Proliferation Of SMCs Research Articles

Flavonoids from Carya cathayensis Sarg. leaves inhibit carotid artery lesion formation induced by low blood flow

The aim of the present study was to investigate the protective effect of the total flavonoids (TFs) from the leaves of Carya cathayensis Sarg. against early development of atherosclerosis. An in vivo model of carotid arterial partial ligation was established in mice, and the effects of TFs were investigated by morphometric measurements, Cell proliferation measurement and immunohistochemistry. The results showed that TFs could reduce neointima area by 41%, and the adventitial thickening induced by partial ligation was remarkable inhibited by TFs treatment. medial SMCs proliferation was significantly inhibited in TFs treated group. Immunohistochemistry analyses demonstrated that mice with TFs treatment have significant less macrophages accumulation in adventitia. These findings indicated that TFs have inhibitory effect in early atherosclerosis lesion formation model and strong action on reduce the inflammation in vivo.

A Functional Variant of SMAD4 Enhances Thoracic Aortic Aneurysm and Dissection Risk through Promoting Smooth Muscle Cell Apoptosis and Proteoglycan Degradation

Recent studies indicate important roles for SMAD4 in SMCs proliferation, extracellular matrix maintenance, and blood vessel remodeling. However, the genetic effects of SMAD4 in the pathogenesis of thoracic aortic aneurysm and dissection (TAAD) are still largely unknown. Here we identified a functional variant of SMAD4 which might be involved in the pathological progression of TAAD. Five tagging SNPs of SMAD4 were genotyped in 202 TAAD cases and 400 controls using MALDI-TOF. rs12455792 CT or TT variant genotypes was associated with an significantly elevated TAAD risk (adjusted OR=1.58, 95%CI=1.09–2.30) under a dominant genetic model. It was located in the 5’UTR and predicted to influence transcription activity and RNA folding of SMAD4. In luciferase reporter assay, rs12455792 T allele markedly decreased luciferase activities. Accordingly, SMAD4 expression in tissues was lower in patients with CT or TT genotypes, compared with CC. Movat's pentachrome showed that rs12455792 T allele enhanced SMCs loss and fibers accumulation. With angiotensin II induction, rate of Apoptotic SMCs was significantly higher while SMAD4 silenced. Moreover, rs12455792 T allele also increased Versican degradation via ADAMTS-4. In conclusion, this variant might promote SMCs apoptosis and proteoglycans degradation, and further facilitate the progress of TAAD. Our findings identified rs12455792 as a predictor for progression of vascular media pathological changes related thoracic aortic disorders.

MiRNA‑504 inhibits p53‑dependent vascular smooth muscle cell apoptosis and may prevent aneurysm formation.

Abdominal aortic aneurysm (AAA) is a common disease that is associated with the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). VSMCs are regulated by microRNAs (miRNA). The aim of the present study was to identify miRNA sequences that regulate aortic SMCs during AAA. miRNA-504 was identified using a miRNA PCR array and by reverse transcription-quantitative polymerase chain reaction analysis, and its expression levels were observed to be downregulated in the aortic cells derived from patients with AAA when compared with controls. Transfection of SMCs with pMSCV-miRNA-504 vector was performed, and cell proliferation and the expression levels of proliferating cell nuclear antigen (PCNA), replication factor C subunit 4 (RFC4), B-cell lymphoma-2 (Bcl-2) and caspase-3/9 were measured by western blotting. The mechanisms underlying the effects of miRNA-504 was then analyzed. The results demonstrated that overexpression of miRNA-504 significantly upregulated the expression levels of PCNA, RFC4 and Bcl-2, while caspase-3/9 expression was significantly inhibited when compared with non-targeting controls. In addition, miRNA-504 overexpression was observed to promote the proliferation of SMCs. The expression level of the tumor suppressor, p53, which is known to be a direct target of miRNA-504, was inhibited following transfection of SMCs with pMSCV-miRNA-504. In addition, the expression of the downstream targets of p53, p21 and Bcl-like protein-4, were significantly reduced following overexpression of miRNA-504. These results revealed the anti-apoptotic role of miRNA-504 in SMCs derived from patients with AAA via direct targeting of p53.

A Novel Urotensin II Receptor Antagonist, KR-36996 Inhibits Smooth Muscle Proliferation through ERK/ROS Pathway.

