Porcine Coronary Artery Smooth Muscle Cells Research Articles

A Biocompatibility Study of Plasma Nanocoatings onto Cobalt Chromium L605 Alloy for Cardiovascular Stent Applications.

The objective of this study was to evaluate the biocompatibility of trimethylsilane (TMS) plasma nanocoatings modified with NH3/O2 (2:1 molar ratio) plasma post-treatment onto cobalt chromium (CoCr) L605 alloy coupons and stents for cardiovascular stent applications. Biocompatibility of plasma nanocoatings was evaluated by coating adhesion, corrosion behavior, ion releasing, cytotoxicity, and cell proliferation. Surface chemistry and wettability were studied to understand effects of surface properties on biocompatibility. Results show that NH3/O2 post-treated TMS plasma nanocoatings are hydrophilic with water contact angle of 48.5° and have a typical surface composition of O (39.39 at.%), Si (31.92 at.%), C (24.12 at.%), and N (2.77 at.%). The plasma nanocoatings were conformal to substrate surface topography and had excellent adhesion to the alloy substrates, as assessed by tape test (ASTM D3359), and showed no cracking or peeling off L605 stent surfaces after dilation. The plasma nanocoatings also improve the corrosion resistance of CoCr L605 alloy by increasing corrosion potential and decreasing corrosion rates with no pitting corrosion and no mineral adsorption layer. Ion releasing test revealed that Co, Cr, and Ni ion concentrations were reduced by 64–79%, 67–69%, and 57–72%, respectively, in the plasma-nanocoated L605 samples as compared to uncoated L605 control samples. The plasma nanocoatings showed no sign of cytotoxicity from the test results according to ISO 10993-05 and 10993-12. Seven-day cell culture demonstrated that, in comparison with the uncoated L605 control surfaces, the plasma nanocoating surfaces showed 62 ± 7.3% decrease in porcine coronary artery smooth muscle cells (PCASMCs) density and had comparable density of porcine coronary artery endothelial cells (PCAECs). These results suggest that TMS plasma nanocoatings with NH3/O2 plasma post-treatment possess the desired biocompatibility for stent applications and support the hypothesis that nanocoated stents could be very effective for in-stent restenosis prevention.

Arsenic Trioxide-Coated Stent Is an Endothelium-Friendly Drug Eluting Stent.

An ideal vascular stent would both inhibit in-stent restenosis (ISR) and promote rapid re-endothelialization. In the current study, the performance of arsenic trioxide (ATO)-drug eluting stent (AES) is compared with the bare metal stent, poly-lactic-co-glycolic acid-coating metal stent, and rapamycin-drug eluting stent (RES). In vivo AES is shown to prevent neointimal hyperplasia more efficiently than the others when implanted into the carotid arteries of rabbits. Moreover, AES promotes endothelial cells proliferation and re-endothelialization more quickly than RES. In vitro ATO exposure significantly increases the viability, proliferation, adhesion, and spreading of primary porcine coronary artery endothelial cells (PCAECs), which are critical for endothelialization. However, ATO exposure reduces the viability of porcine coronary artery smooth muscle cells (PCASMCs). The evaluation of mitochondrial morphology, membrane potential, and function demonstrates that ATO at 2 µmol L-1 causes enlargement of the mitochondrion, enhancement of mitochondrial membrane potential, and adenosine triphosphate (ATP) production in PCAECs but not in PCASMCs. Thus, both in vivo and in vitro studies demonstrate that AES is an effective strategy for rapid re-endothelialization and inhibition of ISR.

The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over control conditions. Lastly, the role of ERK1/2 was determined by applying the MEK inhibitor, PD98059, in isometric tension studies and detecting phospho-ERK1/2 in immunoblotting. PD98059 potentiated G-1-induced relaxation response, but blocked G-1-enhanced ET-1-induced contraction. By western blot, G-1 treatment decreased phospho-ERK1/2, however, in the presence of the adenylyl cyclase inhibitor, SQ22536, G-1 significantly increased ERK1/2 phosphorylation in PCASMC. These data demonstrate that activation of GPER induces relaxation via cAMP as well as contraction via a mechanism involving transactivation of EGFR and the phosphorylation of ERK1/2 in porcine coronary arteries.

