Cultured Rat Vascular Smooth Muscle Cells Research Articles

Effects of magnesium on the production of extracellular matrix metalloproteinases in cultured rat vascular smooth muscle cells

The precise correlation between magnesium and cardiovascular disease remains to be established. Matrix metalloproteinases (MMPs) are expressed in coronary arterial atherosclerotic lesions. MMP production in vascular smooth muscle cells (VSMCs) is stimulated by growth factors such as platelet-derived growth factor (PDGF). To assess the association between magnesium and MMPs, we examined the effects of different extracellular magnesium concentrations (0–3.0 mmol/l) on MMPs production in cultured rat VSMCs under basal and PDGF-stimulated conditions using gelatin zymography and western blotting. As magnesium is called a natural calcium antagonist, we further compared the effects of magnesium with some calcium antagonists. Magnesium reduced MMP-2 production dose-dependently at basal and PDGF-stimulated conditions in VSMCs. However, neither verapamil nor nifedipine influenced MMP-2 production under any conditions examined. The effect of magnesium on the production of MMP-2 was inhibited by two tyrosine kinase inhibitors-genistein and herbimycin A. The results of this study indicate that extracellularly added magnesium decreased MMPs secretion, which appears to be associated with protein tyrosine kinase.

Effects of apigenin on the serum- and platelet derived growth factor-BB-induced proliferation of rat aortic vascular smooth muscle cells.

The inhibitory effects of apigenin on the growth factor-induced proliferative responses, and expression of mitogen-activated protein (MAP) kinase and its downstream c-fos in rat aortic vascular smooth muscle cells (VSMCs) were investigated. Apigenin significantly inhibited both 5 % fetal bovine serum (FBS)- and 50 ng/mL platelet derived growth factor-BB (PDGF-BB)-induced proliferation on primary cultured rat VSMCs in a concentration-dependent manner. In addition, apigenin resulted in a significant inhibition of the FBS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and expression of c-fos mRNA. These results suggest that apigenin inhibits FBS- and PDGF-BB-induced VSMC proliferation, and its activity may be mediated, at least in part, by down regulation of ERK 1/2 and its downstream c-fos mRNA.

Phosphatidylinositide 3-kinase regulates angiotensin II-induced cytosolic phospholipase A2 activity and growth in vascular smooth muscle cells

Angiotensin (Ang) II via the AT 1 receptor acts as a mitogen in vascular smooth muscle cells (VSMC) through stimulation of multiple signaling mechanisms, including tyrosine kinases and mitogen-activated protein kinase (MAPK). In addition, cytosolic phospholipase A 2 (cPLA 2)-dependent release of arachidonic acid (AA) is linked to VSMC growth and we have reported that Ang II stimulates cPLA 2 activity via the AT 1 receptor. The coupling of Ang II to the activation of cPLA 2 appears to involve mechanisms both upstream and downstream of MAPK such that AA stimulates MAPK activity which phosphorylates cPLA 2 to further enhance AA release. However, the upstream mechanisms responsible for activation of cPLA 2 are not well-defined. One possibility includes phosphatidylinositide 3-kinase (PI3K), since PI3K has been reported to participate in the upstream signaling events linked to activation of MAPK. However, it is not known whether PI3K is involved in the Ang II-induced activation of cPLA 2 or if this mechanism is associated with the Ang II-mediated growth of VSMC. Therefore, we used cultured rat VSMC to examine the role of PI3K in the Ang II-dependent phosphorylation of cPLA 2, release of AA, and growth induced by Ang II. Exposure of VSMC to Ang II (100 nM) increased [ 3H]thymidine incorporation, cell number, and the release of [ 3H]AA. Also, using Western analysis, Ang II increased the phosphorylation of MAPK and cPLA 2 which were blocked by the MAPK kinase inhibitor PD98059 (10 μM/L) . Similarly, the PI3K inhibitor LY294002 (10 μM/L) abolished the Ang II-mediated increase in MAPK phosphorylation, as well as phosphoserine-PLA 2. Further, inhibition of PI3K blocked the Ang II-induced release of AA and VSMC mitogenesis. However, exogenous AA was able to restore VSMC growth in the presence of LY294002, as well as reverse the inhibition of MAPK and cPLA 2 phosphorylation by LY294002. Thus, it appears from these data that Ang II stimulates the PI3K-sensitive release of AA which stimulates MAPK to phosphorylate cPLA 2 and enhance AA release. This mechanism may play an important role in the Ang II-induced growth of VSMC.

