Rat Aortic Cells Research Articles

The 14q32 MicroRNA-487b Targets the Antiapoptotic Insulin Receptor Substrate 1 in Hypertension-Induced Remodeling of the Aorta

To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease. MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology. Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells. MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival. Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.

MicroRNA-218 regulation in phenotype transformation, proliferation and migration of rat aortic vascular smooth muscle cells

Objective To observe microRNA (miR)-218 regulation in phenotype transformation,proliferation and migration of rat aortic vascnlar smooth muscle cells (VSMCs) and explore the mechanism of miR-218 affecting the biological behaviors of VSMCs.Methods Rat aortic VSMCs were cuhured in 20％ fatal bovine serun(FBS),and transfected with miR-218 mimics,miR-218 inhibitor and miR-nc for negative control.The expression of miR-218 and α-actin and Calponin-1 in VSMCs was detected.In VSMCs cultured in 10％ FBS,VSMCs proliferation and migration in both miR-218 mimics and miR-nc groups were evaluated,and results were compared to miR-218 inhibitor group.Results In VSMCs cultured in 20％ FBS for 3 days and 7 days,the expression levels of miR-218 were down-regulated more than 90％ (P ＜0.01),and those of α-actin and Calponin-1 were down-regulated significantly simultaneously.There was no significant down-regulation of α-actin and Calponin-1 in miR-218 mimics group as compared with miR-nc group.In VSMCs cultured in by 10％ FBS for 2-3 days,cell proliferatiou and migration were significantly inhibited by 28％ in miR-218 mimics group (P ＜ 0.05),and they were significantly enhanced by 20％ (P ＜ 0.05) and 53％ (P ＜ 0.01) in miR-218 inhibitor group respectively.In VSMCs cultured in 10％ FBS 2-4 days,the expression of cyclin dependent kinase 6 (CDK6),the potential target gene of miR-218,was significantly down-regulated in miR-218 mimics group.Conclusion Our results suggest that up-regulation of miR-218 inhibits phenotype transformation,proliferation and migration of rat VSMCs,which may be achieved partly through targeting the CDK6 expression. Key words: microRNA-218; Vascular smooth muscle cells; Phenotype transformaton; Proliferation

Cytotoxicity effects of tert‐butyl hydroperoxide‐induced p53 ‐ mediated cell‐cycle arrest, apoptosis and aging in rat aortic endotheial cells and aorta: inhibition by Angelica sinesis extracts

Endotheial dysfunction is one of the underlying causes for vascular diseases. tert‐butyl hydroperoxide (t‐BHP), a short‐chain lipid hydroperoxide analog, has been referred to cause cellular senescence in different systems. This study aims to investigate whether the potent antioxidant Angelica sinesis (AS) extracts, often used in traditional Chinese medicine, could protect against the t‐BHP induced toxicity in rat aortic endothelium. Primary cultured endothelial cells (ECs) were treated with vehicle or t‐BHP. Exposure of ECs to lower doses t‐BHP caused cellular senescence and apoptosis, and higher doses initiated cell‐cycle arrest, telomerase acitivty diminished, and cellular inflammation. However, these adverse effects could be antagonized by AS extracts pretreatment via p53‐dependent mitochondrial signaling pathways. In terms of in vivo study, male rats at 6‐or 24‐week of age were administrated with vehicle or t‐BHP. Significanty increased TUNEL‐ and SA‐beta‐gal‐positive cells, provoked caspase‐3 activities, and inhibition of telomerase activities in aorta from t‐BHP‐treated groups. These results suggest t‐BHP impaired vascular cell survival at least partially by activating the p53‐mediated signaling pathway and AS can counteract these actions of t‐BHP to protect the aortic endotheium from t‐BHP induced dysfunction.(Supported by research grants from NSC992313B343001‐MY3, Taiwan)

Promoting effects and mechanisms of prostaglandin E1 on proliferation and migration of endothelial cells