Urotensin II (UII) is a mitogenic and hypertrophic agent that can induce the proliferation of vascular cells. UII inhibition has been considered as beneficial strategy for atherosclerosis and restenosis. However, currently there is no therapeutics clinically available for atherosclerosis or restenosis. In this study, we evaluated the effects of a newly synthesized UII receptor (UT) antagonist, KR-36996, on the proliferation of SMCs in vitro and neointima formation in vivo in comparison with GSK-1440115, a known potent UT antagonist. In primary human aortic SMCs (HASMCs), UII (50 nM) induced proliferation was significantly inhibited by KR-36996 at 1, 10, and 100 nM which showed greater potency (IC50: 3.5 nM) than GSK-1440115 (IC50: 82.3 nM). UII-induced proliferation of HASMC cells was inhibited by U0126, an ERK1/2 inhibitor, but not by SP600125 (inhibitor of JNK) or SB202190 (inhibitor of p38 MAPK). UII increased the phosphorylation level of ERK1/2. Such increase was significantly inhibited by KR-36996. UII-induced proliferation was also inhibited by trolox, a scavenger for reactive oxygen species (ROS). UII-induced ROS generation was also decreased by KR-36996 treatment. In a carotid artery ligation mouse model, intimal thickening was dramatically suppressed by oral treatment with KR-36996 (30 mg/kg) which showed better efficacy than GSK-1440115. These results suggest that KR-36996 is a better candidate than GSK-1440115 in preventing vascular proliferation in the pathogenesis of atherosclerosis and restenosis.

Blockade of Urotensin II Receptor Prevents Vascular Dysfunction

Urotensin II (UII) is a potent vasoactive peptide and mitogenic agent to induce proliferation of various cells including vascular smooth muscle cells (VSMCs). In this study, we examined the effects of a novel UII receptor (UT) antagonist, KR-36676, on vasoconstriction of aorta and proliferation of aortic SMCs. In rat aorta, UII-induced vasoconstriction was significantly inhibited by KR-36676 in a concentration-dependent manner. In primary human aortic SMCs (hAoSMCs), UII-induced cell proliferation was significantly inhibited by KR-36676 in a concentration-dependent manner. In addition, KR-36676 decreased UII-induced phosphorylation of ERK, and UII-induced cell proliferation was also significantly inhibited by a known ERK inhibitor U0126. In mouse carotid ligation model, intimal thickening of carotid artery was dramatically suppressed by oral treatment with KR-36676 (30 mg/ kg/day) for 4 weeks compared to vehicle-treated group. From these results, it is indicated that KR-36676 suppress UII-induced proliferation of VSMCs at least partially through inhibition of ERK activation, and that it also attenuates UII-induced vasoconstriction and vascular neointima formation. Our study suggest that KR-36676 may be an attractive candidate for the pharmacological management of vascular dysfunction.

The isolation and characterization of systemic sclerosis vascular smooth muscle cells: enhanced proliferation and apoptosis resistance

Background Vascular dysfunction is a major pathologic component of systemic sclerosis (SSc). The role of vascular smooth muscle cells (vSMCs) in the development of vascular dysfunction is still unknown. In this study, we describe a method for isolation of dermal vSMCs from skin biopsies, and we outline their functional phenotype. Methods We obtained 4 mm punch-skin biopsies from three SSc patients and matched controls. After trypsin digestion, cells were cultured for 14 days. vSMCs were isolated by first depleting CD31+ cells (endothelial cells), followed by positive selection of CD146+ cells. The CD31- CD146+ cells were then cultured in media optimized for SMCs proliferation. We evaluated cell proliferation, viability and apoptosis in normal and in low serum culture conditions. Cytoplasmic and nuclear expression levels of β-catenin were also investigated. Results The CD31- CD146+ cell population expressed smooth muscle MYH11, Desmin and Vimentin but did not express NG2. Flow cytometry confirmed the high purity of CD31- CD146+ MYHC11+ cell population that was maintained for up to the eleventh passage. SSc-vSMCs exhibited increased cell proliferation and viability compared to control cells. Under serum starvation conditions, SSc-vSMCs exhibited more proliferative capacity, and resistance to apoptosis compared to control-vSMCs. Furthermore, a cytoplasmic to nuclear translocation of β-catenin was seen in SSc-vSMCs but not in control-vSMCs. Conclusions This is the first report of successful isolation and initial characterization of SSc-vSMCs. It is likely that increased proliferation of SSc-vSMCs in association with resistance to apoptosis can adversely impact the vascular lesion in SSc.

