Platonin inhibited PDGF-BB-induced proliferation of rat vascular smooth muscle cells via JNK1/2-dependent signaling

Aim:

To examine the inhibitory actions of the immunoregulator platonin against proliferation of rat vascular smooth muscle cells (VSMCs).

Methods:

VSMCs were prepared from the thoracic aortas of male Wistar rats. Cell proliferation was examined using MTT assays. Cell cycles were analyzed using flow cytometry. c-Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, AKT, and c-Jun phosphorylation or p27 expression were detected using immunoblotting.

Results:

Pretreatment with platonin (1–5 μmol/L) significantly suppressed VSMC proliferation stimulated by PDGF-BB (10 ng/mL) or 10% fetal bovine serum (FBS), and arrested cell cycle progression in the S and G₂/M phases. The same concentrations of platonin significantly inhibited the phosphorylation of JNK1/2 but not ERK1/2 or AKT in VSMCs stimulated by PDGF-BB. Furthermore, platonin also attenuated c-Jun phosphorylation and markedly reversed the down-regulation of p27 expression after PDGF-BB stimulation.

Conclusion:

Platonin inhibited VSMC proliferation, possibly via inhibiting phosphorylation of JNK1/2 and c-Jun, and reversal of p27 down-regulation, thereby leading to cell cycle arrest at the S and G₂/M phases. Thus, platonin may represent a novel approach for lowering the risk of abnormal VSMC proliferation and related vascular diseases.     

Endothelin-1-induced signaling pathways in vascular smooth muscle cells

Endothelin-1 (ET-1), a vasoactive peptide, is believed to contribute to the pathogenesis of vascular abnormalities such as hypertension, atherosclerosis, hypertrophy and restenosis. ET-1 elicits its biological effects through the activation of two receptor subtypes, ET-A and ET-B that belong to a large family of transmembrane guanine nucleotide-binding protein-coupled receptors (GPCRs). ET-1 receptor activation results in the stimulation of several signaling pathways including mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB). An intermediary role of Ca(2+)/calmodulin-dependent protein kinases (CaMK), protein kinase C (PKC) as well as receptor and non-receptor protein tyrosine kinases in triggering the activation of MAPK and PI3-K/PKB signaling in response to ET-1 has been suggested. Activation of these pathways by ET-1 is intimately linked with the regulation of cellular hypertrophy, growth, proliferation and cell survival. Here we provide an overview of these signaling pathways in vascular smooth muscle cells (VSMCs) with an emphasis on their potential role in vascular pathophysiology.     

血管平滑肌的ATP敏感钾通道

ＡＴＰ敏感钾通道（ＫＡＴＰ）是一类较广泛分布的内向整流钾通道。在生理状态及某些病理条件下ＫＡＴＰ参与血管张力的调节。ＫＡＴＰ活性受多种因素的调控,胞内二磷酸核苷酸（ＮＤＰｓ）、钾通道开放剂（ＫＣＯｓ）等可激活通道,而ＡＴＰ和硫脲类药物则特异性抑制通道的开放。分子生物学研究证明ＫＡＴＰ由Ｋｉｒ６０和硫脲类受体（ＳＵＲ）共同组成,Ｋｉｒ６０构成Ｋ＋可穿透的通道核心,ＳＵＲ受体构成通道的调节单位。血管平滑肌中的ＫＡＴＰ是由Ｋｉｒ６１和ＳＵＲ２Ｂ组成的四聚体结构。但两种亚单位如何联结成有功能的多聚体还需进一步的证明。     

Celecoxib induces heme oxygenase-1 expression in macrophages and vascular smooth muscle cells via ROS-dependent signaling pathway