Kaempferol enhances endothelium‐dependent relaxation in the porcine coronary artery through activation of large‐conductance Ca2+‐activated K+ channels

Kaempferol, a plant flavonoid present in normal human diet, can modulate vasomotor tone. The present study aimed to elucidate the signalling pathway through which this flavonoid enhanced relaxation of vascular smooth muscle. The effect of kaempferol on the relaxation of porcine coronary arteries to endothelium-dependent (bradykinin) and -independent (sodium nitroprusside) relaxing agents was studied in an in vitro organ chamber setup. The whole-cell patch-clamp technique was used to determine the effect of kaempferol on potassium channels in porcine coronary artery smooth muscle cells (PCASMCs). At a concentration without direct effect on vascular tone, kaempferol (3 × 10(-6) M) enhanced relaxations produced by bradykinin and sodium nitroprusside. The potentiation by kaempferol of the bradykinin-induced relaxation was not affected by N(ω)-nitro-L-arginine methyl ester, an inhibitor of NO synthase (10(-4) M) or TRAM-34 plus UCL 1684, inhibitors of intermediate- and small-conductance calcium-activated potassium channels, respectively (10(-6) M each), but was abolished by tetraethylammonium chloride, a non-selective inhibitor of calcium-activated potassium channels (10(-3) M), and iberiotoxin, a selective inhibitor of large-conductance calcium-activated potassium channel (KCa 1.1; 10(-7) M). Iberiotoxin also inhibited the potentiation by kaempferol of sodium nitroprusside-induced relaxations. Kaempferol stimulated an outward-rectifying current in PCASMCs, which was abolished by iberiotoxin. The present results suggest that, in smooth muscle cells of the porcine coronary artery, kaempferol enhanced relaxations caused by endothelium-derived and exogenous NO as well as those due to endothelium-dependent hyperpolarization. This vascular effect of kaempferol involved the activation of KCa 1.1 channels.

Decreased Expression of Vitamin D Receptors in Neointimal Lesions following Coronary Artery Angioplasty in Atherosclerotic Swine

BackgroundInflammatory cytokines, such as TNF-α, play a key role in the pathogenesis of occlusive vascular diseases. Activation of vitamin D receptors (VDR) elicits both growth-inhibitory and anti-inflammatory effects. Here, we investigated the expression of TNF-α and VDR in post-angioplasty coronary artery neointimal lesions of hypercholesterolemic swine and examined the effect of vitamin D deficiency on the development of coronary restenosis. We also examined the effect of calcitriol on cell proliferation and effect of TNF-α on VDR activity and expression in porcine coronary artery smooth muscle cells (PCASMCs) in-vitro.Methodology/Principal FindingsExpression of VDR and TNF-α and the effect of vitamin D deficiency in post-angioplasty coronary arteries were analyzed by immunohistochemistry and histomorphometry. Cell proliferation was examined by thymidine and BrdU incorporation assays in cultured PCASMCs. Effect of TNF-α-stimulation on the activity and expression of VDR was analyzed by luciferase assay, immunoblotting and immunocytochemistry. In-vivo, morphometric analysis of the tissues revealed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased expression of TNF-α in neointimal lesions. Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media. Indeed, post-balloon angioplasty restenosis was significantly higher in vitamin D-deficient hypercholesterolemic swine compared to vitamin D-sufficient group. In-vitro, calcitriol inhibited both serum- and PDGF-BB-induced proliferation in PCASMCs and TNF-α-stimulation significantly decreased the expression and activity of VDR in PCASMCs.Conclusions/SignificanceThese data suggest that significant downregulation of VDR in proliferating smooth muscle cells in neointimal lesions could be due to atherogenic cytokines, including TNF-α. Vitamin D deficiency potentiates the development of coronary restenosis. Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR. This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

Suppressor of cytokine signaling-3 and intimal hyperplasia in porcine coronary arteries following coronary intervention