The role of atorvastatin on the restenosis process post-PTA in a diabetic rabbit model

BackgroundRestenosis remains to be a major limitation of percutaneous transluminal angioplasty (PTA) for diabetic patients with peripheral vascular disease (PVD). Despite of stations routine implements to prevent such progress, its exact effect is unclear.Methods and resultsIn our study, balloon was successfully implanted in the iliac artery of atherosclerotic rabbit. Patency of the narrowed artery was interrogated using ultrasound. Atorvastatin or vehicle was administered orally to rabbits from day 0 to day 28 after double-injury surgery. On day 7, day 14, and day 28, restenotic arteries were harvested and processed for histopathlogical analysis. Our data show that, after double-injury surgery, the intima was composed mostly by SMCs at all time course in rabbits undergoing surgery process. Significant increases in stenosis rates were noted from day 7 to day 14 (from 21 ± 5.85 % to 60.93 ± 12.46 %). On day 28 after double-injury surgery, severe restenosis was observed and daily administration of atorvastatin cannot prevent restenosis’ formation (88.69 ± 3.71 % vs. 90.02 ± 3.11 %, P > 0.05). The PCNA index and SMCs proliferation were correlated with the scores of the vascular pathology.ConclusionsOur results indicate that double-injury model can mimic clinical restenosis, based on this model, atorvastatin showed no therapeutic effect on restenosis process in diabetic rabbits after PTA.

Inactivation of semicarbazide-sensitive amine oxidase induces the phenotypic switch of smooth muscle cells and aggravates the development of atherosclerotic lesions

Background and aimsClinical studies have demonstrated that serum semicarbazide-sensitive amine oxidase (SSAO) activities positively correlate with the progression of atherosclerosis. The aim of the present study is to investigate the effect of SSAO inactivation on the development of atherosclerosis. MethodsFemale LDLr knockout (KO) mice were given the Western-type diet for 6 and 9 weeks to induce the formation of early and advanced lesions, and semicarbazide (SCZ, 0.125%) was added into the drinking water to inactivate SSAO in vivo. ResultsDespite no impact on plasma total cholesterol levels, abrogation of SSAO by SCZ not only resulted in the enlargement of both early (1.5-fold, p = 0.0043) and advanced (1.8-fold, p = 0.0013) atherosclerotic lesions, but also led to reduced/increased lesion contents of macrophages/smooth muscle cells (SMCs) (macrophage: ∼0.74-fold, p = 0.0002(early)/0.0016(advanced); SMC: ∼1.55-fold, p = 0.0003(early)/0.0001(advanced)), respectively. Moreover, SSAO inactivation inhibited the migration of circulating monocytes into peripheral tissues and reduced the amount of circulating Ly6Chigh monocytes (0.7-fold, p = 0.0001), which may account for the reduced macrophage content in lesions. In contrast, the increased number of SMCs in lesions of SCZ-treated mice is attributed to an augmented synthetic vascular SMC phenotype switch as evidenced by the increased proliferation of SMCs and accumulation of collagens in vivo. ConclusionSSAO inactivation by SCZ promotes the phenotypic switch of SMCs and the development of atherosclerosis. The enzymatic activity of SSAO may thus represent a potential target in the prevention and/or treatment of atherosclerosis.

Histological changes of the unligated vein wall adjacent to the central venous catheter after open cutdown in rats

