Rat Aortic Vascular Smooth Muscle Cells Research Articles

Involvement of large conductance Ca2+-activated K+ channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation

The large conductance Ca(2+)-activated K(+) (BK(Ca)) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BK(Ca) channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BK(Ca) channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BK(Ca) protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BK(Ca) in SS-induced inhibition of proliferation was examined by BK(Ca) inhibition using a BK(Ca) specific blocker, iberiotoxin (IBTX), and by BK(Ca) transfection in BK(Ca) non-expressing CHO cells. The changes in [Ca(2+)](i) were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC(4)(3). We found that laminar SS inhibited VSMC proliferation and stimulated BK(Ca) channel expression. Furthermore, laminar SS induced an increase in [Ca(2+)](i) and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BK(Ca) channel activity on cell proliferation in response to SS was also confirmed in BK(Ca)-transfected CHO cells showing a decline in proliferation. Blocking BK(Ca) channel reversed its inhibitory effect, providing additional support for the involvement of BK(Ca) in SS-induced proliferation reduction. Our results suggest, for the first time, that BK(Ca) channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.

Proprotein convertase furin enhances survival and migration of vascular smooth muscle cells via processing of pro-nerve growth factor

Maturation of nerve growth factor (NGF) in neuronal cells requires endoproteolytic processing of the precursor protein proNGF to β-NGF by the proprotein convertase furin. Pro- and β-NGF elicit opposite biological functions by differential neurotrophin-receptor binding, leading to apoptosis via sortilin or survival via neurotrophic tyrosine kinase receptor type-1 (TrkA), respectively. The present study was done to investigate the impact of furin-dependent proNGF processing on vascular smooth muscle cell (VSMC) function. We found that β-NGF mRNA and protein expression was upregulated in platelet-derived growth factor-BB/transforming growth factor-β1-stimulated, proliferating rat aortic VSMCs. Although β-NGF itself did not affect VSMC proliferation, it promoted VSMC motility in an autocrine fashion via TrkA/Akt-dependent integrin inside-out signalling. The β-NGF-induced migration of VSMCs required proNGF processing by furin, which was co-regulated with NGF. Furin-inhibition increased proNGF and reduced β-NGF secretion, leading to apoptosis rather than migration. In line with our in vitro demonstration, we found co- and upregulation of NGF, its convertase furin and its high-affinity receptor TrkA in the neointima of balloon-injured rodent arteries. These results indicate that furin determines the balance between proNGF and β-NGF in proliferating VSMCs, thus impacting on VSMC survival and migration and is also important in neointima formation.

Identification of prh as a novel inhibitor of smooth muscle cell proliferation

In 30-50% of coronary artery disease patients receiving saphenous vein grafts or bare-metal stents reoccurrence of symptoms (restenosis) occurs due to intimal thickening involving vascular smooth muscle cell (VSMC) proliferation. By investigating regulators of VSMC proliferation we aim to reduce restenosis. The Proline-Rich Homeodomain protein (PRH/Hhex) is a negative regulator of cellular growth in hematopoietic and several other cell types and modulates Wnt/β-catenin signalling through more than one mechanism. We have shown Wnt/β-catenin signalling regulates VSMC proliferation and intimal thickening. This study aimed to determine whether PRH regulates VSMC proliferation and Wnt/β-catenin signalling. Western blotting revealed that induction of proliferation with foetal calf serum (FCS) significantly attenuated PRH protein levels in human saphenous vein VSMCs (0.39±0.04 vs.0.63±0.09, n=3, p<0.05 paired-Student's t-test). Similar results were observed when proliferation was induced with platelet-derived growth factor and basic fibroblast growth factor in both human saphenous vein and rat aortic VSMCs. MG132 revealed the loss of PRH protein was due to enhanced proteosomal degradation. Using AMAXA nucleofection of specific siRNA, PRH mRNA and protein levels were significantly reduced (20±12% and 60±10% n=3, p<0.05 Student's t-test, respectively), and proliferation was significantly enhanced (21±3 vs. 12±2% proliferation, n=3, p<0.05 paired-Student's t-test) in human VSMCs. We also observed that enhancing PRH protein levels using an adenovirus encoding PRH significantly reduced Wnt/β-catenin signalling to 68±2%, n=3, p<0.05, Student's t-test. In summary, we showed that PRH inhibited VSMC proliferation in part via retarding Wnt/β-catenin signalling. Design of novel treatments to increase PRH levels may be useful for retarding restenosis.