Angiotensin II type 2 receptor gene transfer downregulates angiotensin II type 1a receptor in vascular smooth muscle cells.

Two distinct subtypes of angiotensin (Ang) II receptors, type 1 (AT(1)) and type 2 (AT(2)), have been identified. Vascular smooth muscle cells (VSMCs) usually express AT(1) receptor. To elucidate the direct effects of the AT(2) receptor on the AT(1) receptor in VSMCs, we transfected AT(2) receptor gene into cultured rat VSMCs. Overexpression of AT(2) receptor significantly decreased expression of AT(1a) receptor at both the mRNA and protein levels in the presence and absence of Ang II in VSMCs. Overexpression of AT(2) receptor increased expression of bradykinin and inducible NO in the presence and absence of Ang II in VSMCs. Bradykinin B(2) receptor antagonist HOE-140 and NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) inhibited the decreases in AT(1a) receptor expression by the overexpression of AT(2) receptor in VSMCs. L-Arginine augmented the decrease in AT(1a) receptor expression. Overexpression of AT(2) receptor suppressed basal DNA synthesis and proliferation of VSMCs and abolished response of DNA synthesis to Ang II in VSMCs. Our results demonstrate that overexpression of the AT(2) receptor downregulates AT(1a) receptor expression in rat VSMCs in a ligand-independent manner that is mediated by the bradykinin/NO pathway. Downregulation of AT(1a) receptor is a novel mechanism by which the AT(2) receptor regulates growth and metabolism of VSMCs.

Lysophosphatidylcholine activates extracellular signal-regulated kinases 1/2 through reactive oxygen species in rat vascular smooth muscle cells.

Lysophosphatidylcholine (lysoPC) acts on vascular smooth muscle cells (VSMCs) to produce a mitogenic response through the activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we examined the importance of reactive oxygen species (ROS) in lysoPC-stimulated ERK1/2 activation in cultured rat VSMCs. Treatment with lysoPC for 3 minutes caused a 2-fold increase in intracellular ROS that was blocked by the NADH/NADPH oxidase inhibitor, diphenylene iodonium (DPI). Antioxidants, N-acetyl-L-cysteine, glutathione monoester, or alpha -tocopherol, inhibited ERK1/2 activation by lysoPC. Almost identical results were obtained in the VSMC line A10. Pretreatment of VSMCs with DPI but not allopurinol or potassium cyanide (KCN) abrogated the activation of ERK1/2. The Flag-tagged p47phox expressed in A10 cells was translocated from the cytosol to the membrane after 2 minutes of stimulation with lysoPC. The overexpression of dominant-negative p47phox in A10 cells suppressed lysoPC-induced ERK activation. The ROS-dependent ERK activation by lysoPC seems to involve protein kinase C- and Ras-dependent raf-1 activation. Induction of c-fos expression and enhanced AP-1 binding activity by lysoPC were also inhibited by DPI and NAC. Taken together, these data suggest that ROS generated by NADH/NADPH oxidase contribute to lysoPC-induced activation of ERK1/2 and subsequent growth promotion in VSMCs.

Signaling mechanisms of heparin-binding epidermal growth factor-like growth factor in vascular smooth muscle cells.

A host of growth factors have been implicated in vascular pathologies; one such factor is heparin-binding epidermal growth factor-like growth factor (HB-EGF). Although HB-EGF has been shown to stimulate mitogenesis and chemotaxis of vascular smooth muscle cells (VSMC), its signaling mechanism remains undefined. In this study, we examined possible signal transduction pathways by which HB-EGF leads to mitogenesis in cultured rat VSMC. HB-EGF induced phosphorylation of the EGF receptor (EGFR) with maximum phosphorylation at 0.5 to 1 minute, whereas erbB4, the other receptor to which HB-EGF binds, was not activated on HB-EGF stimulation. HB-EGF induced a time- and concentration-dependent phosphorylation of mitogen-activated protein kinase (MAPK; p42/44 MAPK, extracellular signal-regulating kinase [ERK] 1/2). It also activated Akt and p70S6 kinase (p70S6K) but not p38 MAPK. HB-EGF-induced phosphorylation of these kinases was blocked by the EGFR kinase inhibitor AG1478. To investigate signaling molecules involved in HB-EGF-induced DNA synthesis, we pretreated VSMC with the specific ERK kinase mitogen-activated kinase (MEK) inhibitor PD98059 and the phosphatidylinositol 3-kinase inhibitor LY294002. These inhibitors significantly blocked HB-EGF-induced DNA synthesis. PD98059 inhibited HB-EGF-induced ERK activation, whereas it had no effect on Akt activation by HB-EGF. By contrast, LY294002 inhibited HB-EGF-induced Akt and p70S6K activation without effecting ERK activation by HB-EGF. These results demonstrate that HB-EGF-induced mitogenesis requires both ERK and phosphatidylinositol 3-kinase (Akt and p70S6K) pathways activated through EGFR, thereby providing a new mechanistic insight by which HB-EGF contributes to vascular remodeling.

