Docosahexaenoic acid, a peroxisome proliferator-activated receptor-alpha ligand, induces apoptosis in vascular smooth muscle cells by stimulation of p38 mitogen-activated protein kinase

Omega-3 fatty acids (n-3 FAs) have been shown to exert a blood pressure-lowering effect in hypertension, possibly in part by influencing vascular structure. We previously demonstrated that n-3 FAs induce vascular smooth muscle cell (VSMC) apoptosis, which could exert an effect on the structure of blood vessels. In the present study, we investigated signaling pathways through which n-3 FAs mediate apoptosis in VSMCs. Cultured mesenteric VSMCs from Sprague-Dawley rats were stimulated with docosahexaenoic acid (DHA), a representative n-3 FAs. Morphological changes in apoptosis and DNA fragmentation were examined with phase-contrast microscopy and fluorescence microscopy with Hoechst 33342 staining. To clarify possible pathways of apoptosis, we evaluated the expression of phosphorylated p38 mitogen-activated protein kinases, bax, bcl-2, cytochrome c, and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) with Western blot analysis. DHA treatment induced cell shrinkage, cell membrane blebbing, and apoptotic bodies in VSMCs. DHA time-dependently activated p38 mitogen-activated protein kinases, bax, PPAR-alpha, and cytochrome c, with maximal effects obtained after 5 and 30 minutes and 1 and 3 hours, respectively. SB-203580 and SB-202190, selective p38 inhibitors, reduced DHA-elicited apoptosis and expression of PPAR-alpha but had no effect on the expression of bax or cytochrome c. The present results indicate that DHA induces apoptosis in VSMCs through >/=2 distinct mechanisms: (1) a p38-dependent pathway that regulates PPAR-alpha and (2) a p38-independent pathway via dissipation of mitochondrial membrane potential and cytochrome c release. The death-signaling pathway stimulated by DHA may involve an integration of these multiple pathways. By triggering VSMC apoptosis, DHA may play a pathophysiological role in vascular remodeling in cardiovascular disease.     

Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway

Previous studies have shown that mitofusin 2 (Mfn-2) (or hyperplasia suppressor gene [HSG]) inhibits vascular smooth muscle cell (VSMC) proliferation. Here, we demonstrate that Mfn-2 is a primary determinant of VSMC apoptosis. First, oxidative stress with H2O2, inhibition of protein kinase C with staurosporine, activation of protein kinase A with forskolin, and serum deprivation concurrently elevate Mfn-2 expression and induce VSMC apoptosis. Second, overexpression of Mfn-2 also triggers apoptosis of VSMCs in culture and in balloon-injured rat carotid arteries, thus contributing to Mfn-2-mediated prevention of neointima formation after angioplasty. Third, Mfn-2 silencing protects VSMCs against H2O2 or Mfn-2 overexpression-induced apoptosis, indicating that upregulation of Mfn-2 is necessary and sufficient for oxidative stress-mediated VSMC apoptosis. The Mfn-2 proapoptotic effect is independent of its role in mitochondrial fusion but mainly mediated by inhibition of Akt signaling and the resultant activation of the mitochondrial apoptotic pathway, as manifested by decreased Akt phosphorylation, increased mitochondrial Bax/Bcl-2 ratio, cytochrome c release, and activation of caspases-9 and caspase-3. Furthermore, Mfn-2-induced apoptosis was blocked by overexpression of an active phosphoinositide 3-kinase mutant or Bcl-xL or inhibition of caspase-9 but not caspases-8. Thus, in addition to its antiproliferative effects, Mfn-2 constitutes a primary determinant of VSMC apoptosis.     

Estrogen and raloxifene induce apoptosis by activating p38 mitogen-activated protein kinase cascade in synthetic vascular smooth muscle cells