Arsenic Increases Pi-Mediated Vascular Calcification and Induces Premature Senescence in Vascular Smooth Muscle Cells

Several mechanisms have been proposed to explain the vascular toxicity of arsenic. Some of them are described in this work, such as stress-induced premature senescence (SIPS), dedifferentiation, and medial vascular calcification, and they all affect vascular smooth muscle cells (VSMC). Rat aortic VSMC were treated with 1-100 µM of either sodium arsenate (As(V)), sodium arsenite (As(III)), monomethylarsonic acid, or dimethylarsinic acid. None of the treatments induced VSMC calcification in the presence of 1mM inorganic phosphate (Pi), but 1 µM As(III) did increase calcification when induced with 2.5mM Pi. A lactate dehydrogenase assay revealed that this increase was explained by a rise in cytotoxicity due to simultaneous incubation with 1 µM As(III) and 2.5mM Pi. This calcification increase was also observed in the aortas of a vascular calcification model: 5/6 nephrectomized rats fed with a high Pi diet and treated with vitamin D(3). Several known mechanisms that might explain arsenic toxicity in our experimental model were discarded: apoptosis, oxidative stress, and inflammasome activation. Nevertheless, both senescence-associated β-galactosidase activity and p21 expression were increased by As(III), which reveals the induction of SIPS. As(III) also caused dedifferentiation of VSMC, as shown by the reduced expression of the VSMC markers SM22α and calponin. Senescence and gene expression were also observed in the aortas of healthy rats treated with 50 ppm As(V) in drinking water for 1 month. In conclusion, both premature senescence in aortic VSMC with phenotypic dedifferentiation and the increase of Pi-induced calcification are novel mechanisms of arsenic vasculotoxicity.

NADPH oxidase 4 is required for interleukin-1β-mediated activation of protein kinase Cδ and downstream activation of c-jun N-terminal kinase signaling in smooth muscle

Reactive oxygen species (ROS) are generated in the vascular wall upon stimulation by proinflammatory cytokines and are important mediators of diverse cellular responses that occur as a result of vascular injury. Members of the NADPH oxidase (NOX) family of proteins have been identified in vascular smooth muscle (VSM) cells as important sources of ROS. In this study, we tested the hypothesis that NOX4 is a proximal mediator of IL-1β-dependent activation of PKCδ and increases IL-1β-stimulated c-Jun kinase (JNK) signaling in primary rat aortic VSM cells. We found that stimulation of VSM cells with IL-1β increased PKCδ activity and intracellular ROS generation. SiRNA silencing of NOX4 but not NOX1 ablated the IL-1β-dependent increase in ROS production. Pharmacological inhibition of PKCδ activity as well as siRNA depletion of PKCδ or NOX4 blocked the IL-1β-dependent activation of JNK. Further studies showed that the IL-1β-dependent upregulation of inducible NO synthase expression was inhibited through JNK inhibition and NOX4 silencing. Taken together, these results indicate that IL-1β-dependent activation of PKCδ is modulated by NOX4-derived ROS. Our study positions PKCδ as an important redox-sensitive mediator of IL-1β-dependent signaling and downstream activation of inflammatory mediators in VSM cells.

Transforming growth factor-β increases vascular smooth muscle cell proliferation through the Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinases pathways