The multiple cytoprotective mechanisms of heme oxygenase (HO)-1 make it a promising therapeutic target. This study investigated whether the selective cyclooxygenase (COX)-2 inhibitor, celecoxib, can upregulate HO-1 expression. Murine J774 macrophages and rat aortic vascular smooth muscle cells (VSMCs) were used to study the effect of celecoxib on HO-1 expression. A signal transduction pathway involving reactive oxygen species (ROS) was also investigated. We found that celecoxib can upregulate HO-1 gene and protein expressions in J774 macrophages and VSMCs. This effect was not diminished by prostaglandin E(2) or 15dPGJ(2), while it was additive to hypoxia-induced HO-1 expression, suggesting an event independent of COX-2 activity or hypoxia-inducible factor-1α. Moreover, celecoxib activated ERK, p38, Akt, and Nrf2 as well as increased ROS production. All these events contributed to the increase in the expression of HO-1 caused by celecoxib. In this study, we also, for the first time, demonstrated that AMP-activated protein kinase (AMPK) can mediate HO-1 expression via the downstream activation of p38 and Akt. However, the HO-1-inducing actions of celecoxib and hypoxia were not associated with AMPK. This study demonstrates a COX-2-independent action of celecoxib in upregulating HO-1 in macrophages and VSMCs. This action is dependent on ROS, Akt, ERK, p38, and Nrf2 activation. These findings provide new insights into the action mechanism of celecoxib with broad implications for anti-inflammation therapy.     

Lercanidipine inhibits vascular smooth muscle cell proliferation and neointimal formation via reducing intracellular reactive oxygen species and inactivating Ras-ERK1/2 signaling

Lercanidipine, a calcium channel antagonist, is currently employed in the treatment of essential hypertension and angina pectoris. The purpose of this study was to elucidate the anti-proliferative effect of lercanidipine and to investigate the molecular role of this agent. Both in vitro studies and in a balloon injury rat carotid artery model were employed to study the effect of lercanidipine on smooth muscle cell proliferation. Lercanidipine-inhibited rat vascular smooth muscle cell (VSMC) proliferation and migration in a dose-dependent manner following stimulation of VSMC cultures with 10% fetal bovine serum (FBS) and 20 ng/ml platelet-derived growth factor (PDGF)-BB. FBS- and PDGF-BB-stimulated intracellular Ras, MEK1/2, ERK1/2, proliferative cell nuclear antigen (PCNA), and Akt activations were significantly inhibited by lercanidipine; however, lercanidipine did not affect FBS- and PDGF-BB-induced STAT3 phosphorylation. Lercanidipine also inhibited PDGF-receptor β chain phosphorylation and reactive oxygen species (ROS) production induced by PDGF-BB. Lercanidipine blocked the FBS-inducible progression through the G₀/G₁ to the S-phase of the cell cycle in synchronized cells. In vivo, 14 days after balloon injury, treatment with 3 and 10 mg/kg lercanidipine resulted in significant inhibition of the neointima/media ratio. Suppression of neointima formation by lercanidipine was dependent on its influence on ERK1/2 phosphorylation. These results demonstrate that lercanidipine can suppress the proliferation of VSMCs via inhibiting cellular ROS, Ras-MEK1/2-ERK1/2, and PI3K-Akt pathways, and suggesting that it may have therapeutic relevance in the prevention of human restenosis.     

Kinetics of relaxation by cGMP/cGKI signaling in fundus smooth muscle

cGMP-dependent kinase I (cGKI) is a major mediator of smooth muscle relaxation and exists in two isoforms, α and β. Both isoforms are supposed to mediate their effects via different intracellular signaling pathways. To verify this concept, the kinetics of relaxation mediated by either isoform was analyzed in gastric fundus smooth muscle from mice. Muscles from mice that express selectively the Iα or Iβ isoform of cGKI in smooth muscle (sm-cGKIα or sm-cGKIβ mice) were compared to muscles from conventional cGKI^−/− mice. Fundus muscles were contracted by carbachol and then relaxed by 8-Br-cGMP or by electrical field stimulation (EFS). The time course of relaxation by 8-Br-cGMP was not different between muscles from sm-cGKIα and sm-cGKIβ mice. EFS induced a fast transient relaxation in muscles from sm-cGKIα and sm-cGKIβ mice that was blocked by the NO synthase inhibitor L-NAME. Recovery from this relaxation was about 4-times slower in muscles from sm-cGKIα mice than in muscles from sm-cGKIβ mice. The different kinetic of recovery from relaxation after EFS in sm-cGKIα and sm-cGKIβ mice suggests that different signaling pathways exist for each cGKI isoform in vivo in fundus muscles.     

