrhetinic Acid Antagonizes Pressure-Induced Venous Remodeling in Mice.

Development of spider veins is caused by the remodeling of veins located in the upper dermis and promoted by risk factors such as obesity or pregnancy that chronically increase venous pressure. We have repeatedly shown that the pressure-induced increase in biomechanical wall stress is sufficient to evoke the formation of enlarged corkscrew-like superficial veins in mice. Subsequent experimental approaches revealed that interference with endothelial- and/or smooth muscle cell (SMC) activation counteracts this remodeling process. Here, we investigate whether the herbal agent glycyrrhetinic acid (GA) is a suitable candidate for that purpose given its anti-proliferative as well as anti-oxidative properties. While basic abilities of cultured venous SMCs such as migration and proliferation were not influenced by GA, it inhibited proliferation but not angiogenic sprouting of human venous endothelial cells (ECs). Further analyses of biomechanically stimulated ECs revealed that GA inhibits the DNA binding capacity of the mechanosensitive transcription factor activator protein-1 (AP-1) which, however, had only a minor impact on the endothelial transcriptome. Nevertheless, by decreasing gelatinase activity in ECs or mouse veins exposed to biomechanical stress, GA diminished a crucial cellular response in the context of venous remodeling. In line with the observed inhibitory effects, local transdermal application of GA attenuated pressure-mediated enlargement of veins in the mouse auricle. In summary, our data identifies GA as an inhibitor of EC proliferation, gelatinase activity and venous remodeling. It may thus have the capacity to attenuate spider vein formation and remodeling in humans.

Rho Kinase inhibitors and hypoxia modulate expression of platelet activating factor (PAF) receptor and gene expression in fetal ovine pulmonary vascular smooth muscle cells.

Hypoxia and PAF augment intracellular calcium flux in fetal ovine pulmonary vascular smooth muscle cells (PVSMC) indicating that high pulmonary vascular tone in fetal pulmonary vessels in utero is consequent on combined effects of hypoxia and PAF receptor (PAFR)‐linked vascular responses. PAFR internalization is said to inactivate PAF effects in PVSMC in vitro. Hypoxia increases PAFR expression, but Rho kinase (ROCK) inhibitors decrease the expression suggesting PAFR internalization may be one pathway to downregulate PAFR‐mediated responses in PVSMC. These findings also suggest that ROCK may act in synergy with PAF to modulate PAFR‐mediated pulmonary vascular responses. We studied effects of PAF, hypoxia and other modulators of pulmonary vascular reactivity in arterial (PA) and venous (PV) smooth muscle cells (SMC) to aid in understanding the signaling pathways of PAFR‐mediated fetal ovine pulmonary vascular reactivity in utero. We hypothesized that ROCK inhibitors attenuate PAFR expression in hypoxia leading to increased expression of the contractile proteins. Cells were cultured in normoxia or hypoxia, with and without 10 nM PAF or ROCK inhibitors (Y27632 and HA 1077, each 10 μM). Proteins were probed for PAFR, and Calponin, MLCK and Desmin, which are contractile protein modulators of PVSMC activity. PAF binding, PAFR protein and gene expression by qRT‐PCR were also determined. In normoxia, PAFR density (Bmax) in PA and PV SMC were 487±37 and 624±17 respectively, which increased by 30–35% in hypoxia, and higher in PV than PA in normoxia and hypoxia. The KD for PAF binding to PASMC was higher than for PVSMC, and then hypoxia decreased the KD of PAFR binding in both cell types. ROCK inhibitors attenuated PAFR binding with comparable effect to cycloheximide, but unlike PAFR inhibitor, CV3988, cycloheximide had no effect on pre‐synthesized proteins. ROCK inhibitors decreased PAFR expression, but increased expression of MLCK2, Calponin and Desmin in PASMC especially in hypoxia, with different effects in PVSMC. Generally, PAFR gene expression studied by qRT‐PCR was higher in PVSMC under all conditions. This study shows that hypoxia in conjunction with ROCK inhibitors modulate PAFR‐mediated signaling in PVSMC in vitro with different effects on cells from PA and PV. Increased expression of the contractive proteins in hypoxia indicates involvement of these proteins in PAF‐induced contractions in PVSMC.Support or Funding InformationLABiomedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Autologous fat transplants to deliver glitazone and adiponectin for vasculoprotection

