Mitogen-activated Protein Kinase In Vascular Smooth Muscle Cells Research Articles

Bisphenol A exposure inhibits vascular smooth muscle cell responses: Involvement of proliferation, migration, and invasion

Previous studies have associated bisphenol A (BPA) with malignant tumor formation, infertility, and atherosclerosis in vitro and in vivo. However, the precise mechanisms through which BPA affects the cardiovascular system under normal conditions remain unclear. Therefore, this study investigated the biological mechanisms through which BPA affects the responses of aortic vascular smooth muscle cells (VSMCs). BPA treatment inhibited the proliferative activity of VSMCs and induced G2/M-phase cell cycle arrest via stimulation of the ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade in VSMCs. Furthermore, BPA treatment upregulated the phosphorylation of mitogen-activated protein kinase (MAPK) pathways such as ERK, JNK, and p38 MAPK in VSMCs. However, the phosphorylation level of AKT was down-regulated by BPA treatment. Additionally, the phosphorylation of ERK, JNK, and p38 MAPK was suppressed when the cells were treated with their respective inhibitors (U0126, SP600125, and SB203580). BPA suppressed MMP-9 activity by reducing the binding activity of AP-1, Sp-1, and NF-κB, thus inhibiting the invasive and migratory ability of VSMCs. These data demonstrate that BPA interferes with the proliferation, migration, and invasion capacities of VSMCs. Therefore, our findings suggest that overexposure to BPA can lead to cardiovascular damage due to dysregulated VSMC responses.

5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Promotes Phenotypic Switching from Dedifferentiated to Differentiated Phenotype via Reactive Oxygen Species and p38 Mitogen-Activated Protein Kinase in Vascular Smooth Muscle Cells

Sonodynamic therapy (SDT) has been found to inhibit in-stent restenosis in animal models. However, the mechanism is not fully elucidated. Here, we investigated the effects of 5-aminolevulinic acid (ALA)-mediated SDT (ALA-SDT) on vascular smooth muscle cells (VSMCs), a cause of restenosis, with a focus on SDT-induced phenotypic switching. Serum-induced dedifferentiated VSMCs were cultured with ALA (1 mm, 24 h) and exposed to ultrasound (0.8 W/cm2) for 5 min. Results indicated that ALA-SDT inhibited the migration and proliferation of VSMCs and enhanced the expression of differentiated phenotypic markers in VSMCs. Additionally, ALA-SDT increased intracellular reactive oxygen species accumulation and phosphorylated p38 mitogen-activated protein kinase in VSMCs. Inhibition of reactive oxygen species elevation or p38 mitogen-activated protein kinase activity abolished the expression of smooth muscle 22α (SM22α) in VSMCs induced by ALA-SDT. Taken together, these results suggest that ALA-SDT promotes transformation of the VSMC phenotype from the dedifferentiated to differentiated status via reactive oxygen species and activated p38 mitogen-activated protein kinase.

Advanced oxidation protein products induce monocyte chemoattractant protein-1 expression via p38 mitogen-activated protein kinase activation in rat vascular smooth muscle cells

Advanced oxidation protein products (AOPPs) are new uremic toxins reported by Witko-Sarsat in 1996, which are associated with the pathogenesis of atherosclerosis. However, the mechanisms by which AOPPs enhance atherosclerosis have not been fully understood. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine which stimulates migration of monocytes and plays a critical role in the development of atherosclerosis. In this study, we investigated the effect of AOPPs on MCP-1 expression in cultured vascular smooth muscle cells (VSMCs). VSMCs were cultured and then co-incubated with AOPP (200 micromol/L, 400 micromol/L) for different times with or without pretreatment with specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. RT-PCR and Western blott were used to detect MCP-1 mRNA and protein expression at different time points after AOPP stimulation in rat smooth muscle cells. Western blot was used to detect the expression of phosphorylated p38 MAPK. Treatment of VSMC with AOPPs resulted in a significant increase of the expression of MCP-1 mRNA and protein in time- and dose-dependent manner, and could activated p38 MAPK. Pretreatment of VSMCs with SB203580 resulted in a dose-dependent inhibition of AOPPs-induced MCP-1 mRNA and protein expression. AOPPs can stimulate MCP-1 expression via p38 MAPK in VSMCs. This suggests that AOPPs might contribute to the formation of atherosclerosis through this proinflammatory effect.