Nisoldipine-induced relaxation in intact and skinned smooth muscles of rabbit coronary arteries

In smooth muscles of the rabbit coronary artery, nisoldipine inhibited the phasic and tonic responses of the contraction induced by 128 mM K (the IC50 values were 4 X 10(-8) M and 1 X 10(-13) M, respectively). This agent also inhibited the tonic response of the acetylcholine (ACh) (10(-5) M)-induced contraction (the IC50 value was 3 X 10(-10) M), but only slightly inhibited the phasic response (in 10(-7) M, 0.86 times the control). Nisoldipine (less than 10(-7) M) had no effect on the K-induced depolarization of the membrane at any given concentration. This drug (5 X 10(-8) M) did inhibit the oscillatory potential changes and spike potential evoked on the ACh-induced slow depolarization. After depletion of stored Ca from the polarized muscles (5.9 mM K), muscle cells accumulated Ca by application of 2.6 mM Ca without generation of contraction, i.e. a subsequently applied 20 mM caffeine produced the contraction in Ca-free solution. Nisoldipine (less than 10(-7) M) had little effect on this accumulation of Ca. The rate of rise and time to reach the maximum amplitude of the 128 mM K- or ACh-induced contraction (in 2.6 mM Ca) depended on the amount of stored Ca in cells. Nisoldipine (10(-8) M) consistently inhibited the Ca-induced contraction evoked in depolarized muscles (128 mM K), regardless of the amount of stored Ca. However, this agent (10(-8) M) did not inhibit the Ca release from storage sites evoked by activation of the muscarinic receptor. After prolonged superfusion (over 120 min) with Na- and Ca-free solution (guanethidine and atropine were present), application of 2.6 mM Ca produced contraction which was inhibited by 10(-8) M nisoldipine, while the depolarization induced by application of these solutions was not inhibited by nisoldipine. In saponin-skinned muscles, nisoldipine had no effect on the contractile proteins, as estimated from the pCa-tension relationship, or on the Ca accumulation into the Ca release from the Ca storage sites, as estimated from the caffeine-induced contraction. It is concluded that nisoldipine possesses a selective inhibitory action on voltage-dependent Ca influx, when the Ca channel is activated by depolarization.     

Cyclic nucleotide phosphodiesterase inhibition and vascular smooth muscle relaxation

The inhibitory effects of theophylline, papaverine and isoquinoleine derivatives and the activating action of imidazole were compared on purified 3′,5′-cylcic nucleotide phosphodiesterase and on isolated rat aorta. A quantitative correlation was found between inhibition of the enzyme activity (K_i) and prevention of isolated rat aorta contractions elicited by barium.     

Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries

Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2alpha (3 microM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC(50)=9.64+/-0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC(50)=9.23+/-0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 microM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC(50)=0.043 microg ml(-1)), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC(50)=0.14 microg ml(-1)) of the cathepsin G effect, whereas neither indomethacin (3 microM) nor the thrombin inhibitor hirudin (5 ATU ml(-1)) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC(50)=0.11 microg ml(-1)), heparin (IC(50)=0.48 microg ml(-1)) and suramin (IC(50)=1.85 microg ml(-1)) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like thrombin and trypsin, cathepsin G is able to induce endothelium-dependent vascular relaxation. It can be released from activated leukocytes at sites of vascular injury and inflammation and, therefore, sufficiently high concentrations might be reached locally in the vascular space to induce vasodilatation.     