BackgroundThe sequelae of a central venous cutdown usually include venous deformity causing venous stenosis or stricture. However, the cellular mechanisms causing these deformities have not been elucidated. MethodsSilicone 2.7-Fr catheters were placed via the right external jugular vein of 16 rats with the cutdown method. After fixation with formalin at scheduled intervals (1week, 2weeks, 4weeks, and 8weeks; 4 rats in each group), the vein segment with the catheter in situ was harvested. Histological changes in the vein wall were studied and serially compared with light microscopy; standard hematoxylin-eosin staining, Masson’s trichrome staining, van Gieson’s elastin stain, and immunohistochemical stain against α-actin. ResultsPericatheter sleeve formation, circumferential smooth muscle cell proliferation and infiltration into the pericatheter sleeve by direct contact were noted in all 4 rats of 1-week model; this indicated the initiation of neointimal hyperplasia. The neointimal hyperplasia was located inside the elastin layer. At 2weeks, the SMCs stained faintly but the components of the vein wall were largely replaced by collagen. The proliferation and infiltration of SMCs stabilized at 4weeks and no SMCs were stained around the catheter. At 8weeks, luminal narrowing was noted and the venous wall was composed mainly of collagen. ConclusionsCircumferential neointimal hyperplasia occurred after surgical cutdown of the external jugular vein in a rat model and was caused by SMC activation, proliferation, and infiltration into the pericatheter sleeve.

In Vitro Evaluation of Cenderitide‐Eluting Stent I —An Antirestenosis and Proendothelization Approach

Despite the success that drug-eluting stents (DESs) have achieved for minimizing in-stent restenosis (ISR), the antirestenotic agents used in DES have been implicated in delayed endothelial healing and impairment of endothelial functions. Cenderitide (CD-NP) is a novel antiproliferation chimeric peptide of semiendothelial origin; thus, this paper aims to demonstrate the selectivity aspect of this new peptide via in vitro evaluation on key players in ISR-smooth muscle cells (SMCs) and endothelial cells. The microbicinchoninic acid protein assay was used to investigate the CD-NP release from films and stents. Cenderitide-containing films blended with poly(ethylene glycol) and its copolymer exhibited higher release kinetics compared with neat poly(ε-caprolactone) (PCL) formulation. Cenderitide-eluting stents (CES) was produced by coating bare metallic stents with CD-NP entrapped PCL using an ultrasonic spray coater. The investigation of CD-NP on in vitro cells revealed that CD-NP inhibits human coronary smooth muscle cells (HCaSMCs) proliferation but exhibits no effects on human umbilical vein endothelial cells (HUVECs) proliferation. Moreover, CD-NP released up to 7 days displayed inhibitory effects on SMCs proliferation. The CES produced in this work shows that the released CD-NP inhibits HCaSMCs proliferation but did not hamper HUVECs proliferation in vitro, suggesting that it has potential to reduce ISR without retarding the endothelialization healing in vivo.

Promoting endothelial recovery and reducing neointimal hyperplasia using sequential-like release of acetylsalicylic acid and paclitaxel-loaded biodegradable stents.

IntroductionThis work reports on the development of a biodegradable dual-drug-eluting stent with sequential-like and sustainable drug-release of anti-platelet acetylsalicylic acid and anti-smooth muscle cell (SMC) proliferative paclitaxel.MethodsTo fabricate the biodegradable stents, poly-L-lactide strips are first cut from a solvent-casted film. They are rolled onto the surface of a metal pin to form spiral stents. The stents are then consecutively covered by acetylsalicylic acid and paclitaxel-loaded polylactide-polyglycolide nanofibers via electrospinning.ResultsBiodegradable stents exhibit mechanical properties that are superior to those of metallic stents. Biodegradable stents sequentially release high concentrations of acetylsalicylic acid and paclitaxel for more than 30 and 60 days, respectively. In vitro, the eluted drugs promote endothelial cell numbers on days 3 and 7, and reduce the proliferation of SMCs in weeks 2, 4, and 8. The stents markedly inhibit the adhesion of platelets on days 3, 7, and 14 relative to a non-drug-eluting stent. In vivo, the implanted stent is intact, and no stent thrombosis is observed in the stent-implanted vessels without the administration of daily oral acetylsalicylic acid. Promotion of endothelial recovery and inhibition of neointimal hyperplasia are also observed on the stented vessels.ConclusionThe work demonstrates the efficiency and safety of the biodegradable dual-drug-eluting stents with sequential and sustainable drug release to diseased arteries.