The insulin sensitizing glitazone drugs, rosiglitazone (ROS) and pioglitazone (PGZ) both have anti-proliferative and anti-inflammatory effects and induce adipose tissue (fat) to produce the vaso-protective protein adiponectin. Stenosis due to intimal hyperplasia development often occurs after placement of arteriovenous synthetic grafts used for hemodialysis. This work was performed to characterize the in vitro and in vivo effects of ROS or PGZ incorporation in fat and to determine if fat/PGZ depots could decrease vascular hyperplasia development in a porcine model of hemodialysis arteriovenous graft stenosis.Powdered ROS or PGZ (6–6000μM) was mixed with fat explants and cultured. Drug release from fat was quantified by HPLC/MS/MS, and adiponectin and monocyte chemotactic protein-1 (MCP-1) levels in culture media were measured by ELISA. The effect of conditioned media from the culture of fat with ROS or PGZ on i) platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferation of human venous smooth muscle cells (SMC) was measured by a DNA-binding assay, and ii) lipopolysaccharide (LPS)-induced human monocyte release of tumor necrosis factor-alpha (TNFα) was assessed by ELISA. In a porcine model, pharmacokinetics of PGZ from fat depots transplanted perivascular to jugular vein were assessed by HPLC/MS/MS, and retention of the fat depot was monitored by MRI. A porcine model of synthetic graft placed between carotid artery and ipsilateral jugular vein was used to assess effects of PGZ/fat depots on vascular hyperplasia development.Both ROS and PGZ significantly induced the release of adiponectin and inhibited release of MCP-1 from the fat. TNF production from monocytes stimulated with LPS was inhibited 50–70% in the presence of media conditioned by fat alone or fat and either drug. The proliferation of SMC was inhibited in the presence of media conditioned by fat/ROS cultures. Fat explants placed perivascular to the external jugular vein were retained, as confirmed by MRI at one week after placement. PGZ was detected in the fat depot, in the external jugular vein wall and in adjacent tissue at clinically relevant levels, whereas levels in plasma were below detection. External jugular vein exposed to fat incorporated with PGZ had increased adiponectin expression compared to vein exposed to fat alone. However, the development of hyperplasia within the arteriovenous synthetic grafts was unchanged by treatment with fat/PGZ depots compared to no treatment.

A single nucleotide polymorphism of cyclin-dependent kinase inhibitor 1B (p27Kip1) associated with human vein graft failure affects growth of human venous adventitial cells but not smooth muscle cells

BackgroundCyclin-dependent kinase inhibitor 1B (p27Kip1) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (rs36228499; hereafter called p27 SNP) has been associated with the clinical failure of peripheral vein grafts, but the functional effects of this SNP have not been demonstrated. MethodsHuman saphenous vein adventitial cells and intimal/medial smooth muscle cells (SMCs) were derived from explants obtained at the time of lower extremity bypass operations. We determined the following in adventitial cells and SMCs as a function of the p27 SNP genotype: (1) p27 promoter activity, (2) p27 messenger (m)RNA and protein levels, and (3) growth and collagen gel contraction. Deoxyribonuclease I footprinting was also performed in adventitial cells and SMCs. Resultsp27 promoter activity, deoxyribonuclease I footprinting, p27 mRNA levels, and p27 protein levels demonstrated that the p27 SNP is functional in adventitial cells and SMCs. Both cell types with the graft failure protective AA genotype had more p27 mRNA and protein. As predicted because of higher levels of p27 protein, adventitial cells with the AA genotype grew slower than those of the CC genotype. Unexpectedly, SMCs did not show this genotype-dependent growth response. ConclusionsThese results support the functionality of the p27 SNP in venous SMCs and adventitial cells, but an effect of the SNP on cell proliferation is limited to only adventitial cells. These data point to a potential role for adventitial cells in human vein graft failure and also suggest that SMCs express factors that interfere with the activity of p27.