To investigate the mechanism(s) that prostaglandin E1 (PGE1) promotes human umbilical vein endothelial cell (HUVEC)proliferation and migration. Western blot, enzyme linked immunosorbent assay, cell proliferation and cell migration tests, and tube formation were used for analyzing the roles and mechanisms of PGE1 on HUVEC; Western blot was used for analyzing the effects of PGE1 on the expression of vascular endothelial growth factor (VEGF) in rat aortic vascular smooth muscle cells (VSMC). PGE1 significantly increased VEGF expression of HUVEC in time and a dose dependent manner with concomitantly increased HUVEC proliferation; treatment of HUVEC with Bevacizumab apparently suppressed PGE1-stimulated VEGF expression, which led to decreased tube formation, reduced cell proliferation and migration by 41% and 38%, respectively, compared with PGE1 treatment alone; PGE1 time-dependently induced both phosphorylation of ERK and p38 in HUVEC, whereas ERK inhibitor, PD98059, or p38 inhibitor, SB203580, blocked PGE1-induced VEGF expression of HUVEC, resulting in dramatically suppression of HUVEC proliferation and migration compared with PGE1 treatment alone (60% and 55% by PD98059, 62% and 51% by SB203580, respectively); in addition, cAMP-dependent protein kinase A inhibitor, H89 or Rp-cAMP blocked PGE1-induced VEGF expression in VSMC. PGE1 promotion of proliferation, migration and tube formation of HUVEC via VEGF further provides a novel theoretical support in efficacy of PGE1 treatment of critical limb ischemia and other related diseases.

Antiangiogenesis and antioxidant activity of ethanol extracts of Pithecellobium jiringa

BackgroundAngiogenesis plays a critical role in embryonic development and various physiological processes. However, excessive angiogenesis is associated with several pathological conditions including cancer. Pithecellobium jiringa (Jack) Prain is a traditional medicinal plant from the family Leguminosae. It is native to the Southeast Asia, where it has been used traditionally for treatment of various ailments such as hypertension and diabetes. The present work is aimed to study antioxidant and antiangiogenesis activities of P. jiringa ethanol extracts.MethodsP. jiringa fruit rinds were extracted with ethanol and 50% ethanol. The antioxidant property was analysed using, 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging assay. Phytochemical analysis was performed using thin layer chromatography and colorimetric methods. Then, cell growth inhibition was studied against a panel of human cell lines by MTT test. In vitro inhibition of angiogenesis was studied by the following assays: isolated rat aortic rings cell viability, colony formation, endothelial cell migration, endothelial tube formation on matrigel, and expression of vascular endothelial growth factor by endothelial cells. In vivo antiangiogenesis effect was studied by utilising fertilised chick embryos assay. The results were statistically analysed by analysis of variance.ResultsEthanolic and 50% hydro-ethanolic extracts showed relatively high concentration of total phenolics associated with potent antioxidant activity. The rat aortic rings study conducted showed potent inhibition of the microvessels outgrowth with IC50s 5.27 ± 0.81 μg/ml (ethanolic) and 4.45 ± 0.63 μg/ml (50% hydro-ethanolic). Both extracts arrested the growth of human endothelial cells via down-regulation of VEGF expression, leading to inhibition of other angiogenesis cascades including migration of endothelial cells, and formation of capillary network on matrigel matrix. The extracts also inhibited the neovascularisation of chick embryo chorioallantoic membrane.ConclusionsP. jiringa extracts inhibit angiogenesis by blocking the VEGF expression thus inhibiting endothelial cells proliferation, migration and differentiation most likely due to presence of the antioxidant phenolics.