We have previously demonstrated that transforming growth factor-β (TGF-β) in the presence of elevated levels of Smad3, its primary signaling protein, stimulates rat vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The mechanism is partly through the nuclear exportation of phosphorylated cyclin-dependent kinase inhibitor p27. The objective of this study is to clarify the downstream pathways through which Smad3 produces its proliferative effect. Specifically, we evaluated the role of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in TGF-β-induced VSMC proliferation. Cultured rat aortic VSMCs were incubated with TGF-β at varying concentrations and times, and phosphorylated ERK was measured by Western blotting. Smad3 was enhanced in VSMCs using an adenovirus expressing Smad3 or inhibited with small interfering RNA (siRNA). For in vivo experiments, male Sprague-Dawley rats underwent carotid balloon injury, followed by intraluminal infection with an adenovirus expressing Smad3. Arteries were harvested at 3 days and subjected to immunohistochemistry for Smad3, phospho-ERK MAPK, and proliferating cell nuclear antigen. In cultured VSMCs, TGF-β induced activation and phosphorylation of ERK MAPK in a time-dependent and concentration-dependent manner. Overexpression of the signaling protein Smad3 enhanced TGF-β-induced activation of ERK MAPK, whereas inhibition of Smad3 with a siRNA blocked ERK MAPK phosphorylation in response to TGF-β. These data suggest that Smad3 acts as a signaling intermediate between TGF-β and ERK MAPK. Inhibition of ERK MAPK activation with PD98059 completely blocked the ability of TGF-β/Smad3 to stimulate VSMC proliferation, demonstrating the importance of ERK MAPK in this pathway. Immunoprecipitation of phospho-ERK MAPK and blotting with Smad3 revealed a physical association, suggesting that activation of ERK MAPK by Smad3 requires a direct interaction. In an in vivo rat carotid injury model, overexpression of Smad3 resulted in an increase in phosphorylated ERK MAPK as well as increased VSMC proliferation as measured by proliferating cell nuclear antigen. Our findings demonstrate a mechanism through which TGF-β stimulates VSMC proliferation. Although TGF-β has been traditionally identified as an inhibitor of proliferation, our data suggest that TGF-β enhances VSMC proliferation through a Smad3/ERK MAPK signaling pathway. These findings at least partly explain the mechanism by which TGF-β enhances intimal hyperplasia. Knowledge of this pathway provides potential novel targets that may be used to prevent restenosis.

Synergy Study of the Inhibitory Potential of Red Wine Polyphenols on Vascular Smooth Muscle Cell Proliferation

Vascular smooth muscle cell (VSMC) proliferation contributes to the development of atherosclerosis. Red wine consumption due to the polyphenol content has been reported to counteract atherosclerosis progression possibly through inhibition of VSMC proliferation, among other mechanisms. In this study we investigate the antiproliferative activity of four wine polyphenols: resveratrol, quercetin, ethyl gallate, and (+)-catechin in rat aortic VSMC. All four polyphenols inhibited serum-induced VSMC proliferation when applied as a single treatment. To further address a potential synergistic action of the investigated polyphenols, the antiproliferative effect of different combinations in equimolar, as well as equipotent ratios were quantified. The IC₅₀ values of single polyphenols regarding the inhibition of VSMC proliferation ranged from 49.58 µM to 86.06 µM. However, apparent inhibitory efficacy of each compound increased by a factor of 10.4 in the quadruple equipotent mixture, as calculated from the dose-reduction index. Thus, the effective IC₅₀ values of each of the four mixture constituents ranged from 4.76 µM to 8.27 µM. The calculated combination index (CI, where CI <, =, or > 1 indicate synergy, additivity, or antagonism, respectively) values of equimolar combinations of the polyphenols indeed indicated mainly synergy (CI ranging from 0.24 ± 0.01 to 1.51 ± 0.13). Optimized equipotent mixture showed enhanced synergy (CI ranging from 0.18 ± 0.04 to 1.36 ± 0.26). In conclusion, we show for the first time that four major polyphenols from wine synergistically inhibit VSMC proliferation.

Enhancement of the large conductance, Ca2+‐activated K+ (BK) channel current by cGMP‐dependent protein kinase requires multiple phosphorylation sites on the C‐terminal tail

Nitric oxide (NO)‐mediated activation of the cGMP‐dependent protein kinase (cGK) leads to relaxation of vascular smooth muscle (VSM) through a process involving phosphorylation of the BK channel. In VSM cells, this modification increases channel open probability, resulting in membrane hyperpolarization and closure of voltage‐gated Ca2+ channels. Although this paradigm is now generally accepted, the target residues of cGK‐mediated phosphorylation on the BK channel have thus far been inadequately characterized at the molecular level. We have scrutinized this BK channel phosphorylation by identifying several putative phosphorylation sites on the C‐terminus via electrophysiological and biochemical assays. Wild type (WT) and Ser to Ala mutated BK channels were expressed in cultured rat aortic VSM cells (A7r5) and studied using the cell‐attached patch clamp technique. The NO donor spermine NONOate (5 μM) and the membrane‐permeant analogue db‐cGMP (0.1 mM) enhanced WT BK channel activity, while several BK channel mutants had diminished responses to either stimulant. Exposure to the Ca2+ ionophore A23187 enhanced channel activity in both WT and mutant channels. These results demonstrate that BK current enhancement by NO/cGK‐mediated phosphorylation requires multiple sites on the C‐terminus tail, in an apparent all‐or‐none fashion. Supported by NSERC (APB) and a Kertland Family Scholarship (BDK).