Abstract 463: The Key Regulatory Elements for SM22 Transcription

Gene transcription is controlled by an array of transcriptional regulatory elements. SM22 gene regulation has been widely used to characterize the molecular mechanisms of smooth muscle cell (SMC) phenotypic modulation during cardiovascular development and in vascular diseases. Our previous studies show that the proximal CArG box (CArGnear) in the SM22 promoter is required for its transcription in arterial smooth muscle cells (SMC). However, the role of the CArG box in visceral SMCs has not yet been explored. Here we aim to determine the role of CArG boxes in regulating S M22 transcription in both vascular and visceral SMCs. Using bacterial chromosome (BAC) recombineering, we knock-in a lacZ reporter into a SM22 BAC to trace SM22 transcriptional activities in transgenic mice. Anatomic/histology analyses show that the lacZ expression patterns in the BAC transgenic mice recapitulate that of the endogenous SM22 transcription during embryogenesis and in adult . Similar to the endogenous SM22 regulation, the expression of lacZ is highly sensitive to vascular remodeling: carotid injury abolishes lacZ expression in the arterial wall. Using seamless BAC recombineering mutagenesis, we generate mutations in the proximal and/or distal CArG box in the SM22-lacZ-BAC. Consistent with our previous results, we find that mutating the CArGnear box disrupts the lacZ expression in the aorta; this mutation also drastically reduces its expression in visceral SMCs including stomach, uterus and bladder. Interestingly, mutating the distal CArG (CArGfar) box does not affect the lacZ expression in arterial, venous and visceral SMCs. Mutating both CArG boxes nearly abolishes lacZ expression in all SMCs. This study provides evidence supporting the generation of SM22 knockout mice by mutating the CArG boxes in the SM22 promoter using the CRISPR technology.

MicroRNA-33 protects against neointimal hyperplasia induced by arterial mechanical stretch in the grafted vein.

Mechanical factors play significant roles in neointimal hyperplasia after vein grafting, but the mechanisms are not fully understood. Here, we investigated the roles of microRNA-33 (miR-33) in neointimal hyperplasia induced by arterial mechanical stretch after vein grafting. Grafted veins were generated by the 'cuff' technique. Neointimal hyperplasia and cell proliferation was significantly increased, and miR-33 expression was decreased after 1-, 2-, and 4-week grafts. In contrast, the expression of bone morphogenetic protein 3 (BMP3), which is a putative target of miR-33, and the phosphorylation of smad2 and smad5, which are potential downstream targets of BMP3, were increased in the grafted veins. miR-33 mimics/inhibitor and dual luciferase reporter assay confirmed the interaction of miR-33 and BMP3. miR-33 mimics attenuated, while miR-33 inhibitor accelerated, proliferation of venous smooth muscle cells (SMCs). Moreover, recombinant BMP3 increased SMC proliferation and P-smad2 and P-smad5 levels, whereas BMP3-directed siRNAs had the opposite effect. Then, venous SMCs were exposed to a 10%-1.25 Hz cyclic stretch (arterial stretch) by using the FX4000 cyclic stretch loading system in vitro to mimic arterial mechanical conditions. The arterial stretch increased venous SMC proliferation and repressed miR-33 expression, but enhanced BMP3 expression and smad2 and smad5 phosphorylation. Furthermore, perivascular multi-point injection in vivo demonstrated that agomiR-33 not only attenuates BMP3 expression and smad2 and smad5 phosphorylation, but also slows neointimal formation and cell proliferation in grafted veins. These effects of agomiR-33 on grafted veins could be reversed by local injection of BMP3 lentivirus. The miR-33-BMP3-smad signalling pathway protects against venous SMC proliferation in response to the arterial stretch. miR-33 is a target that attenuates neointimal hyperplasia in grafted vessels and may have potential clinical applications.