Abstract 18326: p38MAPK is a Novel Negative Regulator for Smooth Muscle Differentiation

p38MAPK belongs to members of the MAPK family which can be activated by various environmental stressors and inflammatory cytokines. While more and more studies have defined the critical roles of p38MAPK in different biological processes involving cell growth, differentiation and apoptosis, its actual contribution to smooth muscle cell (SMC) phenotypic modulation is largely unexplored. Using the mouse complete carotid artery ligation model, we found both total p38 and phosphorylated p38 to be predominantly located in the neointima of the vessel wall where SMCs undergo hyper-proliferation and SMC differentiation is compromised, suggesting a negative regulatory role of p38MAPK in SMC differentiation. To test this hypothesis, we applied siRNA studies in primary human coronary artery SMC (HCASM) cultures and examined SMC contractile gene expression. Depletion of p38MAPK evoked a dramatic increase in SMC contractile gene expression. The stimulatory effect of SMC contractile gene expression upon knockdown of p38MAPK was reproduced in a variety of SMC culture models including human aortic SMCs, rat aortic SMCs, PAC1 cells and primary human myofibroblasts. Consistent with the siRNA studies, cotransfecting p38 with its upstream kinase MKK3 or MMK6 abolished promoter activity of several SMC-restricted genes, while little effect was found with the dominant negative mutants of p38. Furthermore, adenovirus carrying the constitutively active MKK6 completely abrogated TGF-beta1 induced SMC differentiation as assessed by Western blotting and immunfluorescence mirocsopy, indicating that phosphorylated p38 is required for the repressive effect of p38MAPK in SMC differentiation. Interestingly, in HCASMs, we found myocardin-related transcription factor A (MRTFA) was mainly located in the nucleus when the endogenous p38MAPK was knocked down; conversely, over-expression of constitutively active MKK6 was found to shift the majority of MRTFA to the cytoplasm of HCASMs, suggesting that phosphorylated p38MAPK is able to block nuclear import of MRTFA in SMCs. Taken together, our studies revealed an unexpected role for p38MAPK in the negative regulation of SMC differentiation.

Diethylstilbestrol induces arrest of rat vascular smooth muscle cell cycle progression through downregulation of cyclin D1 and cyclin E

Cardiovascular disease is associated with a multitude of pathophysiologic conditions, including vascular smooth muscle cell (VSMC) proliferation in response to vessel injury. Diethylstilbestrol (DES) was previously prescribed for at-risk pregnancies to prevent abortion, miscarriage, and premature labor. Our aim in this study was to elucidate the effects and molecular mechanism of DES on proliferation and cell cycle progression of platelet-derived growth factor (PDGF)-BB-stimulated rat aortic VSMCs. Treating the cells with DES (1-7 μM) dramatically inhibited cell proliferation in a dose-dependent manner without any cytotoxic effects. In addition, DES blocked cell cycle progression from PDGF-BB-stimulated cells, which we found was related to down-regulation of the cell cycle regulatory factors, cyclin D1, and cyclin E. Our data demonstrate that DES inhibits rat aortic VSMC proliferation and cell cycle progression through regulation of cell cycle-related proteins. Therefore, our observations may explain, in part, the mechanistic basis underlying the therapeutic effects of DES in cardiovascular disease.

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Nonsteroidal Anti-inflammatory Drugs Inhibit Vascular Smooth Muscle Cell Proliferation by Enabling the Ca2+-dependent Inactivation of Calcium Release-activated Calcium/Orai Channels Normally Prevented by Mitochondria

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca(2+) homeostasis and Ca(2+)-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca(2+) entry (SOCE) and voltage-operated Ca(2+) entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca(2+) released-activated Ca(2+) (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca(2+)-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca(2+) uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca(2+) uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca(2+) uptake, suggesting that they favor the Ca(2+)-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca(2+) buffering conditions but not in strong Ca(2+) buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca(2+)-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca(2+).