Proliferation of vascular smooth muscle cells (VSMC) plays a major role as an initiating event of atherosclerosis. Although estrogen directly inhibits the proliferation of VSMC, the mechanism has not been firmly established. In addition, the effect of raloxifene on VSMC remains unknown. 17Beta-estradiol (E(2)) and raloxifene significantly inhibited the growth of VSMC under growth-stimulated conditions. Since mitogen-activated protein (MAP) kinases have been implicated in VSMC proliferation, the role of MAP kinases in both the E(2)- and raloxifene-induced growth inhibition of VSMC was studied. Both E(2) and raloxifene caused rapid, transient phosphorylation and activation of p38 that was not affected by actinomycin D and was blocked by ICI 182,780. In contrast with p38 phosphorylation, extracellular signal-regulated protein kinase (ERK) phosphorylation was significantly inhibited and c-Jun N-terminal kinase (JNK) phosphorylation was not changed by E(2). Because VSMC expressed both estrogen receptor (ER) alpha and ERbeta, it is not known which of them mediates the E(2)-induced phosphorylation of p38. Although E(2) did not affect the p38 phosphorylation in A10 smooth muscle cells, which express ERbeta but not ERalpha, transfection of ERalpha expression vector into A10 cells rendered them susceptible to induction of p38 phosphorylation by E(2). We then examined whether E(2) and raloxifene induce apoptosis through a p38 cascade. Both E(2) and raloxifene induced apoptosis under growth-stimulated conditions. The p38 inhibitor SB 203580 completely blocked the E(2)-induced apoptosis. Our findings suggest that both E(2)- and raloxifene-induced inhibition of VSMC growth is due to induction of apoptosis through a p38 cascade whose activation is mediated by ERalpha via a nongenomic mechanism.     

Bone morphogenic protein-4 induces endothelial cell apoptosis through oxidative stress-dependent p38MAPK and JNK pathway

The expression of bone morphogenic protein 4 (BMP4), a new pro-inflammatory marker, is increased by disturbed flow in endothelial cells (ECs). BMP4 stimulates production of reactive oxygen species (ROS) and causes endothelial cell dysfunction. The present study examined BMP4-induced apoptosis in ECs and isolated arteries from rat, mouse, and human, and the signaling pathways mediating BMP4-induced apoptosis. Apoptosis was assessed by flow cytometry to detect Annexin-V positive cells, and terminal deoxynucleotidyl transferase dUTP nick end (TUNEL) labeling. The superoxide production was measured by dihydroethidium fluorescence. BMP4 induced EC apoptosis in human mesenteric arteries, mouse aortic endothelium, rat primary ECs, and human ECs. BMP4-induced EC apoptosis was mediated through ROS production by activation of NADPH oxidase, which led to cleaved caspase-3 expression. BMP4 also induced sequential activation of p38 MAPK and JNK which was upstream of caspase 3 activation. Knockdown of BMP receptor 1A by lentiviral shRNA or NOX4 siRNA transfection inhibited BMP4-induced ROS production, p38 and JNK phosphorylation, and caspase-3 activation in ECs. JNK siRNA inhibited BMP4-induced JNK phosphorylation and caspase-3 activation. The present study delineates that BMP4 causes EC apoptosis through activation of caspase-3 in a ROS/p38MAPK/JNK-dependent signaling cascade.     

Overexpression of CREG attenuates atherosclerotic endothelium apoptosis via VEGF/PI3K/AKT pathway

Aims

Cellular repressor of E1A-stimulated genes (CREG) is a homeostasis-modulating gene abundantly expressed in adult artery endothelium. Previous studies have demonstrated a protective effect of CREG against atherosclerosis through prevention of vascular smooth muscle cell apoptosis. However, the role of CREG in endothelial cells (ECs) apoptosis and the underlying signaling mechanisms are unknown.

Method and results

We ascertained that CREG expression was decreased in atherogenesis-prone endothelium in apolipoprotein E-null (apoE^−/−) mice compared with their wild-type littermates using in situ immunofluorescent staining. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining and caspase-3 activity assays determined that treatment of apoE^−/− mice arteries with staurosporine (STS) significantly induced endothelial apoptosis associated with a reduction of CREG expression. Gain- and loss-of-function analyses revealed that silencing CREG expression significantly enhanced ECs apoptosis, whereas CREG overexpression abrogated apoptosis stimulated by STS or etoposide (VP-16). Blocking assays using the neutralizing antibody for vascular endothelial growth factor (VEGF) and the specific inhibitor of phosphoinositide 3-kinase (PI3K), such as LY294002 or wortmannin, demonstrated that the protective effect of CREG on ECs apoptosis was mainly mediated by activation of the VEGF/PI3K/AKT signaling pathway.

Conclusions

These data demonstrate that CREG plays a critical role in protecting the vascular endothelium from apoptosis, and the protective effort of CREG against ECs apoptosis is through the activation of the VEGF/PI3K/AKT signaling pathway.     