Norepinephrine-induced stimulation of p38 mitogen-activated protein kinase is mediated by arachidonic acid metabolites generated by activation of cytosolic phospholipase A(2) in vascular smooth muscle cells.

p38 mitogen-activated protein kinase (MAPK) is activated by norepinephrine (NE) in the vasculature and is implicated in vascular smooth muscle hypertrophy, contraction, and cell migration. NE promotes influx of Ca(2+) and activates cytosolic phospholipase A(2) (cPLA(2)) in vascular smooth muscle cells (VSMC). The purpose of this study was to determine the contribution of cPLA(2)-generated arachidonic acid (AA) and its metabolites to the activation of p38 MAPK measured by its phosphorylation, in response to NE in rabbit VSMC. NE-induced p38 MAPK activation was found to be mediated through the stimulation of alpha-1 and alpha-2 adrenergic receptors, was dependent on extracellular Ca(2+), and was attenuated by an inhibitor of cPLA(2) (pyrrolidine-1). Moreover, the cPLA(2) product, AA, activated p38 MAPK in VSMC. p38 MAPK activation elicited by NE was decreased significantly by the lipoxygenase (LO) inhibitor baicalein, and to a lesser extent by the cytochrome P450 inhibitor 17-octadecynoic acid, but was not affected by the cyclooxygenase inhibitor indomethacin. The LO metabolites of AA, namely 5(S)-hydroxyeicosatetraenoic acid (HETE), 12(S)-HETE, and 15(S)-HETE and the cytochrome P450 metabolite 20-HETE, activated p38 MAPK. NE-induced p38 MAPK stimulation was found to be independent of phospholipase D (PLD) activation in rabbit VSMC. Transactivation of the epidermal growth factor receptor (EGFR) by NE also did not contribute to p38 MAPK activation. These data suggest that cPLA(2)-generated AA and its LO metabolites mediate NE-induced p38 MAPK stimulation in rabbit VSMC by a mechanism that is independent of PLD and EGFR activation.

Localization of extracellular matrix and mitogen-activated protein kinase (MAPK) in aorta of streptozotocin treated Mongolian gerbils.

To evaluate the relationship among the extracellular matrix (ECM) and mitogen-activated protein kinase (MAPK) family for the vascular damages in hyperglycemia, we injected Mongolian gerbils intravenously with 150 mg/kg streptozotocin (STZ) and observed over the next one year the resulting aortic changes by immunohistochemical techniques. After STZ treatment, hyperglycemia was confirmed. At 4 weeks after STZ administration morphological observation revealed increased stromal components among the vascular smooth muscle cells (SMCs). Immunohistochemically, extracellular matrices such as fibronectin and laminin were localized in the aorta at 4 weeks and one year after STZ administration. The reaction products of MAPK in vascular SMCs were more increased at one year than at 4 weeks after STZ administration. After STZ administration, the increase of ECM and MAPK was observed in the aorta, which suggests these factors play important roles in the pathogenesis of macrovasculopathy in diabetes mellitus.

Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells: mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.

Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs.

MAP kinases and vascular smooth muscle function.

The mitogen-activated protein (MAP) kinase family members are ubiquitously expressed protein kinases activated in response to a variety of extracellular stimuli and shown to be involved in cell growth, transformation, differentiation and apoptosis. MAP kinases have been implicated in both growth and apoptosis of vascular smooth muscle cells (VSMC) which suggests that they play important roles in cardiovascular diseases such as essential hypertension, atherosclerosis, and restenosis followed angioplasty. The MAP kinases are themselves components of specific kinase cascades characterized by activation by specific stimuli, families of related serine and threonine kinases and downstream substrates that include other kinases, transcription factors, membrane receptors and other cell mediators. Cross-talk among the different MAP kinases results in direct modulation of signal transduction. In addition, increased expression and activation of MAP kinase phosphatases plays an important role in MAP kinase inactivation. Our laboratory has used angiotensin II (AngII), a potent activator of all MAP kinases in VSMC, to study mechanisms by which MAP kinases are regulated by vasoactive peptides. In this review, we describe the mechanisms by which AngII activates MAP kinases, and potential roles for MAP kinases in AngII-dependent effects on VSMC function.

Arachidonic acid activates Jun N-terminal kinase in vascular smooth muscle cells.