Mechanical stretch-induced mitogen-activated protein kinase activation is mediated via angiotensin and endothelin systems in vascular smooth muscle cells.

We previously reported that pressure loading of the vascular wall can activate mitogen-activated protein kinases (MAPKs), enzymes believed to be involved in the pathway for cell proliferation, partly via the vascular angiotensin system in isolated perfused rat aorta. In this study, we examined whether cyclic stretching of vascular smooth muscle cells (VSMC) also produces activation of p42 and p44 MAPKs in cultured rat VSMC and whether stretch-induced MAPK activation is mediated via angiotensin and endothelin systems in VSMC. Cyclic stretching of VSMC produced an elongation-dependent and frequency-dependent increase in p42 and p44 MAPK activity. The stretch-induced p42 and p44 MAPK activation was inhibited by the angiotensin receptor antagonist losartan and by the angiotensin-converting enzyme inhibitor, captopril. The MAPK activation was also inhibited by the endothelin receptor antagonist cyclo(D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl) (BQ123) and by the endothelin-converting enzyme inhibitor phosphoramidon. Replacement of medium with culture medium of stretched cells caused MAPK activation, which was inhibited by losartan and BQ123. The results of the present study suggest that cyclic stretching of VSMC can activate p42 and p44 MAPKs and that the MAPK activation is mediated via angiotensin and endothelin systems in VSMC.

The Anti-Platelet Agent, Ticlopidine, Upregulates Interleukin-1-Beta-Stimulated Nitric Oxide Production in Cultured Rat Vascular Smooth Muscle Cells

Background: Hemodialysis patients who had been treated with anti-platelet aggregation drugs, including ticlopidine, sometimes developed hypotension. The mechanism by which ticlopidine lowers the blood pressure in hemodialysis patients is unclear. To elucidate the mechanism of the action of this drug, we investigated cytokine-stimulated nitric oxide (NO) metabolism by ticlopidine in cultured rat vascular smooth muscle cells (VSMC). Methods: Nitrite, a stable metabolite of NO, and intracellular cAMP and cGMP contents were assayed by the Griess method and enzyme immunoassay, respectively. iNOS mRNA and protein expressions were analyzed by Northern blotting and Western blotting. Results: Ticlopidine enhanced interleukin-1β (IL-1β)-induced nitrite production in a dose- and time-dependent manner. The mRNA and protein expressions of inducible NO synthase were upregulated by ticlopidine in a dose- and time-dependent manner. IL-1β alone stimulated both intracellular cAMP and cGMP contents, and the addition of ticlopidine further enhanced their contents. KT 5720, a selective inhibitor of protein kinase A, but not KT 5823, a selective inhibitor of protein kinase G, abolished the enhancement of IL-1β-induced nitrite production by ticlopidine. In addition, a phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced IL-1β and ticlopidine induced nitrite production. Conclusion: We concluded that ticlopidine enhanced the IL-1β-induced NO production via cAMP and subsequent activation of protein kinase A in cultured rat VSMC.