Anti‐proliferative effect of rosiglitazone on angiotensin II‐induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.

Matrine Inhibits Disturbed Flow–Enhanced Migration via Downregulation of ERK1/2–MLCK Signaling Vascular Smooth Muscle Cells

To investigate the effects of matrine on the vascular smooth muscle cell (VSMC) migration modulated by disturbed flow and their underlying molecular mechanisms in vitro. Isolated rat aortic VSMCs were grown to confluence on 20- × 80-mm fibronectin-coated glass cover slides, and then, denuded zones were made at the position calculated to be the oscillating flow-reattachment zone and also in the downstream laminar flow region. VSMCs were treated with different doses of matrine (0, 10, 20, 30, and 40 mg/L), or PD98059 (30 μM), ML-7 (10 μM) combined with matrine (40 mg/L) for 30 minutes before and during the experiments. Then, the wounded monolayers were kept under static conditions or were subjected to laminar or disturbed flow for 21 hours or 10 hours. The VSMC migration was assessed by microscopic images. The extracellular signal-regulated kinase 1/2 (ERK1/2) and myosin light chain kinase (MLCK) proteins were determined by Western blot. Disturbed flow significantly increased phosphorylation of ERK1/2. Selective inhibition of ERK1/2 phosphorylation by inhibitor PD98059 and matrine significantly suppressed VSMC migration under disturbed flow. Disturbed flow significantly enhanced phosphorylation of MLCK, whereas both matrine and PD98059 inhibited the phosphorylation of MLCK under disturbed flow. The complete inhibition of MLCK phosphorylation using the selective MLCK inhibitor ML-7 significantly inhibited VSMC migration under disturbed flow. Matrine inhibits VSMC migration under disturbed flow, in part, by downregulation of ERK1/2-MLCK signaling pathway.

ERK and Akt signaling pathways are involved in advanced glycation end product-induced autophagy in rat vascular smooth muscle cells

Advanced glycation end products (AGEs) play an important role in the proliferation of vascular smooth muscle cells (VSMCs) and accelerate atherosclerosis in diabetic patients. Autophagy, a life-sustaining process, is stimulated in atherosclerotic plaques by oxidized lipids, inflammation and metabolic stress conditions. In our studies, we utilized MTT assays to show that autophagy is involved in AGE-induced proliferation of VSMCs. Furthermore, treatment with AGEs (100 μg/ml) could induce autophagy in a time- and dose-dependent manner in rat aortic VSMCs. These results were further substantiated by electron microscopy and immunofluorescence imaging. Treatment with AGEs activated ERK, JNK and p38/MAPK, but inhibited Akt. Pretreatment with an ERK inhibitor and an Akt activator inhibited AGE-induced autophagy, demonstrating that AGEs induce autophagy in VSMCs through the ERK and Akt signaling pathways. In addition, RNA interference of RAGE decreased autophagy, indicating that RAGE is pivotal in the process of AGE-induced autophagy. Therefore, AGE-induced autophagy contributes to the process of AGE-induced proliferation of VSMCs, which is related to atherosclerosis in diabetes.

Abstract P341: MRTF-A, a Rho-Dependent Coactivator of SRF, Plays a Critical Role in Vascular Remodeling

Objective: Rho-ROCK pathway is involved in the progression of pathological vascular remodeling through the regulation of cell proliferation and migration by controlling gene transcription. While a transcriptional co-activator Myocardin-related transcription factor(MRTF)-A transduces Rho-actin signaling to SRF activation in the nucleus, roles of MRTF-A in the process underlying vascular diseases remain unknown. Methods and Results: We found that MRTF-A mRNA and protein expression in femoral arteries 2weeks after wire injury was significantly higher than that in sham-operated arteries of mice. In contrast, the expression of myocardin or MRTF-B was significantly decreased in injured arteries. We then evaluated the role played by MRTF-A in pathological vascular remodeling by creating several vascular disease models of mice lacking MRTF-A (MRTF-A -/- ). Neointima formation induced by ligation in carotid arteries of MRTF-A -/- was significantly smaller than that of MRTF-A +/- . Neointima formation induced by wire injury in femoral arteries of MRTFA -/- was significantly smaller than that of MRTF-A +/+ . Atherosclerotic lesions of MRTF-A -/- ;ApoE -/- was also significantly smaller than those of MRTF-A +/+ ;ApoE -/ . The expression of SRF-target genes involved in cellular migration, such as MMP9 and vinculin,within injured arteries of MRTF-A -/- was significantly attenuated compared to that of MRTF-A +/+ . In cultured, de-differentiated rat aortic vascular smooth muscle cells (RAVSMC), the expression of these genes was decreased by knocking down MRTF-A. Indeed, the promoter activity of vinculin gene was controlled by MRTF-A in RAVSMC. In addition, knocking down MRTF-A in RAVSMC resulted in a significant impairment in migration capacity. Furthermore, treatment with a small molecule CCG1423 inhibiting MRTF-A significantly reduced neointima formation induced by wire injury in femoral arteries of mice. Conclusions: Our study revealed that in de-differentiated vascular smooth muscle cells, in which myocardin and MRTTF-B expression is decreased, increased expression of MRTF-A contributes to the acquisition of migration capacity by maintaining SRF-target genes transcription, thereby promoting stress-induced vascular remodeling.