Aims The growth and differentiation of cells is regulated by cytokines by binding to cell-surface receptors and activating intracellular signal transduction cascade. Suppressor of cytokine signaling (SOCS)-3 is a negative regulator of cytokines. In this study we examined the expression of SOCS-3 in porcine coronary artery smooth muscle cells (PCASMCs) in vitro and in proliferating smooth muscle cells of neointimal lesions after coronary artery intervention in a swine model. Methods and results PCASMCs were cultured and stimulated with TNF-α and/or IGF-1 individually or in combination. Protein expression of SOCS-3 was examined using Western blot. For in vivo studies, six female Yucatan miniswine were fed with special high cholesterol diet for 8 months. At 4 months of high cholesterol diet, animals underwent coronary balloon angioplasty. At the end of 8 months animals were euthanized, coronary arteries were isolated and morphological and histological studies were performed. Western blot data revealed significantly high SOCS-3 expression in PCASMCs in the presence of either TNF-α or IGF-1 (5–6 fold) alone. However, in the presence of both TNF-α and IGF-1 the SOCS-3 expression was significantly decreased (4–5 fold). Results from morphological studies including, H&E and Masson's trichrome stain showed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased proliferating cell nuclear antigen (PCNA) in neointimal lesion. Interestingly, there was significantly decreased expression of SOCS-3 in smooth muscle cells of neointima as compared to control. Conclusions These data suggest that SOCS-3 expression is decreased in proliferating smooth muscle cells of neointimal lesions. This leads to uncontrolled growth of vascular smooth muscle cells in injured arteries leading to restenosis. Therefore, local delivery of SOCS-3 gene at the site of injury after coronary artery intervention could regulate the proliferation of vascular smooth muscle cells and help in preventing the neointimal hyperplasia and restenosis.

Long-term viability of coronary artery smooth muscle cells on poly(L-lactide-co-epsilon-caprolactone) nanofibrous scaffold indicates its potential for blood vessel tissue engineering.

Biodegradable polymer nanofibres have been extensively studied as cell culture scaffolds in tissue engineering. However, long-term in vitro studies of cell-nanofibre interactions were rarely reported and successful organ regeneration using tissue engineering techniques may take months (e.g. blood vessel tissue engineering). Understanding the long-term interaction between cells and nanofibrous scaffolds (NFS) is crucial in material selection, design and processing of the tissue engineering scaffolds. In this study, poly(L-lactide-co-epsilon-caprolactone) [P(LLA-CL)] (70:30) copolymer NFS were produced by electrospinning. Porcine coronary artery smooth muscle cells (PCASMCs) were seeded and cultured on the scaffold to evaluate cell-nanofibre interactions for up to 105 days. A favourable interaction between this scaffold and PCASMCs was demonstrated by cell viability assay, scanning electron microscopy, histological staining and extracellular matrix (ECM) secretion. Degradation behaviours of the scaffolds with or without PCASMC culture were determined by mechanical testing and gel permeation chromatography (GPC). The results showed that the PCASMCs attached and proliferated well on the P(LLA-CL) NFS. Large amount of ECM protein secretion was observed after 50 days of culture. Multilayers of aligned oriented PCASMCs were formed on the scaffold after two months of in vitro culture. In the degradation study, the PCASMCs were not shown to significantly increase the degradation rate of the scaffolds for up to 105 days of culture. The in vitro degradation time of the scaffold could be as long as eight months by extrapolating the results from GPC. These observations further supported the potential use of the P(LLA-CL) nanofibre in blood vessel tissue engineering.

Effects of Dopamine on ATP-Sensitive Potassium Channels in Porcine Coronary Artery Smooth-Muscle Cells

Dopamine is reported to be a coronary vasodilator; however, the exact mechanism of dopamine action in the coronary circulation remains unclear. In this study, we hypothesized that dopamine-induced activation of coronary ATP-sensitive potassium (KATP) channels may be associated with coronary vasodilation. We therefore investigated the direct effects of dopamine on coronary KATP-channel activity. We used patch-clamp configurations to investigate the effects of dopamine on coronary KATP-channel activity. Application of dopamine (10 to 10 M) to the bath solution during cell-attached recordings induced a concentration-dependent increase in KATP-channel activity. In contrast, dopamine failed to activate KATP channels in inside-out patches. Dopamine-induced coronary KATP-channel currents in cell-attached patches were inhibited by pretreatment with the selective D1-like antagonist, Sch-23390, but they were not influenced by the selective D2-like antagonist, domperidone, or the beta-adrenergic receptor antagonist, propranolol. The selective D1-like agonist, SKF-38393, and the adenylyl cyclase activator, forskolin, mimicked the dopamine effects on coronary KATP channels. Furthermore, pretreatment with an inhibitor of protein kinase A, Rp-cAMPS, abolished the dopamine-induced KATP-channel activation. This study demonstrates that dopamine activates coronary KATP channels via signal transduction involving the D1-like dopaminergic receptor-protein kinase A-signaling pathway.