RORα suppresses proliferation of vascular smooth muscle cells through activation of AMP-activated protein kinase

BackgroundRetinoic acid-related orphan receptor α (RORα) has been implicated in the progression of atherosclerosis, but its role in the proliferation of vascular smooth muscle cells (vSMCs) has not been fully examined. We previously reported that RORα activates AMP-activated protein kinase (AMPK), which is associated with the suppression of vSMC proliferation. Therefore, we investigated the suppressive function of RORα on the proliferation of vSMCs and the molecular mechanisms involved. ResultsFirst, RORα and its activator, cholesterol sulfate (CS), induced the activation of AMPK in both human aortic SMCs and rat A7r5 cells, which was accompanied by the suppression of mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1. Second, RORα and CS modulated the expression of cell-cycle-regulating factors, such as p53, p27, and cyclin D in vSMCs. Consistent with this, the overexpression of RORα or CS treatment suppressed the proliferation of human aortic SMCs and rat A7r5 cells, possibly through G1 arrest. RORα and CS also inhibited the migration of A7r5 cells in two-dimensional and three-dimensional cell migration assays. Finally, we demonstrated that the infusion of adenovirus encoding RORα into arteries suppressed neointima formation after balloon injury in rats. ConclusionThese results demonstrate that RORα inhibits vSMC proliferation through AMPK-induced mTOR suppression, and suggest that RORα is a therapeutic target for the cardiovascular diseases associated with vSMC proliferation.

MiR-663 and the miRaculous Vascular Smooth Muscle Phenotypic Switch

Cardiovascular diseases, such as ischemic heart disease and stroke, represent the number one cause of death worldwide.1 In most cases, atherosclerosis and hypertension constitute the underlying causes for cardiovascular disease because it leads to arterial occlusion and impaired cardiac function, respectively. Therapeutic compensation of atherosclerotic artery occlusion by bypass grafting, angioplasty, or stenting bears the risk of intimal hyperplasia and subsequent restenosis.2 In general, a prerequisite for any pathological and physiological vascular remodeling process is the phenotypic switch of vascular smooth muscle cells (VSMCs).3 Even in adult blood vessels, VSMCs retain a remarkable plasticity that is essential for any changes in the vessel wall architecture. Contractile VSMCs representing the majority of VSMCs in healthy vessels guarantee maintenance of vascular tone and thereby the systemic blood pressure and blood flow by controlling the blood vessel diameter. As outlined in the Figure, a plethora of humoral factors, such as platelet-derived growth factor-BB and biomechanical stimuli, especially chronic changes in blood flow and wall stress, is capable to trigger the phenotypic switch of VSMCs from a quiescent, contractile differentiated state to a synthetic, proliferative, and dedifferentiated state.3,4 Figure. The effect of microRNA (miR)-633 on vascular homeostasis and remodeling. Major stimuli, transcription factors, and miRs that are involved in the differentiation and phenotypic switch of vascular smooth muscle cells (VSMCs) are depicted. miR-143/miR-145 promotes the contractile state by inhibiting suppressors of myocardin/serum response factor (SRF) activity (eg, ETS domain-containing protein-1 [Elk-1] and Kruppel-like factor [KLF] 4/5). Interestingly, myocardin activity seems to be regulated by an intrinsic loop because it stabilizes the expression of miR-145, whereas miR-143 seems to inhibit myocardin expression. miR-633 is a novel player with a dual effect on the VSMC phenotype not only by attenuating proliferation, migration, and proteolytic activity, but also by limiting their …

GW24-e0507 Role of wnt signal pathway in proliferation and collagen secretion of smooth muscle cells from rats aorta induced by AngII

ObjectivesTo evaluate whether Wnt signalling pathway modulates proliferation and collagen secretion of vascular smooth muscle cells and try to find novel gene target of anti-vascular restenosis.MethodsCultured vascular smooth muscle cells...

Abstract 277: Downregulation of PDGF-D is Associated with Increased Collagen Production in Abdominal Aortic Aneurysm