Cytokine-activated Jak-2 is involved in inducible nitric oxide synthase expression independent from NF-κB activation in vascular smooth muscle cells

Inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, activate nuclear factor-kappa B (NF-κB) which transactivates inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains obscure whether cytokine-mediated iNOS expression in VSMCs requires signaling pathway(s) other than NF-κB activation. The present study was designed to elucidate whether protein tyrosine kinases (PTKs) are involved in the cytokine-induced NF-κB activation and iNOS expression in cultured rat VSMCs. Both IL-1β and TNF-α stimulated NF-κB activity, iNOS mRNA and protein expression with massive nitrite/nitrate (NOx) production in rat VSMCs. PTK inhibitors (genistein, herbimycin A) dose-dependently inhibited the cytokine-stimulated NOx production and iNOS mRNA expression. However, neither genistein nor herbimycin A affected the cytokine-stimulated phosphorylation and degradation of IκB-α, or NF-κB activation, whereas they completely blocked the cytokine-stimulated iNOS transcriptional activity. Tyrphostin B42 (AG490), a Jak-2 tyrosine kinase inhibitor, similarly blocked the cytokine-induced NOx production, iNOS expression and its promoter activity without affecting NF-κB-dependent transcription. Transfection of a dominant-negative Jak-2 mutant antagonized the cytokine-induced NOx production and iNOS expression, while wild-type Jak-2 expressing construct was without effect. These data indicate that the cytokine-induced iNOS expression involves activation of Jak-2 signaling pathway independent from NF-κB activation in rat VSMCs.

Effect of red wine polyphenols on vascular smooth muscle cell function—molecular mechanism of the ‘French paradox’

Red wine polyphenols (RWP) have been shown to have an anti-atherogenic activity mainly through anti-oxidative effects on low-density lipoprotein (LDL) oxidation. Though proliferation of vascular smooth muscle cells (VSMC) is critical to atherosclerosis formation, the effect of RWP on VSMC proliferation has not been elucidated. In this study, we investigated whether RWP, which extracted from red wine using column chromatography, could affect the 10% serum-stimulated VSMC proliferation. Treatment with RWP showed a potent inhibitory effect on the proliferation and DNA syntheses is in cultured rat VSMC. In contrast, the inhibitory effect of RWP on the proliferation of bovine vascular endothelial cells (EC) was only observed at much higher doses. Moreover, RWP significantly inhibited the proliferation and DNA synthesis of human VSMC but no human vascular EC in a dose-dependent manner. To elucidate the molecular mechanisms of these anti-proliferative effects of RWP on VSMC, but not on vascular EC, we investigated the effects of RWP on the cell cycle regulation. RWP downregulated the expression and promoter activity of cyclin A gene, one of cell cycle regulators. In addition, RWP inhibited the binding of nuclear proteins to the activating transcription factor (ATF) site in the cyclin A promoter, and downregulated the expression of transcription factors, cyclic AMP-responsive element binding protein (CREB) and ATF-1. In conclusion, these results demonstrate one possible finding that the anti-proliferative effect of RWP on VSMC may be associated with the downregulation of cyclin A gene expression through the inhibition of transcription factor expression.

Taurine prevents the decrease in expression and secretion of extracellular superoxide dismutase induced by homocysteine: amelioration of homocysteine-induced endoplasmic reticulum stress by taurine.

Hyperhomocysteinemia is an independent risk factor for atherosclerosis. Homocysteine has been shown to induce endoplasmic reticulum (ER) stress in vascular endothelial cells. ER stress is a condition in which glycoprotein trafficking is disrupted and unfolded proteins accumulate in the ER. ER molecular chaperons, such as GRP78, are induced and an ER resident kinase, PERK, is activated when cells are subjected to ER stress. Conversely, taurine is reported to have antiatherogenic effects by unknown mechanisms. To elucidate the mechanisms by which homocysteine induces atherosclerosis and taurine prevents it, we examined whether homocysteine and taurine affect the expression and secretion of extracellular superoxide dismutase (EC-SOD), a glycoprotein secreted from vascular smooth muscle cells (VSMCs) that protects the vascular wall from oxidative stress. We assessed the expression of EC-SOD and GRP78 mRNA in cultured rat VSMCs by Northern blot analysis. The EC-SOD protein secreted into the culture medium was examined by Western blot analysis. Homocysteine (5 mmol/L) and other ER stress inducers, including A23187, were found to decrease EC-SOD mRNA expression and protein secretion. Furthermore, they upregulated GRP78 mRNA expression and activated PERK. Taurine (0.5 to 10 mmol/L), conversely, prevented these actions induced by homocysteine. Homocysteine induces ER stress and reduces the secretion and expression of EC-SOD in VSMCs, leading to increased oxidative stress in the vascular wall. Taurine restores the secretion and expression of EC-SOD by ameliorating ER stress induced by homocysteine.

Characterization of platelet-derived growth factor-induced p38 mitogen-activated protein kinase activation in vascular smooth muscle cells.