The cAMP responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down regulation of RhoA activity

This study reports a new mechanism of cAMP mediated relaxation of Ca2+sensitized force, in smooth muscle (SM) through Epac, a GTP exchange factor for the small GTPase Rap1 which results in suppression of RhoA activity. We find that Epac selective cAMP analogue, 8-pCPT-2′-O-Me-cAMP (007), significantly reduced agonist-induced contractile force, in both intact and permeabilized vascular, gut and airway SM. Responses to 007 were independent of PKA and PKG. Activation of Epac resulted in increased Rap1·GTP accompanied by a significant decrease in RhoA activity and reductions in phosphorylation of RLC20 and MLCP. Transcriptional regulation of SM α-actin and SM22, known to be regulated by RhoA, was also significantly decreased by activation of Epac. Forskolin, the phosphodiesterase inhibitor IBMX and isoproterenol significantly increased Rap1·GTP in rat aortic SM cells. Over-expression of wild-type Epac but not dominant negative Epac1R279E increased Rap1 activation after 007 stimulation. LPA-induced activation of RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts. All together, our findings show a novel signaling mechanism whereby activation of Epac via cAMP results in PKA independent, Rap1 dependent Ca2+ desensitization of force in SM.

Antiangiogenic and antitumor activity of LP-261, a novel oral tubulin binding agent, alone and in combination with bevacizumab.

LP-261 is a novel tubulin targeting anticancer agent that binds at the colchicine site on tubulin, inducing G2/M arrest. Screening in the NCI60 cancer cell lines resulted in a mean GI50 of approximately 100 nM. Here, we report the results of testing in multiple mouse xenograft models and angiogenesis assays, along with bioavailability studies. To determine the antiangiogenic activity of LP-261, both in vitro and ex vivo experiments were performed. Human umbilical vein endothelial cells (HUVECs) were incubated with LP-261 at 50 nM to 10 μM. LP-261 was also tested in a rat aortic ring assay, from 20 nM to 10 μM. Multiple mouse xenograft studies were performed to assess in vivo antitumor activity. LP-261 was tested as a single agent in colon adenocarcinoma (SW620) and prostate cancer (LNCaP and PC3) xenografts, evaluating several different dosing schedules. LP-261 was also used in combination with bevacizumab in the SW620 xenograft model. LP-261 also exhibited high oral bioavailability and apparent lack of efflux by intestinal transporters such as ABCB1. LP-261 is a very potent inhibitor of angiogenesis, preventing microvessel outgrowth in the rat aortic ring assay and HUVEC cell proliferation at nanomolar concentrations. Complete inhibition of tumor growth was achieved in the PC3 xenograft model and shown to be schedule dependent. Excellent inhibition of tumor growth in the SW620 model was observed, comparable with paclitaxel. Combining oral, low dose LP-261 with bevacizumab led to significantly improved tumor inhibition. Oral LP-261 is very effective at inhibiting tumor growth in multiple mouse xenograft models and is well tolerated.

INHIBITORY EFFECT OF D3 DOPAMINE RECEPTORS ON INSULIN RECEPTOR EXPRESSION AND FUNCTION IN VASCULAR SMOOTH MUSCLE CELLS: PP.13.472