We have previously demonstrated that arachidonic acid activates extracellular signal-regulated protein kinases (ERKs) group of mitogen-activated protein kinases (MAPKs) in vascular smooth muscle cells (VSMC). To understand the role of arachidonic acid in cellular signaling events, we have now studied its effect on jun N-terminal kinases (JNKs) group of MAPKs in VSMC. Arachidonic acid activated JNK1 in a time- and concentration-dependent manner with maximum effects at 10 min and 50 microM. Induced activation of JNK1 by arachidonic acid is specific as other fatty acids such as linoleic and stearic acids had no such effect. Indomethacin and nordihydroguaiaretic acid (NDGA), potent inhibitors of the cyclooxygenase (COX) and the lipoxygenase (LOX)/monooxygenase (MOX) pathways, respectively, had no effect on arachidonic acid activation of JNK1 suggesting that the observed phenomenon is independent of its metabolism through either pathway. However, 12-hydroperoxyeicosatetraenoic acid (12-HpETE), the LOX metabolite of arachidonic acid significantly induced JNK1 activity. Protein kinase C (PKC) depletion by prolonged treatment of VSMC with phorbol 12-myristate 13-acetate (PMA) resulted in partial decrease in the responsiveness of JNK1 to arachidonic acid suggesting a role for both PKC-dependent and -independent mechanisms in the activation of JNK1 by this important fatty acid. On the other hand, the responsiveness of JNK1 to 12-HpETE was completely abolished in PKC-depleted cells, suggesting a major role for PKC in 12-HpETE-induced JNK1 activation. IL-1beta and TNF-alpha activated JNK1 in a time-dependent manner with maximum effect at 10 min. Desensitization of JNK1 by arachidonic acid significantly reduced its responsiveness to both the cytokines. In addition, 4-bromophenacyl bromide (4-BPB), a potent and selective inhibitor of phospholipase A2 (PLA2), significantly attenuated the cytokine-induced activation of JNK1. Together, these results show that (1) arachidonic acid and its LOX metabolite, 12-HpETE, activate JNK1 in VSMC, (2) PKC-dependent and -independent mechanisms play a role in the activation of JNK1 by arachidonic acid and 12-HpETE, and (3) arachidonic acid mediates, at least partially, the cytokine-induced activation of JNK1.

Oxidized LDL stimulates mitogen-activated protein kinases in smooth muscle cells and macrophages.

It has been proposed that oxidized LDL is more atherogenic than native LDL. However, the mechanisms by which native LDL and oxidized LDL alter function of cells in the vessel wall remain undefined. A signal transduction pathway that mediates many changes in cell function is the mitogen-activated protein (MAP) kinase cascade. We therefore examined the effect of native LDL and oxidized LDL on MAP kinase activity in cultured vascular smooth muscle cells (VSMC), endothelial cells, and macrophages by using an in-gel-kinase assay and anti-phosphotyrosine MAP kinase antibodies. Native LDL and LDL oxidized by the addition of Cu2+ (Cu(2+)-oxidized LDL) stimulated MAP kinase in a time- and dose-dependent manner in baboon and rat VSMC but not in bovine endothelial cells. Cu(2+)-oxidized LDL stimulated MAP kinase in human monocyte-derived macrophages, but the effect was much greater in cells cultured for 7 days compared with 1 day, suggesting dynamic regulation of the cellular response to oxidized LDL. In rat VSMC, the maximal MAP kinase response to Cu(2+)-oxidized LDL was significantly greater than the response to native LDL. Cu(2+)-oxidized LDL was more potent, with half-maximal activation at 15 micrograms/mL versus 30 micrograms/mL for native LDL. Stimulation of MAP kinase appeared to involve protein kinase C, since phorbol ester pretreatment for 24 hours blocked MAP kinase activation. Oxidation of LDL by other methods showed that activation of MAP kinase was not well correlated with lipid peroxides or aldehydes, suggesting that other components present in oxidized LDL were responsible. The active moiety appeared to be lipid based on extraction of oxidized LDL with organic solvents. These data indicate that LDL stimulates MAP kinase in VSMC, oxidation of LDL potentiates the effect, a lipid moiety is involved, and Cu(2+)-oxidized LDL activation of MAP kinase is cell-type specific. These findings suggest a role for MAP kinase in the pathways by which oxidized LDL contributes to altered cellular function associated with atherogenesis.

Mechanism of catecholamine-induced proliferation of vascular smooth muscle cells.

Catecholamines have been shown to aggravate atherosclerosis in animals and humans, and abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the early stage of atherosclerosis. Catecholamines may be involved in such cell growth. Therefore, a series of experiments using cultured VSMC was performed to elucidate their possible mitogenic effect. We examined the mitogenic effect of catecholamines using rat aortic smooth muscle cells (VSMC) by measuring [3H]thymidine incorporation, checking with flow cytometry, and counting the cell number directly. Furthermore, the catecholamine-activated signal transduction pathway was assessed by measurement of the formation of inositol 1, 4, 5-triphosphate, intracellular Ca2+ concentration, mitogen-activated protein kinase (MAPK) activity, and mitogenic gene expression. Norepinephrine (NE) and phenylephrine stimulated [3H]thymidine incorporation and cell growth. Clonidine and isoproterenol showed little of such effects. Prazosin was more effective than either yohimbine or propranolol in suppressing the mitogenic effect of NE, indicating that catecholamine-induced VSMC proliferation is mediated by alpha 1-adrenoceptors. The alpha 1-adrenoceptor activation was coupled to pertussis toxin-insensitive Gq-protein and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C and MAPK in VSMC. In response to NE, both 42- and 44-kD MAPK were activated and tyrosine was phosphorylated. alpha 1-Adrenoceptor stimulation with NE also caused accumulation of c-fos, c-jun, and c-myc mRNA. Chloroethylclonidine completely blocked the alpha 1-adrenoceptor-mediated mitogenesis. The effect of catecholamines appears to be mediated via the activation of the chloroethylclonidine-sensitive alpha 1-adrenoceptors that triggers the phosphoinositide hydrolysis and activates the MAPK pathway, leading to DNA synthesis and cell proliferation.