Inhibition of vascular smooth muscle cell proliferation,migration, and survival by the tumor suppressor protein PTEN

Phosphatidylinositol (PI) 3-kinase signaling regulates numerous cellular processes, including proliferation, migration, and survival, which are required for neointimal hyperplasia and restenosis. The effectors of PI 3-kinase are activated by the phospholipid products of PI 3-kinase. In this report, we investigated the hypothesis that overexpression of the tumor suppressor protein PTEN, an inositol phosphatase specific for the products of PI 3-kinase, would inhibit the vascular smooth muscle cell (VSMC) responses necessary for neointimal hyperplasia and restenosis. Effects of PTEN were assessed in primary rabbit VSMCs after overexpression with a recombinant adenovirus and compared with uninfected or control virus-infected cells. PTEN was expressed endogenously in VSMCs, and PTEN overexpression inhibited PDGF-induced phosphorylation of p70(s6k), Akt, and glycogen synthase kinase-3-alpha and -beta but not ERK1 or -2. Overexpression of PTEN significantly inhibited both basal and PDGF-mediated VSMC proliferation and migration, the latter possibly due in part to downregulation of focal adhesion kinase. Moreover, PTEN overexpression induced cleavage of caspase-3 and significantly increased apoptosis compared with control cells. Taken together, these results demonstrate that PTEN overexpression potently inhibits the VSMC responses required for neointimal hyperplasia and restenosis. Adenovirus-expressed PTEN may therefore provide a useful tool for the local treatment of these and other vascular proliferative disorders.     

软脂酸通过丝裂原活化蛋白激酶通路促进血管内皮细胞凋亡

目的探讨软脂酸(PA)诱导的血管内皮细胞凋亡中丝裂原活化蛋白激酶(MAPK)通路的作用。方法将人脐静脉内皮细胞(HUVEC)分对照组、PA组、MAPK通路干预组[分别先用p38抑制剂SB203580、氨基末端激酶(JNK)抑制剂PD98059、细胞外信号调节激酶(ERK)抑制剂SP600125干预]再分为PA+SB组、PA+PD组、PA+SP组。流式细胞仪检测细胞凋亡率;Western blot法检测caspase-3、磷酸化p38、JNK和ERK1/2表达水平;分光光度法检测caspase-3的活性。结果与对照组比较,PA组、PA+SB组、PA+PD组、PA+SP组HUVEC凋亡及caspase-3表达和活性明显增加,PA组磷酸化p38MAPK表达明显增加(P<0.05)。与PA组比较,PA+SB组HUVEC细胞凋亡率、caspase-3表达和活性明显降低(P<0.05);而PA+PD组和PA+SP组HUVEC凋亡率、caspase-3表达和活性无明显变化(P>0.05)。结论 PA通过p38MAPK通路促进内皮细胞凋亡。     

Frequency-dependent phenotype modulation of vascular smooth muscle cells under cyclic mechanical strain

Phenotype transformation of vascular smooth muscle cells (VSMCs) is known to be modulated by mechanical strain. The present study was designed to investigate how different frequencies of mechanical strain affected VSMC phenotype. VSMCs were subjected to the strains of 10% elongation at 0, 0.5, 1 and 2 Hz for 24 h using a Flexercell strain unit. VSMC phenotype was assessed by cell morphology, measurement of two-dimensional cell area, Western blotting for protein and RT-PCR for mRNA expression of differentiation markers. Possible protein kinases involved were evaluated by Western blotting with their specific antibodies. The strains at certain frequencies could induce a contractile morphology in VSMC with almost perpendicular alignment to the strain direction. The strains also regulated protein and mRNA expression of several differentiation markers, as well as the activation of extracellular signal-regulated kinases (ERKs), p38 MAP kinase and protein kinase B (Akt) in a frequency-dependent manner. Furthermore, the inhibition of the p38 pathway could block the frequency-induced phenotype modulation of VSMCs, but not inhibition of ERK or Akt pathways. These results indicate that the frequency of cyclic strain can result in the differentiated phenotype of VSMCs, and it is mediated at least partly by the activation of the p38 pathway.     