Involvement of calcium-sensing receptor in oxLDL-induced MMP-2 production in vascular smooth muscle cells via PI3K/Akt pathway

Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs) and up-regulated in atherosclerotic lesion by various stimuli, such as oxidized low-density lipoprotein (oxLDL). Calcium-sensing receptor (CaSR) is also expressed in VSMCs, but it remains unclear whether CaSR is associated with overproduction of MMP-2 in VSMCs. In this study, the expression of MMP-2 was detected by real-time PCR and Western blot analysis, and the gelatinolytic activity of MMP-2 was measured using gelatin zymography. Our results showed that oxLDL enhanced MMP-2 expression and activity in rat aortic VSMCs in a time- and dose-dependent manner. In addition, CaSR expression was up-regulated by oxLDL. Manipulating CaSR function in these cells by NPS2390 (an antagonist of CaSR) or GdCl(3) (an agonist of CaSR) affected the oxLDL-induced MMP-2 production. In VSMCs, oxLDL stimulated the rapid activation of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, as determined by Western blot analysis. Phosphorylation of Akt and MMP-2 production stimulated by oxLDL were attenuated by LY294002 (a specific inhibitor of PI3K). Activation of Akt was suppressed by NPS2390 but enhanced by GdCl(3). In contrast, oxLDL had no stimulatory effect on the phosphorylation of JNK, and pretreatment with SP600125 (an inhibitor of JNK) produced no significant effect on oxLDL-induced MMP-2 production. These results suggest that CaSR mediates oxLDL-induced MMP-2 production in VSMCs via PI3K/Akt signal pathway.

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.

Nifedipine inhibits vascular smooth muscle cell proliferation and reactive oxygen species production through AMP-activated protein kinase signaling pathway

The dihydropyridine calcium channel blocker nifedipine induces specific pharmacological effects by binding to L-type calcium channels, which results in a reduced calcium influx in vascular smooth muscle cells (VSMCs) and is currently employed in antihypertensive drug. Dihydropyridine calcium channel blocker is reported to reduce oxidative stress and exhibits anti-proliferative effect in VSMCs. VSMCs are useful in the study of atherosclerosis because they show cell proliferation and reactive oxygen species (ROS) production with growth factor. To determine the mechanisms involved in these effects, we investigated the influence of nifedipine-induced AMP-activated protein kinase (AMPK) activation on VSMC proliferation and ROS production by using rat aortic VSMCs in vitro and in vivo. Nifedipine induced phosphorylation of AMPK in a dose-and time-dependent manner, and inhibited rat VSMC proliferation and ROS production following stimulation with 15% fetal bovine serum (FBS). Nifedipine also blocked the FBS-stimulated cell cycle progression through the G0/G1 arrest. Compound C, a specific inhibitor of AMPK, or AMPK siRNA reduced the nifedipine-mediated inhibition of VSMC proliferation. As an upstream kinase, LKB1 is required for nifedipine-induced AMPK activation in VSMCs. 7days oral administration of 1mg/kg nifedipine resulted in activation of LKB1 and AMPK in vivo. These data suggest that nifedipine suppress the VSMC proliferation and ROS production via activating LKB1-AMPK pathway.