Mitogen-activated Protein (MAP) Kinase Is Regulated by the MAP Kinase Phosphatase (MKP-1) in Vascular Smooth Muscle Cells: EFFECT OF ACTINOMYCIN D AND ANTISENSE OLIGONUCLEOTIDES

Angiotensin II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC) and activates many growth-promoting kinases such as mitogen-activated protein (MAP) kinase. A novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1), is induced by angiotensin II in VSMC and selectively dephosphorylates MAP kinase in vitro. Using actinomycin D and antisense oligonucleotides targeted to MKP-1, we demonstrate that MKP-1 regulates MAP kinase in VSMC. Both actinomycin D and MKP-1 antisense oligonucleotides inhibited MKP-1 mRNA expression and caused prolonged activation of the p42 and p44 MAP kinases as measured by in-gel-kinase assays and Western blot. For example, MAP kinase activity 120 min after angiotensin II treatment was 30% (range 25-35%), 79%, and 74% of maximum in control, actinomycin D-treated (3 micrograms/ml, 30 min), and antisense oligonucleotide-treated (300 nM, 6 h) cells, respectively. A sense oligonucleotide was without effect (34%). MKP-1 antisense oligonucleotides did not affect the activity of MEK indicating that sustained activation of MAP kinase was due to inhibition of MKP-1 expression. These findings demonstrate that inactivation of MAP kinase by angiotensin II is mediated predominantly by MKP-1, suggesting an important role for MKP-1 and other related phosphatases in the regulation of MAP kinases in VSMC.

Activation of mitogen-activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells.

Previous studies from this laboratory and others suggest that arachidonic acid and its metabolites play important roles in a variety of biological processes such as signal transduction, contraction, chemotaxis, and cell growth and differentiation. Here we studied the effect of arachidonic acid on mitogen-activated protein (MAP) kinases in vascular smooth muscle cells (VSMC). Arachidonic acid activated MAP kinases in VSMC in a time- and dose-dependent manner. Nordihydroguaiaretic acid (NDGA), a potent inhibitor of the lipoxygenase system, significantly blocked the arachidonic acid-induced activation of MAP kinases, whereas indomethacin, an inhibitor of cyclooxygenase, had no effect. In VSMC, arachidonic acid was converted to 15-hydroxyeicosatetraenoic acid (15-HETE); NDGA inhibited the formation of this HETE. Exogenous addition of 15-HETE to VSMC caused stimulation of MAP kinases. Depletion of protein kinase C attenuated both the arachidonic acid- and 15-HETE-induced activation of MAP kinases in VSMC. Together these results suggest that 1) arachidonic acid activates MAP kinases in VSMC; 2) 15-HETE, a 15-lipoxygenase product of arachidonic acid, at least in part, mediates the arachidonic acid effect on MAP kinases; and 3) protein kinase C appears to be important in arachidonic acid activation of MAP kinases. Therefore, MAP kinases may play an important role in arachidonic acid signaling of VSMC growth and function.

Involvement of MAP kinase activators in angiotensin II-induced activation of MAP kinases in cultured vascular smooth muscle cells

In cultured vascular smooth muscle cells (VSMC) angiotensin II (ang II) induces tyrosine and serine/threonine phosphorylation and activation of two mitogen-activated protein (MAP) kinases, When extracts of ang II-stimulated VSMC were fractionated by Mono Q anion-exchange column chromatography, three peaks of the activities which in vitro activate inactive MAP kinases were detected. These MAP kinase activator activities were not detected in extracts of unstimulated VSMC. In vitro activation of MAP kinases by the MAP kinase activators was accompanied by tyrosine and serine/threonine phosphorylation of MAP kinases. These results suggest that the MAP kinase activators are involved in the ang II-induced phosphorylation and activation of MAP kinases in VSMC.