高磷通过SET8调控p53/Bcl-2/Caspase信号通路促进血管平滑肌细胞钙化

摘要：目的 探讨组蛋白赖氨酸甲基转移酶SET8在高磷诱导血管平滑肌细胞（VSMCs）钙化中的作用及机制。 方法 选取80～100 g的清洁雄性SD大鼠6只,取其胸主动脉,分离VSMCs后进行原代培养。VSMCs传至第3～4 代,铺6孔板,并给予相应刺激。将VSMCs随机分为正常组和高磷组（10 mmol/L β-甘油磷酸）,培养4 d后茜素红染 色观察钙结节形成情况,甲基麝香草酚蓝比色法钙含量,流式细胞数检测细胞凋亡,RT-PCR 和 Western blot 检测 SET8、p53、Bcl-2、Bax、Caspase3的mRNA和蛋白的表达水平。之后用脂质体将SET8-shRNA质粒与NS-shRNA转染 VSMCs 作为 SET8-shRNA 组和空质粒组,未转染组作为正常对照组,RT-PCR 和 Western blot 检测 p53、Bcl-2、Bax、 Caspase3的mRNA和蛋白的表达水平。结果 （1）高磷组钙盐沉积较正常组明显增加（P＜0.05）。（2）流式细胞仪检 测结果显示,与正常组相比,高磷组VSMCs的细胞凋亡率明显增加（P＜0.05）。（3）与正常组相比,高磷组Bcl-2、SET8 的mRNA和蛋白表达水平下降;p53、Bax、Caspase3的mRNA和蛋白表达水平升高（P＜0.05）。（4）干扰SET8基因表达 后钙盐沉积增加（P＜0.05）,Bcl-2的mRNA和蛋白表达水平下降;p53、Bax、Caspase3的mRNA和蛋白表达水平升高 （P＜0.05）。结论 高磷通过SET8调控p53/Bcl-2/Caspase信号通路,下调抗凋亡蛋白Bcl-2表达,上调促凋亡蛋白 Bax和Caspase3的表达,进而参与调控VSMCs的钙化。     

Ezetimibe suppresses cholesterol accumulation in lipid-loaded vascular smooth muscle cells in vitro via MAPK signaling

Aim:

To investigate the mechanisms of anti-atherosclerotic action of ezetimibe in rat vascular smooth muscle cells (VSMCs) in vitro.

Methods:

VSMCs of SD rats were cultured in the presence of Chol:MβCD (10 μg/mL) for 72 h, and intracellular lipid droplets and cholesterol levels were evaluated using Oil Red O staining, HPLC and Enzymatic Fluorescence Assay, respectively. The expression of caveolin-1, sterol response element-binding protein-1 (SREBP-1) and ERK1/2 were analyzed using Western blot assays. Translocation of SREBP-1 and ERK1/2 was detected with immunofluorescence.

Results:

Treatment with Chol:MβCD dramatically increased the cellular levels of total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC) in VSMCs, which led to the formation of foam cells. Furthermore, Chol:MβCD treatment significantly decreased the expression of caveolin-1, and stimulated the expression and nuclear translocation of SREBP-1 in VSMCs. Co-treatment with ezetimibe (3 μmol/L) significantly decreased the cellular levels of TC, CE and FC, which was accompanied by elevation of caveolin-1 expression, and by a reduction of SREBP-1 expression and nuclear translocation. Co-treatment with ezetimibe dose-dependently decreased the expression of phosphor-ERK1/2 (p-ERK1/2) in VSMCs. The ERK1/2 inhibitor PD98059 (50 μmol/L) altered the cholesterol level and the expression of p-ERK1/2, SREBP-1 and caveolin-1 in the same manner as ezetimibe did.