Targeting Connexin 43 Prevents Platelet-Derived Growth Factor-BB–Induced Phenotypic Change in Porcine Coronary Artery Smooth Muscle Cells

We previously reported that reducing the expression of the gap junction protein connexin (Cx)43 in mice restricts intimal thickening formation after acute vascular injury by limiting the inflammatory response and the proliferation and migration of smooth muscle cells (SMCs) toward the damaged site. SMC populations isolated from porcine coronary artery exhibit distinct phenotypes: spindle-shaped (S) and rhomboid (R). S-SMCs are predominant in the normal media, whereas R-SMCs are recovered in higher proportion from stent-induced intimal thickening, suggesting that they participate in the restenotic process. Here, we further investigate the relationship between connexin expression and SMC phenotypes using porcine coronary artery SMCs. Cx40 was highly expressed in normal media of porcine coronary artery in vivo, whereas Cx43 was barely detectable. In contrast, Cx40 was downregulated and Cx43 was markedly upregulated in stent-induced intimal thickening. In vitro, S-SMCs expressed Cx40 and Cx43. In R-SMCs, Cx43 expression was increased and Cx40 was absent. We confirmed that S-SMCs treated with platelet-derived growth factor-BB acquire an R phenotype. This was accompanied by an upregulation of Cx43 and a loss of Cx40. Importantly, platelet-derived growth factor-BB-induced S-to-R phenotypic change was prevented by a reduction of Cx43 expression with antisense, ie, S-SMCs retained their typical elongated appearance and the expression of alpha-smooth muscle actin, a well-known SMC differentiation marker, whereas the expression of S100A4, a typical marker of R-SMCs, was prevented. In conclusion, limiting Cx43 expression in S-SMCs prevents platelet-derived growth factor-BB-induced S-to-R modulation. This suggests that Cx43 may be an additional target for local delivery strategies aimed at reducing restenosis.

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca(2+)-activated-K(+) (BK(Ca)) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BK(Ca) channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca(2+) concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca(2+) abolished STOCs activity. Ca(2+) ionophore A23187 (10 micromol/L) increased STOCs activity significantly. Verapamil (20 micromol/L) and CdCl(2) (200 micromol/L), two kinds of organic L-type voltage-dependent Ca(2+) channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 micromol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) by 2APB (40 micromol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BK(Ca) channels. Extracellular Ca(2+) is essential for STOCs activity, while Ca(2+) entry through L-VDCCs has little effect on STOCs. Intracellular Ca(2+) release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.

Involvement of p38-mitogen-activated protein kinase in adenosine receptor-mediated relaxation of coronary artery