Phase Ib Safety, Two-Dose Study of MultiGeneAngio in Patients with Chronic Critical Limb Ischemia

Critical limb ischemia (CLI) is the most severe presentation of peripheral arterial disease. We developed cell-based therapy entailing intra-arterial injection of autologous venous endothelial cells (ECs) modified to express angiopoietin 1, combined with autologous venous smooth muscle cells (SMCs) modified to express vascular endothelial growth factor. This combination promoted arteriogenesis in animal models and was safe in patients with limiting claudication. In an open-label,phase Ib study, we assessed the safety and efficacy of this therapy in CLI patients who failed or were unsuitable for surgery or intravascular intervention. Of 23 patients enrolled, 18 with rest pain or non-healing ulcers (Rutherford categories 4 and 5) were treated according to protocol, and 5 with significant tissue loss (Rutherford 6) were treated under compassionate treatment. Patients were assigned randomly to receive 1× 107 or 5× 107 (EC-to-SMC ratio, 1:1) of the cell combination. One-year amputation-free survival rate was 72% (13/18) for Rutherford 4 and 5 patients; all 5 patients with Rutherford 6 underwent amputation. Of the 12 with unhealing ulcers at dosing, 6 had complete healing and 2 others had >66% reduction in ulcer size. Outcomes did not differ between the dose groups. No severe adverse events were observed related to the therapy.

Abstract 144: A Single Nucleotide Polymorphism of p27 Kip1 Associated with Vein Graft Patency Regulates Expression of p27 Kip1 in Both Venous Adventitial Cells and Smooth Muscle Cells but Selectively Regulates Proliferation of Adventitial Cells

Introduction: p27 Kip1 (p27) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (SNP) accounts for ~40% of the risk of peripheral vein graft failure. However, whether this SNP is functional has not been definitely established. Methods: To determine functionality, we investigated paired adventitial cells and smooth muscle cells (SMC) derived from fresh human saphenous veins (N≥7 lines per group). After growth arrest in 2% serum followed by stimulation with 10 ng/ml PDGF-BB, we measured p27 mRNA, p27 protein, and cell proliferation using qRT-PCR, Western blotting, and cell counts, respectively. Results: The SNP genotype was associated with 72 hour adventitial cell growth, but not SMC growth. Adventitial AA cells grew 33% slower than those with the CC genotype (Figure 1A, B, p=.004). We expected AA adventitial cells to produce more p27 mRNA, but paradoxically, p27 mRNA was lower in both cell types of the AA genotype (Figure 1C, D, P<.01). However, levels of p27 protein (a single ~27 kD band in both cell types) were ~2 fold higher in AA adventitial cells and SMCs compared to the CC cells at both 0 and 72 hours (Figure 1E, 72 hours; P<.001 for genotype; 0 h data not shown). Adventitial cells and SMCs made comparable amounts of p27 protein. Conclusion: The p27 Kip1 -838C>A SNP regulates levels of p27 in both venous adventitial cells and SMCs. There is an effect of genotype on the growth of adventitial cells, but not on SMCs. AA cells produce ~2 fold higher p27 protein than CC cells, despite AA cells having ~2 fold lower levels of p27 mRNA. These data demonstrate that the p27 SNP is functional, that different cell types within the vein wall respond uniquely to this genetic polymorphism, and suggest an important role in graft failure for the precursor of the cultured adventitial cell.

Varicose Remodeling of Veins Is Suppressed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors.

BackgroundDespite the high prevalence of chronic venous insufficiency and varicose veins in the Western world, suitable pharmaceutical therapies for these venous diseases have not been explored to date. In this context, we recently reported that a chronic increase in venous wall stress or biomechanical stretch is sufficient to cause development of varicose veins through the activation of the transcription factor activator protein 1.Methods and ResultsWe investigated whether deleterious venous remodeling is suppressed by the pleiotropic effects of statins. In vitro, activator protein 1 activity was inhibited by two 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors, rosuvastatin and atorvastatin, in stretch‐stimulated human venous smooth muscle cells. Correspondingly, both statins inhibited venous smooth muscle cell proliferation as well as mRNA expression of the activator protein 1 target gene monocyte chemotactic protein 1 (MCP1). In isolated mouse veins exposed to an increased level of intraluminal pressure, statin treatment diminished proliferation of venous smooth muscle cells and protein abundance of MCP1 while suppressing the development of varicose veins in a corresponding animal model by almost 80%. Further analyses of human varicose vein samples from patients chronically treated with statins indicated a decrease in venous smooth muscle cell proliferation and MCP1 abundance compared with samples from untreated patients.ConclusionsOur findings imply that both atorvastatin and rosuvastatin effectively inhibit the development of varicose veins, at least partially, by interfering with wall stress–mediated activator protein 1 activity in venous smooth muscle cells. For the first time, this study reveals a potential pharmacological treatment option that may be suitable to prevent growth of varicose veins and to limit formation of recurrence after varicose vein surgery.