Cadmium-induced apoptosis and necrosis in human osteoblasts: role of caspases and mitogen-activated protein kinases pathways

Cadmium is a widespread environmental pollutant which induces severe toxic alterations, including osteomalacia and osteoporosis, likely by estrogen receptor-dependent mechanisms. Indeed, cadmium has been described to act as an endocrine disruptor and its toxicity is exerted both in vivo and in vitro through induction of apoptosis and/or necrosis by not fully clarified intracellular mechanism(s) of action. Aim of the present study was to further investigate the molecular mechanism by which cadmium might alter homeostasis of estrogen target cells, such as osteoblast homeostasis, inducing cell apoptosis and/or necrosis. Human osteoblastic cells (hFOB 1.19) in culture were used as an in vitro model to characterize the intracellular mechanisms induced by this heavy metal. Cells were incubated in the presence/ absence of 10-50 μM cadmium chloride at different times and DNA fragmentation and activation of procaspases- 8 and -3 were induced upon CdCl(2) treatment triggering apoptotic and necrotic pathways. Addition of caspase-8 and -3 inhibitors (Z-IETD-FMK and Z-DQMD-FMK) partially blocked these effects. No activation of procaspase-9 was observed. To determine the role of mitogen-activated protein kinases (MAPK) in these events, we investigated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation which were activated by 10 μM CdCl(2). Chemical inhibitors of JNK, p38, and ERK1/2, SP600125, SB202190, and PD98059, significantly reduced the phosphorylation of the kinases and blunted apoptosis. In contrast, caspase inhibitors did not reduce the cadmium-induced MAPK phosphorylation, suggesting an independent activation of these pathways. In conclusion, at least 2 pathways appear activated by cadmium in osteoblasts: a direct induction of caspase-8 followed by activation of caspase-3 and an indirect induction by phosphorylation of ERK1/2, p38, and JNK MAPK triggering activation of caspase-8 and -3.     

Adipocyte-derived factors regulate vascular smooth muscle cells through mineralocorticoid and glucocorticoid receptors

Adipose tissue influences vascular function through adipocyte-derived factors, including components of the renin-angiotensin-aldosterone system. Molecular mechanisms underlying these phenomena are elusive. We investigated the role of adipocyte-derived factors on mitogen-activated protein kinases (MAPKs), proinflammatory status, apoptosis, and mitogenic signaling in vascular smooth muscle cells (VSMCs) and questioned whether these effects involve mineralocorticoid receptor (MR), glucocorticoid receptor (GR), and angiotensin II type 1 receptor (AT(1)R). Cultured mouse VSMCs were exposed to adipocyte-conditioned medium (ACM) from differentiated 3T3-L1 adipocytes. ACM induced phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase, p38MAPK, and extracellular signal-regulated kinase 1/2 and increased expression of proinflammatory and proliferative markers in VSMCs. Eplerenone (MR antagonist), mifepristone (GR antagonist), and candesartan (AT(1)R antagonist) inhibited ACM-induced effects on extracellular signal-regulated kinase 1/2, p38MAPK, and proliferating cell nuclear antigen, without influencing apoptosis (Bax, Bcl, and caspase 3). Stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation was inhibited by mifepristone and candesartan but not by eplerenone. ACM-induced increase of fibronectin, vascular cell adhesion molecule 1, and plasminogen activator inhibitor 1 expression was blocked by MR and AT(1)R antagonism but not by GR inhibition. ACM has no effect on GR, MR, and AT(1)R expression. Our data show that adipocyte-derived factors influence MAPK signaling, leading to VSMC proinflammatory and profibrotic responses through distinct pathways. Although ACM stimulates p38MAPK and extracellular signal-regulated kinase 1/2 phosphorylation through MR, GR, and AT(1)R, activation of stress-activated protein kinase/c-Jun N-terminal kinase involves GR and AT(1)R. These findings suggest that adipocyte-derived factors regulate VSMC function through specific MAPKs linked to MR, GR, and AT(1)R, a posttranslational phenomenon, because ACM did not influence receptor expression. Such cross-talk between adipocytes and VSMCs may provide a potential molecular mechanism linking renin-angiotensin-aldosterone system, adipocytes, and vascular function.     

Viral vector-mediated 12/15-lipoxygenase overexpression in vascular smooth muscle cells enhances inflammatory gene expression and migration

Increased expression and activity of 12/15-lipoxygenase (12/15-LO) in vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetes and vascular complications. However, the consequences of 12/15-LO overexpression for VSMC migration and inflammatory gene expression are not known. In this study, 12/15-LO was overexpressed using adeno- and baculoviral vectors in human VSMC (HVSMCs) and proatherogenic responses compared with control enhanced green fluorescent protein (EGFP)-expressing cells. HVSMCs transduced with 12/15-LO viruses expressed high levels of enzymatically active protein and produced increased levels of the LO product, 12(S)-hydroxyeicosatetraenoic acid. 12/15-LO-overexpressing HVSMCs exhibited increased oxidant stress, activation of p38 mitogen-activated protein kinase, migration and inflammatory gene expression relative to HVSMCs expressing EGFP. Furthermore, inflammatory gene expression induced by 12/15-LO overexpression was abolished by anti-oxidants, siRNAs targeting p65 (nuclear factor-kappaB), or new-generation baculoviruses expressing inhibitory IkappaBalpha or IkappaBalpha superrepressor mutant. Thus, we have used novel viral vector delivery systems, including baculoviruses, for the first time to deliver foreign genes into VSMCs and thereby demonstrated that 12/15-LO overexpression increases oxidant stress, mitogen-activated protein kinase activation, migration and inflammatory genes in VSMCs and that NF-kappaB is a key downstream effector. Enhanced proatherogenic responses in VSMCs triggered by increased 12/15-LO levels under pathological conditions may contribute to vascular dysfunction.     