Conclusion:

Ezetimibe suppresses cholesterol accumulation in rat VSMCs in vitro by regulating SREBP-1 and caveolin-1 expression, possibly via the MAPK signaling pathway.     

Oleic acid enhances vascular smooth muscle cell proliferation via phosphatidylinositol 3-kinase/Akt signaling pathway.

Nonesterified fatty acids are acutely liberated during lipolysis and are chronically elevated in pathological conditions such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The present study was designed to investigate the effects of oleic acid (OA), an 18-carbon cis-monosaturated fatty acid on proliferation of vascular smooth muscle cells (VSMC). Incubation of a rat VSMC (A10 cells) with OA (50 microM) resulted in an increase of cells entering the S phase of the cell cycle. In consistent with the effects on cell cycle distribution, OA stimulated VSMC proliferation in a dose-dependent manner. The mitogenic effect of OA was significantly reduced by pretreatment of LY294002 (5 microM) or wortmannin (1 microM), potent, and specific inhibitors of phosphatidylinositol 3-kinase (PI3K). OA also induced activation of Akt/protein kinase B (PKB) in a time-dependent manner. OA-induced activation of Akt/PKB was inhibited by either LY294002 or wortmannin. Taken together, these experiments show that the enhanced phosphorylation of Akt/PKB by OA is dependent on PI3K and suggest that this signaling event may be important for the regulation of OA-induced VSMC proliferation.     

NK1 receptor-mediated endothelium-dependent relaxation and contraction with different sensitivity to post-receptor signaling in pulmonary arteries

In rabbit intrapulmonary arteries, substance P (SP) has been reported to induce endothelium-dependent relaxation (EDR) and endothelium-dependent contraction (EDC) via tachykinin NK₁ receptors, and endothelium-independent contraction (EIC) via tachykinin NK₂ receptors. The present study pharmacologically examined whether these opposite responses (EDR and EDC) are mediated by the same NK₁ receptor. Five tachykinin agonists, including septide, a reportedly atypical NK₁ agonist, caused concentration-dependent EDR in the presence of NK₂ antagonist (SR-48968) + TXA₂ synthetase inhibitor (ozagrel), which blocked EIC and EDC, in pre-contracted arteries, and concentration-dependent EDC in the presence of NK₂ antagonist (SR-48968) + nitric oxide synthase inhibitor (l-N^G-nitro-arginine methyl ester), which blocked EIC and EDR, in non-contracted arteries. The EC₅₀ values of these agonists for EDR were smaller than those for EDC, indicating that the affinities of NK₁ agonists to NK₁ receptors are different between EDR and EDC. However, the rank order of their potency for EDR and EDC was the same: SP = septide > SP methyl ester (SPME) > neurokinin A > neurokinin B. [Ala⁵, β-Ala⁸]-α-neurokinin fragment 4–10 (NK₂ agonist) and senktide (NK₃ agonist) caused no responses. Two structurally different NK₁ antagonists, CP-99994 and SR-140333, shifted the concentration–EDC and –EDR curves of SPME, a selective NK₁ agonist, and septide rightward and suppressed their maximal responses in a similar concentration-dependent manner, indicating that the affinities of NK₁ antagonists to NK₁ receptors are similar between EDR and EDC. U-73122, a phospholipase C inhibitor, and thapsigargin, 2,5-di-tert-butylhydroquinone, and ruthenium red, all intracellular Ca²⁺ release blockers, inhibited SP-induced EDR and EDC. Effective concentrations of ionomycin (Ca²⁺ ionophore) causing EDR were also lower than those causing EDC. Taken together, SP-induced EDR and EDC are mediated by activation of the same NK₁ receptor followed by an increase in intracellular Ca²⁺, and sensitivity to Ca²⁺ may be higher in the EDR than EDC pathway.     

Desensitization of beta-adrenoceptor- and prostaglandin E1 receptor-mediated human vascular smooth muscle relaxation.