TRPC6 is required for hypoxia-induced basal intracellular calcium concentration elevation, and for the proliferation and migration of rat distal pulmonary venous smooth muscle cells.

Hypoxia induces pulmonary vasoconstriction and reconstruction in the pulmonary arteries and pulmonary veins (PVs), and elevation of intracellular calcium concentration ([Ca2+]i) is a primary factor of these processes. In the present study, the role of transient receptor potential cation channels (TRPCs) in mediating the hypoxia-induced elevation of [Ca2+]i in rat distal pulmonary venous smooth muscle cells (PVSMCs) was investigated. Rats with chronic hypoxic pulmonary hypertension (CHPH) were used for in vivo experiments, and PVSMCs were isolated for in vitro experiments. [Ca2+]i was measured using fura-2-based fluorescence calcium imaging. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of TRPCs. Methyl thiazolyl tetrazolium and Transwell assays were used to investigate the proliferation and migration of PVSMCs, respectively. The results of the present study demonstrated that TRPC6 was increased in the distal PVs of CHPH rats, and in PVSMCs exposed to hypoxic conditions (4% O2, 72 h); however, TRPC1 was not. The 1-oleoyl-2-acetyl-sn-glycerol-induced [Ca2+]i elevation was increased in PVSMCs isolated from CHPH rats and in PVSMCs cultured under hypoxic conditions (4% O2, 72 h). Hypoxia induced PVSMC [Ca2+]i elevation, proliferation and migration. These alterations were inhibited following TRPC6 knockdown. Results from the present study suggest that TRPC6 expression is increased during chronic hypoxia, which contributes to Ca2+ entry into the cell, thus promoting proliferation and migration of PVSMCs.

Hypoxia Differentially Regulates Arterial and Venous Smooth Muscle Cell Migration.

ObjectiveIntimal hyperplasia (IH) is a clinical concern leading to failure of up to 50% of vein grafts and 10% of arterial grafts after 10 years with no known current treatment. Recent studies have shown that hypoxia differentially regulates proliferation of vein derived smooth muscle cells (V-SMC) compared to artery derived smooth muscle cells (A-SMC). The objective of this study is to evaluate the effect of hypoxia on cellular migration and the mechanisms underlying the differential effects of hypoxia on A-SMC and V-SMC migration.Methods and ResultsHypoxic treatment (3–5% O2) of Smooth Muscle Cells (SMC) resulted in differential migration in scratch wound and electric cell substrate impedance sensing (ECIS) assays. Hypoxia led to greater migration compared to normoxia with venous derived wound closure (V-SMC 30.8% Normoxia to 67% Hypoxia) greater than arterial wound closure (A-SMC 6.2% Normoxia to 24.7% Hypoxia). Paracrine factors secreted by hypoxic endothelial cells induced more migration in SMC compared to factors secreted by normoxic endothelial cells. Migration of V-SMC was greater than A-SMC in the presence of paracrine factors. Neutralizing antibody to Vascular Endothelial Growth Factor Receptor -1 (VEGFR-1) completely inhibited V-SMC migration while there was only partial inhibition of A-SMC migration. A-SMC migration was completely inhibited by Platelet Derived Growth Factor BB (PDGF-BB) neutralizing antibody. p38 Mitogen Activated Protein kinase (p38 MAPK) inhibitor pre-incubation completely inhibited migration induced by paracrine factors in both A-SMC and V-SMC.ConclusionOur study determines that SMC migration under hypoxia occurs via both an autocrine and paracrine mechanism and is dependent on Vascular Endothelial Growth Factor-A (VEGF-A) in V-SMC and PDGF-BB in A-SMC. Migration of both A-SMC and V-SMC is inhibited by p38 MAPK inhibitor. These studies suggest that pharmacotherapeutic strategies directed at modulating p38 MAPK activity can be exploited to prevent IH in vascular grafts.