美托洛尔下调CaMKIIδC-p38通路对异丙肾上腺素诱导心力衰竭的保护机制

目的研究美托洛尔下调CaMKIIδC—p38通路对异丙肾上腺素诱导心力衰竭的保护机制。方法40只SD大鼠随机分成四组，正常对照组（Control）、异丙肾上腺组（Iso）、异丙肾上腺＋美托洛尔组（Iso＋Meto）和美托洛尔组（Meto），每组10只，所有动物均自由进食进水。（1）Iso组大鼠背部皮下注射Iso5my／（kg·d），连续10d；对照组背部皮下注射相同体积的生理盐水；Iso＋Meto组大鼠背部皮下注射Iso5mg／（kg·d）．连续10d，在背部皮下注射Iso前1天开始Meto10mg／（kg·d）灌胃，连续4固；Meto组给予10mg／（kg·d），连续4周灌胃；（2）所有大鼠饲养4周后，采用MillarP-V Loop导管经颈动脉插管至左心室，使用Powerlab生理记录系统测量血流动力学相关指标；统计各组大鼠心脏重量和心脏重量／体重比值；（3）TUNEL法和Caspase-3活性检测心肌细胞凋亡；（4）ELISA分析CAMKII活性；（5）Westernblot检测CaMKIl8、p-CaMKIIδ、CaMKIIδC和MAPKs家族（p-38、JNK、ERK）、和凋亡相关基因Bcl-2／Bax的蛋白表达水平。结果40只sD大鼠实验过程精神状态好，进食进水正常，无呼吸困难及水肿。（1）Iso和Meto干预SD大鼠心脏重塑和血流动力学指标有显著改变，与Control组相比．Iso组心脏重量和心脏重量指数明显增加（P〈0．05）；而Iso＋Meto组心脏重量和心脏重量指数明显低于Iso组（P〈0．05）；大鼠体重、肝重和肺重四组间也无明显差异。大鼠血流动力学指标心率（HR）、平均动脉血压（MBP）和亢事舒张末压（LVEDP）Iso组明显高于Control组；但左室压力变化速率（LV±dp/dt max）Iso组明显低于Control组（P〈0．05）；而Iso＋Meto组HR、MBP和LVEDP明显低于Iso组（P〈0．05），但Iso＋Meto组±dp／dtmax明显高于Iso组（P〈0．05）；（2）Iso组SD大鼠心肌细胞TUNEL阳性细胞数和Caspase-3活性明显高于Control组（P〈0．05）；Western杂?     

The chemotactic and mitogenic responses of vascular smooth muscle cells to insulin-like growth factor-I require the activation of ERK1/2

Insulin-like growth factors (IGFs) play an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. IGFs exert these biological actions through the activation of the IGF-I receptor and its downstream signaling network. While the involvement of the IRS-PI3 kinase-Akt pathway in mediating the chemotactic and mitogenic actions of IGFs is clear, the role of the mitogen-activated protein kinase (MAPK) signaling pathway is still under debate. In this study, the role of ERK1 and 2 in mediating the chemotactic and mitogenic actions of IGF-I in cultured porcine VSMCs was investigated. IGF-I treatment caused a significant increase in the phosphorylation state, as well as the kinase activity, of ERK1 and 2. Compared to the strong and sustained MAPK activation induced by platelet-derived growth factor-BB, the IGF-I-induced MAPK activation was weaker and more transient. Specific inhibition of the MAPK activation by PD98059 or U0126, two selective MEK inhibitors, significantly inhibited IGF-I-stimulated cell proliferation, and reduced the number of cells that migrated towards IGF-I. The p38 MAPK inhibitor SB203580 had no such effect. Likewise, depletion of ERK1/2 using antisense oligonucleotides abolished the IGF-I-induced VSMC migration and proliferation. These results suggest that the chemotactic and mitogenic responses of VSMCs to IGF-I require the activation of ERK1 and 2.     