Identification of Ostruthin from Peucedanum ostruthium Rhizomes as an Inhibitor of Vascular Smooth Muscle Cell Proliferation

Inhibition of vascular smooth muscle cell (VSMC) proliferation is of substantial interest in combating cardiovascular disease. A dichloromethane extract from the rhizomes of Peucedanum ostruthium, a traditionally used Austrian medicinal plant with anti-inflammatory properties, was examined for a putative antiproliferative activity in rat aortic VSMC. This extract inhibited serum (10%)-induced VSMC proliferation concentration dependently. Further identification and biological testing of its major constituents revealed that the coumarin ostruthin (7) is the major antiproliferative substance. In summary, a new bioactivity of P. ostruthium rhizomes is described, and 7 has been identified as the responsible compound.

2′,3′-cAMP, 3′-AMP, and 2′-AMP inhibit human aortic and coronary vascular smooth muscle cell proliferation via A2B receptors

Rat vascular smooth muscle cells (VSMCs) from renal microvessels metabolize 2',3'-cAMP to 2'-AMP and 3'-AMP, and these AMPs are converted to adenosine that inhibits microvascular VSMC proliferation via A(2B) receptors. The goal of this study was to test whether this mechanism also exists in VSMCs from conduit arteries and whether it is similarly expressed in human vs. rat VSMCs. Incubation of rat and human aortic VSMCs with 2',3'-cAMP concentration-dependently increased levels of 2'-AMP and 3'-AMP in the medium, with a similar absolute increase in 2'-AMP vs. 3'-AMP. In contrast, in human coronary VSMCs, 2',3'-cAMP increased 2'-AMP levels yet had little effect on 3'-AMP levels. In all cell types, 2',3'-cAMP increased levels of adenosine, but not 5'-AMP, and 2',3'-AMP inhibited cell proliferation. Antagonism of A(2B) receptors (MRS-1754), but not A(1) (1,3-dipropyl-8-cyclopentylxanthine), A(2A) (SCH-58261), or A(3) (VUF-5574) receptors, attenuated the antiproliferative effects of 2',3'-cAMP. In all cell types, 2'-AMP, 3'-AMP, and 5'-AMP increased adenosine levels, and inhibition of ecto-5'-nucleotidase blocked this effect of 5'-AMP but not that of 2'-AMP nor 3'-AMP. Also, 2'-AMP, 3'-AMP, and 5'-AMP, like 2',3'-cAMP, exerted antiproliferative effects that were abolished by antagonism of A(2B) receptors with MRS-1754. In conclusion, VSMCs from conduit arteries metabolize 2',3'-cAMP to AMPs, which are metabolized to adenosine. In rat and human aortic VSMCs, both 2'-AMP and 3'-AMP are involved in this process, whereas, in human coronary VSMCs, 2',3'-cAMP is mainly converted to 2'-AMP. Because adenosine inhibits VSMC proliferation via A(2B) receptors, local vascular production of 2',3'-cAMP may protect conduit arteries from atherosclerosis.

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [ 3H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

Insights into the effect of nitric oxide and its metabolites nitrite and nitrate at inhibiting neointimal hyperplasia

Objective Periadventitial delivery of the nitric oxide (NO) donor PROLI/NO following arterial injury effectively inhibits neointimal hyperplasia. Given the short half-life of NO release from PROLI/NO, our goal was to determine if inhibition of neointimal hyperplasia by PROLI/NO was due to NO, or its metabolites nitrite and nitrate. Methods and results In vitro, the NO donor DETA/NO inhibited proliferation of rat aortic vascular smooth muscle cells (RASMC), but neither nitrite nor nitrate did. In vivo, following rat carotid artery balloon injury or injury plus the molar equivalents of PROLI/NO, nitrite, or nitrate ( n = 8–11/group), PROLI/NO was found to provide superior inhibition of neointimal hyperplasia (82% inhibition of intimal area, and 44% inhibition of medial area, p < 0.001). Only modest inhibition was noted with nitrite or nitrate (45% and 41% inhibition of intimal area, and 31% and 29% inhibition of medial area, respectively, p < 0.001). No effects on blood pressure were noted with any treatment groups. In vivo, only PROLI/NO inhibited cellular proliferation and increased arterial lumen area compared to injury alone ( p < 0.001). However, all three treatments inhibited inflammation ( p < 0.001). Conclusions PROLI/NO was more effective at inhibiting neointimal hyperplasia following arterial injury than nitrite or nitrate. However, modest inhibition of neointimal hyperplasia was observed with nitrite and nitrate, likely secondary to anti-inflammatory actions. In conclusion, we have demonstrated that the efficacy of NO donors is primarily due to NO production and not its metabolites, nitrite and nitrate.