BACKGROUND Abdominal aortic aneurysm (AAA) is characterized by loss of aortic smooth muscle cells (aoSMCs) and remodeling of extracellular matrix, such as collagen. Platelet-derived growth factor-D (PDGF-D) is the latest discovered member of the PDGF/VEGF family and involved in proliferation and migration of SMCs. Here we analyzed the expression of PDGF-D in human and murine AAA. Furthermore, we studied the potential role of PDGF-D in regulation of collagen in cultured aoSMC. METHOD AND RESULTS Gene expression analysis of abdominal aortas revealed a significant lower expression of PDGF-D (-85%) and its receptor PDGFR-β (-73%) in human aneurysmal wall compared to non-aneurysmal control aortas. PDGF-D and PDGFR-β was mainly localized to SMC and endothelial cells as determined by immunohistochemical analysis and was expressed weaker in aneurysmal samples compared with controls. In mice, luminal aortic diameter was increased by 60% (P<0.001) in aortas treated with 0.5M CaCl 2 compared to saline treated control aortas. In agreement with human AAA, PDGF-D expression was significantly decreased in mice with AAA (-60%) compared to controls. A decrease in the expression of PDGF-D was strongly correlated to both α-actin (r=0.95, P<0.001) and SM22α (r=0.94, P<0.001) expression. A decreased expression of PDGF-D (r=-0.76, P<0.05) and α-actin (r=-0.88, P<0.01) was correlated with increased expression of the apoptosis marker caspase-3. To investigate the potential inhibitory role of PDGF-D in collagen remodeling, cultured aoSMCs were stimulated with 20 ng/ml of human recombinant PDGF-D or PBS as control for 24 and 48 hours. Treatment of aoSMC with rh-PDGF-D resulted in a decrease in the synthesis of collagen-III (P=0.023) and -IV (P=0.01) as well as prolyl 4-hydroxylase (P=0.01) and lysyl oxidase (P=0.02) after 48 hours. The synthesis of collagen-I was unaffected with increased PDGF-D. CONCLUSION Our findings imply that PDGF-D functions as a negative regulator of collagen synthesis and that lack of PDGF-D could therefore be important for the AAA process.

RAGE regulation and signaling in inflammation and beyond

RAGE is a key molecule in the onset and sustainment of the inflammatory response. New studies indicate that RAGE might represent a new link between the innate and adaptive immune system. RAGE belongs to the superfamily of Ig cell-surface receptors and is expressed on all types of leukocytes promoting activation, migration, or maturation of the different cells. RAGE expression is prominent on the activated endothelium, where it mediates leukocyte adhesion and transmigration. Moreover, proinflammatory molecules released from the inflamed or injured vascular system induce migration and proliferation of SMCs. RAGE binds a large number of different ligands and is therefore considered as a PRR, recognizing a structural motif rather than a specific ligand. In this review, we summarize the current knowledge about the signaling pathways activated in the different cell types and discuss a potential activation mechanism of RAGE, as well as putative options for therapeutic intervention.

Cytotoxic Effects of Transforming Growth Factor-α Conjugated with Cytotoxin Saporin on Proliferating Vascular Smooth Muscle Cells

Abstract Objective To testify the special cytotoxicity of TGF-alpha-SAP on proliferating vascular smooth muscle cells and endothelial cells. Methods Conjugation of saporin to TGF-alpha was accomplished after derivatization of saporin and TGF-alpha with N-succinimidyl-3 (2-pyridyldithio) proprionate and final purification of the conjugate was achieved within Eppendorf Centrifugal Filter Cytotoxicity assays were measured by cell count. The studies of influence of TGF-alpha-SAP on values of Thymidine and leucine incorporation into SMCs and ECs were measured by 3H-thymidine uptake and 3H-leucine uptake, respectively. and receptor competition studies of TGF-alpha-SAP are measured by adding excess TGF-alpha in SMCs exposed for TGF-SAP. Results Cytotoxicity assays testified TGF-alpha-SAP conjugate could inhibit remarkably proliferation of SMCs in culture. The values of thymidine of TGF-alpha-SAP group (10-9M and 10-7M) in comparison significantly decreased to 60.9% and 56.0% of the control group respectively, suggesting that cellular DNA synthesis obviously decreased as TGF-alpha-SAP was added. But Saporin did not affect cellular DNA synthesis at higher level. The rate of 3H-leucine incorporation of TGF-alpha-SAP group significantly decreased to 47.3% of the control group, suggesting that SMCs protein synthesis obviously decreased as TGF-alpha-SAP was added. But TGF-alpha-SAP at the same level did not affect DNA synthesis and protein synthesis of ECs compared with the control group. Conclusion The results indicated that TGF-alpha-SAP possesses the more effective cytotoxicity than Saporin and the more special citotoxicity on proliferating vascular smooth muscle cells than on proliferating endothelial cells. Key words：cytotoxicity;selective; Drug-eluting stents;proliferation;saporin