The mitogen-activated protein (MAP) kinase super-family plays a crucial role in cell growth and differentiation and even in programmed cell death in response to diverse extracellular stimuli. The platelet-derived growth factor (PDGF)-BB is well known to promote the proliferation of vascular smooth muscle cells (VSMC) via extracellular-regulated protein kinases (ERKs), leading to the development of cardiovascular diseases. However, it has not yet been clarified whether PDGFs that include other isoforms can activate the other parallel signal transduction pathways, c-jun NH2-terminal protein kinase (JNK) and p38 MAP kinase (p38), in VSMC. In this study, we investigated the effect of PDGFs on p38 activation in cultured rat VSMC. After stimulation by PDGFs with SB-203580 or PD-98059, the cells were solubilized, and the expressions of MAP kinases, MAP kinase kinases (MKKs), phosphorylated DNA-binding proteins, and cyclooxigenases (COXs) were examined by immunoblot analysis. PDGFs activated p38 phosphorylation dose-dependently, and the phosphorylations were specifically inhibited by SB-203580 but not by PD-98059. PDGFs also activated the phosphorylation of MKK 3/MKK 6 but not that of either stress-activated protein kinase/ERK kinase or JNK. PDGFs affected the activation of a cyclic AMP response-element binding protein, which was inhibited by SB-203580. However, the activating transcription factor-2 was not activated by PDGFs. Interestingly, the stimulation of PDGFs for 72 h enhanced the level of COX-2, and these levels were decreased by SB-203580. These results have clarified that PDGFs activate the p38 cascade via an MKK 3/6 pathway, independently of the ERK cascade, and subsequently regulate the level of COX-2 in rat VSMC, providing that PDGFs influence the inflammatory process in the vascular wall.

A pyrrolidinone derivative inhibits cytokine-induced iNOS expression and NF-kappaB activation by preventing phosphorylation and degradation of IkappaB-alpha.

We previously showed that 1-[3-(3-pyridyl)-acryloyl]-2-pyrrolidinone hydrochloride (N2733) inhibits lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-alpha secretion and improves the survival of endotoxemic mice. Since overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMCs) is largely responsible for the development of endotoxemic shock, and iNOS gene expression is mainly regulated by LPS and inflammatory cytokines, we studied whether or not N2733 affects interleukin (IL)-1beta-induced iNOS gene expression, NF-kappaB activation, and NF-kappaB inhibitor (IkappaB)-alpha degradation in cultured rat VSMCs. N2733 dose-dependently (10-100 microM) inhibited IL-1beta-stimulated NO production, and decreased IL-1beta-induced iNOS mRNA and protein expression, as found on Northern and Western blot analyses, respectively. Gel shift assay and an immunocytochemical study showed that N2733 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation. Western blot analyses involving anti-IkappaB-alpha and anti-phospho IkappaB-alpha antibodies showed that IL-1beta induced transient degradation of IkappaB-alpha preceded by the rapid appearance of phosphorylated IkappaB-alpha, both of which were markedly blocked by N2733. N2733 blocked IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of a proteasome inhibitor (MG115). Immunoblot analysis involving anti-IkappaB kinase (IKK)-alpha and anti-phosphoserine antibodies revealed that N2733 inhibited IL-1beta-induced IKK-alpha phosphorylation, whereas N2733 had no inhibitory effect on IL-1beta-stimulated p42/p44 MAP kinase or p38 MAP kinase activity. Our results suggest that the inhibitory action of N2733 toward IL-1beta-induced NF-kappaB activation and iNOS expression is due to its blockade of the upstream signal(s) leading to IKK-alpha activation, and subsequent phosphorylation and degradation of IkappaB-alpha in rat VSMCs.

Erythropoietin modulates angiotensin II‐ or noradrenaline‐induced Ca2+ mobilization in cultured rat vascular smooth‐muscle cells