The purpose of this study was to explore the involvement of adenosine receptor(s) in porcine coronary artery (PCA) relaxation and to define the role of MAPK signaling pathways. Isometric tensions were recorded in denuded PCA rings. 5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine receptor agonist, induced a concentration-dependent relaxation (EC(50) = 16.8 nM) of PGF(2alpha) (10 microM)-preconstricted arterial rings. NECA-induced relaxation was completely blocked by 0.1 microM SCH-58261 (A(2A) antagonist) at lower doses (1-40 nM) but not at higher doses (80-1,000 nM). MRS-1706 (1 microM, A(2B) antagonist) was able to shift the NECA concentration-response curve to the right. CGS-21680 (selective A(2A) agonist) induced responses similarly to NECA, whereas N(6)-cyclopentyladenosine (A(1) agonist) and Cl-IB-MECA (A(3) agonist) did not. Furthermore, the effect of NECA was attenuated by the addition of SB-203580 (10 microM, p38 MAPK inhibitor) but not by PD-98059 (10 microM, MEK inhibitor). Interestingly, SB-203580 had no effect on CGS-21680-induced relaxation. Western blot analysis demonstrated that PGF(2alpha) and adenosine agonists stimulated p38 MAPK at a concentration of 40 nM in PCA smooth muscle cells. MRS-1706 (1 microM) significantly reduced NECA-induced p38 MAPK phosphorylation. Addition of NECA and SB-203580 alone or in combination inhibited PGF(2alpha)-induced p38 MAPK. Western blot data were further confirmed by p38 MAPK activity measurement using activating transcription factor-2 assay. Our results suggest that the adenosine receptor subtype involved in causing relaxation of porcine coronary smooth muscle is mainly A(2A) subtype, although A(2B) also may play a role, possibly through p38 MAPK pathway.

Differences in positional esterification of 14,15-epoxyeicosatrienoic acid in phosphatidylcholine of porcine coronary artery endothelial and smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are readily incorporated into phospholipids of smooth muscle cells (SMC) and endothelial cells (EC). Incorporation of EETs into intact porcine coronary arteries potentiates EC-dependent relaxation, but not vasorelaxation induced by agents that act solely on SMC. To explore the potential mechanisms responsible for this difference, porcine coronary artery SMC and EC preloaded with [ 3 H ]14,15-EET were treated with calcium ionophore A23187. Although the amount of EET incorporated into EC and SMC was similar, A23187 stimulated a five-fold increase in release of radioactivity from EC, but only a 21% increase in release from SMC. Thin layer chromatography (TLC) examination of cell lipids demonstrated that >70% of the incorporated radioactivity was present in phosphatidylcholine (PC) in both SMC and EC. After treatment of EC PC with PLA 2, TLC analysis indicated that ≅75% of radioactivity was present as free EET, and 25% of radioactivity was present as lyso-PC. Therefore, most of the 14,15-EET was esterified into the sn-2 position of PC in EC. However, in SMC, ≅70% of radioactivity was present as lyso-PC after PLA 2 treatment, indicating that the EET was predominately esterified into the sn-1 position. In contrast, all of the 14,15-EET was esterified into the sn-2 position of PI in both EC and SMC. These results suggest that the preferential incorporation of 14,15-EET into the sn-1 position of PC in SMC may help to explain the greater retention of the compound in SMC, while incorporation into the sn-2 position of PC in EC may facilitate agonist-induced 14,15-EET release and potentiation of EC-dependent porcine coronary artery relaxation.

Control of Smooth Muscle Cell Proliferation and Phenotype by Integrin Signaling through Focal Adhesion Kinase

Extracellular matrix proteins such as fibronectin (FN) and laminin (LM) are known to help control the growth and phenotype of vascular smooth muscle cells (VSMCs). Here we have analyzed the relationship between growth factor and integrin signaling pathways in VSMCs. Culturing porcine coronary artery smooth muscle cells (PCASMCs) on FN and LM leads to distinct effects on cell proliferation and contractile protein expression. PCASMCs cultured on FN proliferate at a higher rate than cells cultured on LM, regardless of the growth factor used to support proliferation. Moreover, cells cultured on LM show higher levels of expression of smooth muscle myosin heavy chain (a marker of smooth muscle cell differentiation) than cells cultured on FN. In contrast to the effects on proliferation and contractile protein expression, both FN and LM supported cell migration in response to PDGF. Also, both FN and LM supported activation of ERK1 and ERK2 in response to PDGF and bFGF. However, FN and LM did show a difference in their ability to support signaling through the focal adhesion kinase (FAK). PCASMCs cultured on FN show robust activation of FAK in response to either PDGF or bFGF, however, cells cultured on LM show little-to-no activation of FAK in response to the growth factors. The results show that integrin signaling pathways have a profound effect on VSMC proliferation and phenotype, and that FAK is an important intermediate in these signaling pathways. The implications of our findings on the mechanisms controlling VSMC proliferation and phenotype in pathological states such as atherosclerosis and restenosis are discussed.