Proteomics analysis of distinct proteins in human atherosclerosis obliterans: identification and verification

To identify distinct proteins involved in human atherosclerosis obliterans (ASO) by a differential proteomic approach. Eight atherosclerotic femoral arteries with a mean age of 68.6 years (6 male and 2 female) and 5 normal femoral arteries with a mean age of 44.2 years (3 male and 2 female) were obtained from high amputation patients. Then the first 2-dimensional maps of the proteome of human femoral arteries was plotted to compare ASO and control specimens. Proteomic profiling was to differentiate and identify histological proteins that were associated with ASO. The differentially expressed proteins were sequenced by matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). The result was verified by immunohistochemistry (IHC) and Western blot. ASO was associated with distinct patterns of protein expression in the femoral arteries. A total of 25 distinct spots corresponding to 13 different proteins were identified by MALDI-TOF-MS using the NCBI and IPI databases. These proteins were mainly involved in the pathogenetic mechanisms such as inflammation, oxidative stress, proliferation and transformation of SMCs. The low level of heat shock protein 27 (HSP27) in ASO was verified by IHC and western-blot in accord with the result of MS. Proteomic analysis can be used to investigate differentially expressed proteins, which may provide new insights into ASO pathogenesis, such as HSP27.

English

Intercellular gap junction (GJ) plays a pivotal role in the proliferation and transformation of vascular smooth muscle cells (VSMCs). This study was designed to test the hypothesis that expressions of the component proteins of gap junctions, connexins40 and 43 (Cx40 and Cx43), are up-regulated in arteries subjected to balloon injury and that this up-regulation can be suppressed by statin therapy. Transmission electron microscopy (TEM) revealed that there were abundant GJ between neointimal SMCs but fewer and smaller GJ after losartan and ramipril treatment. Reverse transcription–polymerase chain reaction and Western blot analysis showed the messenger ribonucleic acid (mRNA) and protein expressions of Cx40 and Cx43 were elevated after injury, and these elevations were suppressed by losartan and ramipril. Immunostaining showed the Cx40 and Cx43 expressions were consistently enhanced in the neointimal area after injury, which was decreased by losartan and ramipril treatment. Balloon injury causes up-regulation of Cx40 and Cx43 in neointimal SMCs. angiotensin-converting enzyme inhibitors (ACEIs) and AT1 antagonist losartan can reduce the proliferation of SMCs, suggesting the rennin-angiotensin system (RAS) system plays an important role in the remodeling of GJ in the VSMCs under pathological conditions.   Key words: Vascular smooth muscle cell, gap junction, connexin, balloon angioplasty, statin.

Underlying mechanism for suppression of vascular smooth muscle cells by green tea polyphenol EGCG released from biodegradable polymers for stent application

Epigallocatechin-3-O-gallate (EGCG), the predominant catechin from tea, is known to exert a variety of cardiovascular beneficial effects by affecting the activity of receptor and signal transduction kinases. In this study, we investigated the suppressive effects of EGCG released from biodegradable poly(L-lactide-co-ε-caprolactone, PLCL) films on the proliferation, cell cycle progression and matrix metalloproteinase-2 (MMP-2) expression of vascular smooth muscle cells (VSMCs). The involvement of phosphorylated Akt (pAkt) and nuclear factor-κB (pNF-κB) as well as the internalization of EGCG into VSMCs was also examined as underlying mechanisms for EGCG-mediated VSMC inhibition. The proliferation of canine aortic SMCs (CASMCs) on EGCG-releasing PLCL (E-PLCL) was significantly inhibited. The culture of CASMCs on E-PLCL resulted in induction of cell cycle arrest at G(0)/G(1) phase and inactivation of pAkt, leading to subsequent apoptosis. Active MMP-2 expression was directly lowered by EGCG released from E-PLCL and indirectly inhibited by the EGCG-mediated suppression of pNF-κB. We also observed the incorporation of fluorescein isothiocyanate-conjugated EGCG into the cytoplasm of CASMCs and its further nuclear translocation, which could lead to the interruption of the exogenous signals directed to genes responsible for cellular responses of CASMCs. Taken together, the attenuated responses of VSMCs to E-PLCL were shown to be mediated through the suppression of pNF-κB, pAkt and each subsequent target genes or proteins by EGCG incorporated into the cells.