Vascular smooth muscle cell (VSMC) proliferation, which is central to development of vascular diseases, including hypertension, is regulated by numerous hormones and humoral factors. Activation of the insulin receptor stimulates the VSMC proliferation while dopamine receptors, via D1 and D3 receptors, inhibit the stimulatory effects of norepinephrine on VSMC proliferation (Am J Physiol Heart Circ Physiol. 2008;294:H2761-8). We hypothesize that there is an interaction between D3 dopamine and insulin receptors in VSMCs; stimulation of D3 receptor inhibits insulin receptor expression and function. We found that stimulation of the D3 dopamine receptor inhibited the insulin receptor expression in a concentration- and time-dependent manner in rat aortic A10 cells, an embryonic thoracic aortic smooth muscle cells from normotensive Berlin-Druckrey IX. The inhibitory effect was evident at 10–7 M, noted as early as 2 hrs and maintained for at least 30rs. The inhibitory effect of D3 receptor on VSMC proliferation was blocked by PKA inhibitor (PKA inhibitor 14–22, 10–6 M), indicating that PKA is involved into the signal pathway. There is a physiological significance of the interaction between D3 receptor and insulin receptors because the D3 receptor agonist, PD128907, by itself had no effect on VSMC proliferation, but reduced the stimulatory effect of insulin VSMC proliferation, determined by MTT assay (control = 0.3±0.004, 10–7 M insulin = 0.6±0.007; 10–7 M PD128907 + 10–7 M insulin = 0.4±0.01, P < 0.05, n = 9). The inhibitory effect of D3 receptor on insulin receptor expression and function also occurred in primary cultured VSMCs from Wistar-Kyoto rats and spontaneously hypertensive rats (SHRs) (WKY: control = 0.3±0.01, 10–7 M insulin = 0.6±0.01; 10–7 M PD128907 + 10–7 M insulin = 0.4±0.01; SHR: control = 0.3±0.03, 10–7 M insulin = 0.7±0.02; 10–7 M PD128907 + 10–7 M insulin = 0.5±0.05, P < 0.05, n = 5). We suggest that the inhibitory effect of the D3 receptor on insulin receptor expression and function may be an alternative to reduce the effect of insulin in hypertension.

Effect of bradykinin on bradykinin-B2 receptor in rat aortic vascular smooth muscle cells and the involved signal transduction pathways

The aim of this paper is to study the effect of bradykinin (BK) on bradykinin-B2 receptor as well as the possible involved signal transduction pathways in cultured rat aortic vascular smooth muscle cells (VSMCs). Rat aortic VSMCs were cultured. Cells after 4–6 passages were used in the experiment. VSMCs were incubated with BK, BK + B2 receptor inhibitor (HOE-140), BK + MEK inhibitor (PD98059), BK + mitogen-activated protein kinase (MAPK) inhibitor (apigenin), BK + phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), and BK + Akt inhibitor to evaluate the expression of B2 receptor and phosphorylation of signaling molecules MAPK, Akt, and PI3K by Western blot. (1) BK markedly up-regulated the expression of B2 receptor in VSMC. (2) Apigenin, PD98059, Akt inhibitor, and LY294002 inhibited upregulation of B2 receptor induced by BK. (3) Signal transduction pathways of MAPK and PI3K were involved in the up-regulation of B2 receptor by BK mediation. Results suggest that bradykinin can up-regulate the expression of B2 receptor in VSMCs.

Modulation of Myocardin Function by the Ubiquitin E3 Ligase UBR5

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

Flt-1 expression influences apoptotic susceptibility of vascular smooth muscle cells through the NF-κB/IAP-1 pathway

Flt-1 is an fms-like tyrosine kinase receptor which binds to vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Ligand activation and blocking of flt-1 influence several vascular smooth muscle cell (SMC) functions, including apoptotic susceptibility. However, downstream signal transduction pathways by which flt-1 regulates SMC apoptosis have still to be investigated. Flt-1 expression and apoptosis in Wistar rat aortic intimal cells 15 days after ballooning were studied by immunohistochemistry, cytometry, cell sorting, western blotting, and PCR. Anti-flt1 blocking antibody effects were compared with those of anti-PlGF and anti-VEGF antibodies. Rat aortic intimal cells 15 days after injury exhibited increased flt-1 protein and mRNA and lower smooth muscle markers compared with normal media SMCs. Immunoreactivity for flt-1 protein was also observed in apoptotic intimal cells. Anti-flt-1 (EC(50) = 16.5 ng/mL) and anti-PlGF (EC(50) = 20.5 ng/mL) antibodies added to intimal cultures reduced serum-deprived apoptosis but not serum- and PDGF-BB-induced proliferation; the anti-VEGF antibody was ineffective. Sorted flt-1(+) cells were more clonogenic than flt-1(-) and whole intimal SMC populations. Increased nuclear factor-kappaB (NF-kappaB) and inhibitor of apoptosis protein-1 (IAP-1) and reduced bax levels associated with the anti-flt-1-induced increase of intimal SMC survival; the latter was prevented by NF-kappaB activity inhibitor and IAP-1 interfering RNA (RNAi). Blocking of NF-kappaB activity reduced IAP-1 expression and prevented IAP-1 RNAi effects. Increased flt-1 immunoreaction was also documented in human atheromatous lesions. Our results show that anti-flt-1 blocking reduces apoptosis through NF-kappaB and the downstream IAP-1 pathway. The close link between flt-1, PlGF, and apoptotic susceptibility of intimal SMCs suggests new potential strategies aimed at influencing post-injury arterial remodelling.