Regulation of beta-adrenoceptors in animal tissues and human cell cultures has been extensively described; on the other hand, relatively little is known about regulation of beta-adrenoceptors in human tissues in vivo. Both beta-adrenoceptors and the prostaglandin E1 (PGE1) receptors stimulate vasodilation. We wondered if prolonged infusion of isoproterenol or PGE1 would cause desensitization of smooth muscle relaxation and used the dorsal hand-vein compliance technique to investigate this question. After constructing a dose-response curve to either the beta-agonist isoproterenol or to PGE1 in a phenylephrine preconstricted vein, isoproterenol (271 ng/min), PGE1 (956 pg/min), or saline was infused for 4 h in separate experiments. There was no change in the ED50 or Emax for either isoproterenol or PGE1 after saline infusion. After a 4-h infusion of isoproterenol, the maximal vasodilator response to isoproterenol was significantly (p less than 0.01) attenuated from 61 +/- 33% to 19 +/- 10%, while the ED50 significantly increased (p less than 0.01) from a geometric mean of 37 to 197 ng/min. After infusion of isoproterenol, the mean maximum PGE1-induced venorelaxation of 129 +/- 29% was modestly but significantly (p less than 0.05) blunted to 96 +/- 35%, while the ED50 of PGE1 increased significantly (p less than 0.01) from a geometric mean of 81 to 398 pg/min. A 4-h infusion of PGE1 significantly (p less than 0.01) attenuated the maximum response to PGE1 from 73 +/- 35 to 28 +/- 16%. The maximal vasodilatory response to isoproterenol was also significantly blunted (p less than 0.05) from 62 +/- 35 to 42 +/- 41%, with no change in ED50.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular effects of propofol: smooth muscle relaxation in isolated veins and arteries

Isolated hepatic portal veins and aorta taken from the rat were used to investigate the direct action of the intravenous anaesthetic propofol. This compound is known to produce a fall in blood pressure in man and animals and it has been suggested that the hypotension may result from a direct vasodilator action on the veins and arterioles. In our experiments propofol caused a dose related decrease of potassium-induced tone in both types of blood vessel. However, the concentrations required to produce this effect in the experiments on veins were significantly lower than those required to produce similar changes in the isolated artery preparation. We conclude that this direct action may contribute towards the hypotensive effects of propofol.     

Endothelin-1 acts via protein kinase C to block KATP channels in rabbit coronary and pulmonary arterial smooth muscle cells

We investigated the effects of the vasoconstrictor endothelin-1 (ET-1) on the whole-cell ATP-sensitive K+ (KATP) currents of smooth muscle cells that were isolated enzymatically from rabbit coronary artery (CASMCs) and pulmonary artery (PASMCs). The size of the KATP current did not differ significantly between CASMCs and PASMCs. ET-1 reduced the KATP current in a concentration-dependent manner, and this inhibition was greater in PASMCs than in CASMCs (half-inhibition values of 12.20 nM and 1.98 nM in CASMCs and PASMCs, respectively). However, the level of inhibition induced by other vasoconstrictors (angiotensin II, norepinephrine, and serotonin) were not significantly different between CASMCs and PASMCs. Pretreatment with the protein kinase C (PKC) inhibitors staurosporine (100 nM) and GF 109203X (1 microM) prevented ET-1-induced inhibition of the KATP current in both arterial smooth muscle cell preparations. The PKC activators phorbol-12,13-dibutyrate (PDBu) and 1-olelyl-2-acetyl-sn-glycerol (OAG) reduced the KATP current in dose-dependent manner. Although the numbers of ET receptors were not significantly different between the 2 arterial smooth muscle cell preparations, the effects of PDBu and OAG were greater on PASMCs. ET-1-induced inhibition of the KATP current was unaffected by the PKA inhibitor Rp-cAMPs (100 microM) and PKA inhibitory peptide (5 microM).