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<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<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<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<0.01, N=6; proliferation: control IgG 3.4 ± 0.3 vs. Anti-FGF2 Ab 2.7 ± 0.3 fold; P<0.01, N=7). Conclusion: The increased migration and proliferation of VSMC compared to NVSMC is largely mediated by FGF2.

Abstract 304: Varicose Vein Development in Mice Is Blocked by Ator- and Rosuvastatin

Venous diseases such as varicose veins and chronic venous insufficiency constitute a substantial source of morbidity. Despite their high prevalence, neither molecular mechanisms initiating these diseases nor pharmaceutic therapies have been explored so far. We recently reported that chronically increased filling pressure or biomechanical venous wall stress is sufficient to cause development of varicose-like veins in mice through a mechanism preceded by activation of the transcription factor activator protein 1 (AP-1). Considering these findings, we hypothesized that pharmacologic inhibition of AP-1 activity may attenuate venous remodeling. To prove this therapeutic concept, human venous smooth muscle cells (SMCs) were subjected to biomechanical stretch (13%, 0.5Hz) and treated with statins - HMG-CoA reductase inhibitors that are supposed to interfere with the activity of AP-1. EMSA and immunofluorescence studies revealed that ator- and rosu- but not simvastatin blocked stretch-induced AP-1 activity. Furthermore, ator- and rosuvastatin inhibited expression of the AP-1 target monocyte chemotactic protein 1 (MCP-1) and proliferation of these cells. In mouse veins exposed to an increased level of intraluminal pressure, atorvastatin diminished the abundance of MCP-1 and MMP-2 by ~90 % and inhibited proliferation of endothelial cells and SMCs (n=5, p<0.05). In line with this observation, growth of varicose-like veins in mice was suppressed by almost ~80 % upon treatment with rosu- and atorvastatin but not simvastatin (n=5, p<0.05) which was accompanied by a decrease in cellular proliferation as well as MMP-2 abundance in remodeling blood vessels. Consequent analyses of human varicose vein samples from patients chronically treated with statins indicated a decrease in SMC proliferation, MCP-1 and MMP-2 abundance (50-60 %, n=10, p<0.05). Collectively, our current findings imply that both ator- and rosuvastatin effectively inhibit the development of varicose veins presumably by interfering with the wall stress-mediated activity of AP-1 in VSMCs. For the first time, this study revealed a possible pharmaceutic treatment that may be suitable to prevent growth of varicose veins and to limit formation of recurrences after varicose vein surgery.

Brilliant blue FCF is a nontoxic dye for saphenous vein graft marking that abrogates response to injury

Injury to saphenous vein grafts during surgical preparation may contribute to the subsequent development of intimal hyperplasia, the primary cause of graft failure. Surgical skin markers currently used for vascular marking contain gentian violet and isopropanol, which damage tissue and impair physiologic functions. Brilliant blue FCF (FCF) is a nontoxic dye alternative that may also ameliorate preparation-induced injury. Porcine saphenous vein (PSV) was used to evaluate the effect of FCF on physiologic responses in a muscle bath. Cytotoxicity of FCF was measured using human umbilical venous smooth muscle cells. Effect of FCF on the development of intimal hyperplasia was evaluated in organ culture using PSV. Intracellular calcium fluxes and contractile responses were measured in response to agonists and inhibitors in rat aorta and human saphenous vein. Marking with FCF did not impair smooth muscle contractile responses and restored stretch injury-induced loss in smooth muscle contractility of PSV. Gentian violet has cytotoxic effects on human umbilical venous smooth muscle cells, whereas FCF is nontoxic. FCF inhibited intimal thickening in PSV in organ culture. Contraction induced by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate and intracellular calcium flux were inhibited by FCF, oxidized adenosine triphosphate, KN-62, and brilliant blue G, suggesting that FCF may inhibit the purinergic receptor P2X7. Our studies indicated that FCF is a nontoxic marking dye for vein grafts that ameliorates vein graft injury and prevents intimal thickening, possibly due to P2X7 receptor inhibition. FCF represents a nontoxic alternative for vein graft marking and a potentially therapeutic approach to enhance outcome in autologous transplantation of human saphenous vein into the coronary and peripheral arterial circulation.