Two separate receptors for insulin and insulinlike growth factors on arterial smooth muscle cells.

Insulin and insulinlike growth factors (IGF) are related polypeptides that have similar biological activities. Both factors produce metabolic effects as well as growth effects. Most cells have separate receptors for IGF and insulin. In the present study we have demonstrated specific IGF receptors in cultured smooth muscle cells of rat aorta. The properties and specificity of these receptors were compared with those of the insulin receptor in the same cell system. The specific binding of both 125I-IGF and 125I-insulin binding in the order of potency: insulin proinsulin IGF. The potency of IGF in displacing 125I-insulin was rapid and reversible. Maximal 125I-IGF binding occurred at 20 degrees C with a specific binding of 10%. At this temperature specific 125I-insulin binding was 1.3% and occurred in biphase. The pH optimum for 125I-IGF binding was between pH 7 and 8. Both receptors show a high degree of specificity. IGF, insulin and proinsulin competed for 125I-IGF binding in the order of potency: IGF proinsulin insulin. The potency of insulin in displacing 125I-IGF was about 2000 times lower than that of IGF itself. In addition, IGF, insulin and proinsulin competed for 125I-insulin was about 450 times lower than that of insulin. These results indicate two separate binding sites for insulin and IGF on arterial smooth muscle cells.

Role of nitric oxide and cytoskeleton in regulation of contractile activity smooth muscle cells

The influence of modulation of cytoskeleton by colchicine, nocodazole and cytochalasine D on contractile reactions of smooth muscle cells caused by depolarization, phenylephrine end sodium nitroprusside has been investigated by the mechanographical method. It was found that the reduction in smooth muscle cells of rat aorta caused by fenilefrin more sensitive to sodium nitroprusside than the reduction induced hyperpotassium solution. We show that microtubules involved in relaxation, indirect nitric oxide of smooth muscle in hyperpotassium solution, whereas the efficiency of relaxing influence of nitric oxide in rat aorta smooth muscle in the action depends on phenylephrine and microfilaments and microtubules.

Proteomic screening of antioxidant effects exhibited by Radix Salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment

Aim of the study Still little is known about the cellular mechanisms that contribute to the attenuated proliferation of aortic smooth muscle cells under the influence of the oxidative stress factors such as homocysteine (Hcy). Thus, we aimed to evaluate whether Salvia miltiorrhiza Bunge (Labiatae), a Chinese medicinal herb widely used in folk medicine for therapy of variety of human cardiovascular disorders would modulate this Hcy promoted growth effect in model animal aortic cells system. Materials and methods The Salvia miltiorrhiza roots aqueous extract (SMAE) containing 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenyl lactic acid and salvianolic acid B, as confirmed by narrow-bore HPLC analyses with binary gradient elution was used in variable concentrations for the treatment of the rat aortic smooth muscle A10 cells under Hcy stimulation. Two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry was applied for the elucidation of protein changes characterizing the response of the rat A10 cells into the Hcy-induced oxidative stress. Results This study showed that a low dose (0.015 mg/mL) of the SMAE significantly inhibited growth (>60%, p < 0.05) of the Hcy stimulated rat A10 cells. In addition, concentration of intracellular reactive oxygen species (ROS) obviously decreased in the rat A10 cells after its incubation with SMAE in terms of catalase increasing activity. Next, marked down-regulation of protein kinase C beta-1 (PKC beta-1) and phosphorylated mitogen-activated protein kinase (p-MAPK) expression suggest that observed inhibitory effect of the polyphenol-rich SMAE on the Hcy-induced growth of rat A10 cells was realized via the PKC/p44/42 MAPK-dependent pathway. The intensity changes of 10 protein spots in response of the rat A10 cells to the Hcy-induced oxidative damage as alpha-4-tropomyosin, vimentin, F1F0-ATP synthase (beta subunit), glucose regulated protein 75 (GRP75), actin (fragment), prohibitin, capping protein, plakoglobin, endoplasmic reticulum protein (ERp29), and peptidylprolyl isomerase A (PPIase A), were detected with statistical significance ( p < 0.05). Meanwhile, it was showed that used here SMAE resist carbonylation of specific cytoskeleton and chaperone proteins as vimentin, alpha-4-tropomyosin and GRP75, respectively, leading to phenotype transformations in the rat A10 cells. Conclusion These data suggest that applied here SMAE exerts its protective effect through circulating ROS suppression and subsequent modulation of protein carbonylation in rat aortic smooth muscle A10 cells. Redox-proteomics protocol highlighted in this study may be applicable in facilitating the assessing potential novel molecular therapeutic targets to reduce cardiovascular risk related with elevated Hcy levels in various human populations and elucidating new mechanisms through which protein functions can be regulated by the redox status with the use of naturally occurring antioxidants.

Analysis of the mechanisms underlying the endothelium-dependent antivasoconstriction of puerarin in rat aorta

Puerarin, a major isoflavonoid compound from the Chinese herb, Ge-gen (Pueraria lobata), has effective treatment on myocardial and cerebral ischemia, glaucoma and sudden deafness in clinical setting in China. Our present work showed that puerarin (50, 150, 450 microM) concentration-dependently inhibited phenylephrine or KCl-induced contraction only in endothelium-intact rat aortic rings. In Ca(2+)-free solution, the antivasoconstriction of puerarin on phenylephrine was totally deprived. N (G)-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, indomethacin and the three K(+) channel blockers, glibenclamide, tetraethylammonium and Ba(2+) displayed significant inhibitory effects on the antivasoconstriction of puerarin. 8-bromo-cGMP significantly strengthened the action of puerarin. Puerarin (10-160 microM) concentration-dependently induced the NO production in the rat aortic cells. These findings suggested that the antivasoconstriction elicited by puerarin is endothelium-dependent. NO/NO-cGMP pathway, PGI(2) and the opening of K(+) channels sensitive to glibenclamide, tetraethylammonium, and Ba(2+), which might be triggered by the extracellular Ca(2+) influx in the endothelium, appear to contribute to the antivasoconstriction of puerarin.

Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

Despite the central physiological function of the myogenic response, the underlying signalling pathways and the identity of mechanosensors in vascular smooth muscle (VSM) are still elusive. In contrast to present thinking, we show that membrane stretch does not primarily gate mechanosensitive transient receptor potential (TRP) ion channels, but leads to agonist-independent activation of G(q/11)-coupled receptors, which subsequently signal to TRPC channels in a G protein- and phospholipase C-dependent manner. Mechanically activated receptors adopt an active conformation, allowing for productive G protein coupling and recruitment of beta-arrestin. Agonist-independent receptor activation by mechanical stimuli is blocked by specific antagonists and inverse agonists. Increasing the AT(1) angiotensin II receptor density in mechanically unresponsive rat aortic A7r5 cells resulted in mechanosensitivity. Myogenic tone of cerebral and renal arteries is profoundly diminished by the inverse angiotensin II AT(1) receptor agonist losartan independently of angiotensin II (AII) secretion. This inhibitory effect is enhanced in blood vessels of mice deficient in the regulator of G-protein signalling-2. These findings suggest that G(q/11)-coupled receptors function as sensors of membrane stretch in VSM cells.