It has been reported that human recombinant erythropoietin (rHuEpo) modulates the sensitivity of the cardiovascular system to angiotensin II (Ang II) or noradrenaline (NA). In the present study, we explored the effect of rHuEpo on the responsiveness of Ang II- or NA-induced cytosolic free calcium ([Ca(2+)]i) mobilization in cultured rat vascular smooth-muscle cells (VSMC). [Ca(2+)]i concentrations in VSMC were measured by using the calcium-sensitive fluorescent dye fura-2. The addition of rHuEpo (250 U/ml) alone induced elevation in [Ca(2+)]i, which remained significantly elevated above basal level for at least 60 min in the presence of extracellular Ca(2+). Pre-incubation with specific protein kinase C (PKC) inhibitor calphostin C (1 micromol/l) significantly reduced the peak and the sustained elevations of [Ca(2+)]i. Pre-treatment with rHuEpo for 60 min increased both basal [Ca(2+)]i and the changes in [Ca(2+)]i by Ang II or NA in a dose-dependent manner in the presence of extracellular Ca(2+). The synergistic effects of rHuEpo with Ang II or NA were also retained when VSMC were bathed in the Ca(2+)-free medium after the pre-incubation of rHuEpo. Conversely, they were diminished in the presence of extracellular Ca(2+) combined with intracellular Ca(2+) release inhibitor 8-(NN-diethylamino)octyl-1,3,4,5-trimethoxybenzoate (TMB-8). The synergistic effects of rHuEpo were also diminished by PKC depletion or by PKC inhibitor. These observations suggest that rHuEpo has synergistic effects on Ang II- or NA-induced [Ca(2+)]i mobilization, particularly on intracellular Ca(2+) release, in VSMC. This may be a potential mechanism contributing to hypertension associated with rHuEpo therapy.

Inhibitors of arachidonic acid metabolism have variable effects on calcium signaling pathways

The metabolic pathways of arachidonic acid (AA) have been shown to be important in the regulation of cellular function. Several studies have demonstrated both direct and indirect effects of products of these pathways in the regulation of vascular actions, and in particular on signaling pathways. Because intracellular calcium concentration is a significant mediator of stimulus-coupled signal transduction, we investigated the effects of AA pathway inhibitors on angiotensin II (Ang II)-induced calcium mobilization in cultured rat vascular smooth muscle cells (VSMC). Thus, specific calcium pools were examined for differential effects resulting from inhibitors of the three major pathways of AA metabolism. Inhibition of lipoxygenase (LO) with 2.5 μmol/L of 5,8,11 eicosatriynoic acid (ETI), cyclooxygenase (CO) with 2 μmol/L of ibuprofen (IBU), and cytochrome P-450 (P450) with 1 μmol/L of 7-ethoxyresorufin (7ER) all reduced total Ang II-induced intracellular calcium transients ([Ca 2+] i) in fura 2-loaded cultured rat VSMC. However, the sites of action affected were unique for each inhibitor. Pretreatment of VSMC with either ETI or IBU inhibited thapsigargin (TG) (1 μmol/L)-sensitive calcium increments (control; 118.0 ± 33.1 nmol/L, n = 9, ETI; 34.7 ± 4.8 nmol/L, n = 9, IBU; 40.3 ± 8.8 nmol/L, n = 8, P < .05 v control). Both caffeine (CAF) and ryanodine (RY) treatment attenuated Ang II-induced [Ca 2+] i; however, pretreatment with ETI, IBU, or 7ER did not alter this effect. In other studies, the LO inhibitor ETI attenuated Ang II-induced Ca 2+ influx, whereas inhibitors of CO and P450 pathways had no effect. These data show that 1) ETI and IBU affect TG-sensitive Ca pools; 2) ETI, but not IBU nor 7ER, inhibited calcium influx; and 3) ETI, IBU, and 7ER affect similar intracellular calcium stores as CAF and RY. Thus, agonist-specific differences in the effects of LO, CO, and P450 inhibition on vascular actions may be due to site-specific effects of these inhibitors on calcium mobilization.

Troglitazone and rosiglitazone induce apoptosis of vascular smooth muscle cells through an extracellular signal-regulated kinase-independent pathway

Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands thiazolidinediones have been reported to induce apoptosis in macrophages and in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis in VSMC, cultured rat VSMC were treated with increasing doses of the thiazolidinedione analogues troglitazone (TRO) and rosiglitazone (RSG). Both ligands induced cell death in a concentration-dependent manner (EC50 12.1+/-3.3 microM and 1.43+/-0.39 microM, respectively), causing almost complete cell death at the highest concentrations (100 microM and 10 microM for TRO and RSG, respectively), along with an expected parallel decrease in [3H]thymidine uptake into cell DNA (EC50 6.7+/-2.4 microM and 0.75+/-0.19 microM, respectively). The cell count was determined by the coulter counter principle. Furthermore two apoptotic markers were measured, the caspase 3 activity and the cytoplasmic histone-associated DNA fragments, both of which were significantly increased when the aforementioned high concentrations were used. This indicates that apoptosis is involved in the TRO- and RSG-induced VSMC growth suppression. The same concentrations of TRO and RSG caused an unexpected stimulation of the extracellular signal-regulated response kinases 1 and 2 (ERK1/2) and stimulated the p38 mitogenic-activated protein (MAP) kinase as determined by Western blotting. In order to establish whether the proapoptotic effects of TRO and RSG are mediated through ERK1/2 activation, we used the selective MAP kinase kinase (MEK) inhibitor PD98059 (20 microM), which suppressed the TRO- and RSG-induced ERK1/2 activation but did not abolish their proapoptotic effects. We conclude that the thiazolidinedione analogues TRO and RSG induce cell death due to apoptosis in VSMC through an ERK1/2-independent pathway.

Cultured rat vascular smooth muscle cells are resistant to methylamine toxicity: no correlation to semicarbazide-sensitive amine oxidase.

Methylamine (MA), a component of serum and a metabolite of nicotine and certain insecticides and herbicides, is metabolized by semicarbazide-sensitive amine oxidase (SSAO). MA is toxic to cultured human umbilical vein and calf pulmonary artery endothelial cells. Endothelial cells, which do not exhibit endogenous SSAO activity, are exposed to SSAO circulating in serum. In contrast, vascular smooth muscle cells (VSMC) do exhibit innate SSAO activity both in vivo and in vitro. This property, together with the critical localization of VSMC within the arterial wall, led us to investigate the potential toxicity of MA to VSMC. Cultured rat VSMC were treated with MA (10-5 to 1 M). In some cultures, SSAO was selectively inhibited with semicarbazide or MDL-72145 [(E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine]. Cytotoxicity was measured via MTT, vital dye exclusion, and clonogenic assays. MA proved to be toxic to VSMC only at relatively high concentrations (LC(50) of 0.1 M). The inhibition of SSAO with semicarbazide or MDL-72145 did not increase MA toxicity, suggesting that the production of formaldehyde via tissue-bound, SSAO-mediated MA metabolism does not play a role in the minimal toxicity observed in isolated rat VSMC. The omission of fetal calf serum (FCS), which contains high SSAO activity, from media similarly showed little effect on cytotoxicity. We conclude that VSMC--in contrast to previous results in endothelial cells--are relatively resistant to MA toxicity, and SSAO does not play a role in VSMC injury by MA.

Nifedipine suppresses neointimal thickening by its inhibitory effect on vascular smooth muscle cell growth via a MEK-ERK pathway coupling with Pyk2.

The aim of this study was to determine whether nifedipine could suppress an atherogenic process such as balloon-injured intimal thickening in vivo and the proliferation of vascular smooth muscle cells (VSMC) in vitro. First, we examined the in vivo effect of nifedipine to determine whether it could suppress intimal thickening induced by balloon catheterization. Sprague-Dawley (SD) rats were divided into three groups (L, nifedipine 0.3 mg kg(-1) day(-1); H, nifedipine 3 mg kg(-1) day(-1); C, no nifedipine), and Alzet((R)) osmotic pumps were implanted in their backs for continuous administration. The neointimal layers were completely occupied by proliferated VSMC, and the area ratios of neointima/media treated with nifedipine significantly decreased dose-dependently compared to those of the control. Neither blood pressure nor lipid levels changed among the three groups. We next evaluated the in vitro effect of nifedipine on the proliferation of cultured rat VSMC. Nifedipine decreased the values of [(3)H]-thymidine incorporation and total cellular protein content as well as the levels of phosphorylated extracellular signal-regulated protein kinase (ERK) 1/2, mitogen-activated protein kinase kinase (MEK) 1/2, and even the phosphorylation of Pyk2, in dose-dependent fashions. In addition, nifedipine suppressed the levels of proliferative cell nuclear antigen (PCNA) dose-dependently in both VSMC and balloon-injured thoracic aortae. These results indicate that nifedipine has an inhibitory effect on intimal thickening by attenuating intimal VSMC proliferation, suggesting that nifedipine could be effective for preventing the progression of atherosclerotic plaque as in restenosis after angioplasty.

Negative feedback regulation of reactive oxygen species on AT1 receptor gene expression.