Angiotensin II and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells

OBJECTIVE: The role of reactive oxygen species (ROS) in mitogen-activated protein kinase (MAPK) signaling by angiotensin (Ang) II and endothelin-1 (ET-1) in human vascular smooth muscle cells (VSMC) was investigated. DESIGN: VSMCs were derived from resistance arteries from healthy subjects. MAPK activity was assessed using phospho-specific antibodies. ROS generation was measured by CMH2DCFDA fluorescence and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by lucigenin chemiluminescence. RESULTS: Ang II and ET-1 increased MAPK phosphorylation (P < 0.01). Pre-treatment with Tiron and Tempol, *O2 scavengers, attenuated agonist-stimulated phosphorylation of p38MAPK, c-Jun N-terminal kinases (JNK) and ERK5, but not of ERK1/2 (extracellular signal-regulated kinases). Apocynin and diphenylene iodinium (DPI), NAD(P)H oxidase inhibitors, decreased Ang II-induced responses 60-70%. ET-1-mediated MAPK phosphorylation was unaffected by apocynin but was reduced (> 50%) by thenoyltrifluoroacetone (TIFT) and carboxyl cyanide-m-chlorophenylhydrazone (CCCP), mitochondrial inhibitors. Allopurinol and N-nitro-l-arginine methyl ester (l-NAME), xanthine oxidase and nitric oxide synthase (NOS) inhibitors, respectively, did not influence MAPK activation. Intracellular ROS generation, was increased by Ang II and ET-1 (P < 0.01). DPI inhibited Ang II- but not ET-1-mediated ROS production. Expression of p22phox and p47phox and activation of NAD(P)H oxidase were increased by Ang II but not by ET-1. CCCP and TIFT significantly attenuated ET-1-mediated ROS formation (P < 0.05), without influencing Ang II effects. CONCLUSIONS: Ang II activates p38MAPK, JNK and ERK5 primarily through NAD(P)H oxidase-generated ROS. ET-1 stimulates these kinases via redox-sensitive processes that involve mitochondrial-derived ROS. These data suggest that redox-dependent activation of MAPKs by Ang II and ET-1 occur through distinct ROS-generating systems that could contribute to differential signaling by these agonists in VSMCs.     

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.     

Study on the role of Cathepsin B and JNK signaling pathway in the development of cerebral aneurysm

Objective: To investigate the correlation between JNK signal and the apoptosis of VSMC as well as the expression of Cathepsin B and to explore the role of JNK signal in the development of cerebral aneurysm. Methods: Rat models of cerebral aneurysm were established and histopathologic changes of cerebral aneurysm and the apoptosis of VSMC were analyzed. Rat models were respectively subject to subcutaneous injection of Cathepsin B si RNA and JNK inhibitor SP600125. Western blot technique was used to detect the expression of proteins like Cathepsin B, Caspase-3, and p-JNK. Spearman's rho was used to examine the correlation between p-JNK and Cathepsin B, as well as the expression of relevant proteins. Results:The success rate of modeling rats with cerebral aneurysm was 88.75%. After the respective injection of Cathepsin B si RNA, SP600125 and their combination, the cell densities of VSMC of rats with cerebral aneurysm all increased significantly(P0.05 or P0.01), but the apoptosis rate of VSMC decreased significantly(P0.01). Compared with normal rats, the expression of Cathepsin B, Caspase-3 and p-JNK in cerebral aneurysm models increased significantly. Effectively intervening Cathepsin B genes with Cathepsin B si RNA could significantly inhibit the expression of Cathepsin B and Caspase-3, but hardly influence the expression of p-JNK. JNK inhibitor SP600125 had no influence on the expression of Cathepsin B and Caspase-3, but effectively inhibited the expression of p-JNK. In cerebral aneurysm tissues, positive correlation was observed between the expression of p-JNK and Cathepsin B, the correlation coefficient was r=0.640. Conclusion: After the attack of cerebral aneurysm, proteins like Cathepsin B, Caspase-3 and p-JNK are all involved in the apoptosis of VSMCs. This process may be realized by Cathepsin B which activates the apoptosis mechanism of Caspase-3 and mediate the apoptosis of VSMC through the JNK signaling pathway. Therefore, silencing Cathepsin B gene or inhibiting the conduction through JNK signaling pathway can mitigate the apoptosis of vascular smooth muscle cells in cerebral aneurysm.