Free radicals as well as the AT1 receptor are involved in the pathogenesis of cardiovascular disease. Both the intracellular mechanisms of AT1 receptor regulation and the effect of free radicals on AT1 receptor expression are currently unknown. This study investigates the role of free radicals in the modulation of AT1 receptor expression and in the angiotensin II-induced AT1 receptor regulation. AT1 receptor mRNA was assessed by Northern blotting and AT1 receptor density by radioligand binding assays, respectively, in vascular smooth muscle cells (VSMC). Free radical release was measured by confocal laser scanning microscopy. AT1 receptor mRNA transcription rate was determined by nuclear run-on assays and AT1 receptor mRNA half-life was measured under transcriptional blockade. Angiotensin II caused a time-dependent decrease of AT1 receptor mRNA expression in rat VSMC in culture (30+/-6% at 4 h with 100 nM angiotensin II). This was followed by a consistent decrease in AT1 receptor density. Angiotensin II caused release of reactive oxygen species in VSMC which was abolished by preincubation with 100 microM diphenylene iodonium (DPI). DPI inhibited partially the down-regulating effect of angiotensin II on the AT1 receptor. Incubation of VSMC with either hydrogen peroxide or xanthine/xanthine oxidase caused a dose-dependent decrease in AT1 receptor mRNA expression which was not mediated by a decreased rate of transcription but rather through destabilization of AT1 receptor mRNA. Experiments which included preincubation of VSMC with various intracellular inhibitors suggested that free radicals caused AT1 receptor downregulation through activation of p38-MAP kinase and intracellular release of calcium. However, angiotensin II-induced AT1 receptor expression was not inhibited by blockade of p38-MAP kinase activation or intracellular calcium release. Free radicals may at least in part mediate angiotensin II-induced AT1 receptor regulation through direct post-transcriptional effects on AT1 receptor mRNA expression which involves intracellular release of calcium and activation of p38-MAP kinase. These findings may help to clarify the intracellular mechanisms involved in AT1 receptor regulation and reveal a novel biological feature for reactive oxygen species.

Pyk2 Is a Redox-Sensitive Tyrosine Kinase in Vascular Smooth Muscle Cells

P162 PYK2 is a focal adhesion kinase-related protein tyrosine kinase (PTK) and is implicated in downstream mitogen-activated protein kinase activation. In cultured vascular smooth muscle cells (VSMCs), Ca 2+ and/or protein kinase C- (PKC) dependent PYK2 activation by angiotensin II and platelet-derived growth factor has been reported. Because reactive oxygen species (ROS) have been shown to mediate mitogenic signals by these agonists in VSMCs, we hypothesized that PYK2 represents a redox-sensitive PTK used by vascular mitogens. Activation of PYK2 was assessed by three distinct methods: (1) immunoblotting with phospho-specific antibody against autophosphorylated PYK2 at Tyr 402 ; (2) immunoprecipitation of PYK2 by anti-PYK2 antibody and immunoblotting with anti-phosphotyrosine antibody; (3) measurement of PYK2’s kinase activity towards the tyrosine kinase specific substrate poly-[Glu 80 Tyr 20 ] after immunoprecipitating with anti-PYK2 antibody. In cultured rat VSMCs, H 2 O 2 concentration- and time- dependently induced PYK2 phosphorylation at its autophosphorylation site Tyr 402 . Maximal phosphorylation occurred using 10-20 μM H 2 O 2 at 5-10 min and the phosphorylated protein co-migrated with a major tyrosine-phosphorylated 120 kDa protein induced by H 2 O 2 . Diamide (1 mM), a thiol-oxidizing agent, gave similar results to that of H 2 O 2 . H 2 O 2 also increased tyrosine phosphorylation of PYK2 immunoprecipitated with anti PYK2 antibody. Moreover, treatment of VSMCs with 10 μM H 2 O 2 for 5 min increased PTK activity immunoprecipitated with anti PYK2 antibody. However, 10 μM H 2 O 2 had no effect on intracellular Ca 2+ concentration detected by the fluorescent dye, fura-2 and GF109203X, a PKC inhibitor, failed to inhibit H 2 O 2 -induced PYK2 phosphorylation indicating that ROS-dependent PYK2 activation does not require Ca 2+ or PKC. These data demonstrated for the first time that PYK2 represents a major redox-sensitive PTK in the vessel wall, suggesting its pivotal role in vascular remodeling.