Mitochondrial dysfunction enhances the migration of vascular smooth muscles cells via suppression of Akt phosphorylation

Background

Atherosclerosis is one of the major complications of diabetes, which may result from insulin resistance via mitochondrial dysfunction. Although a strong association between insulin resistance and cardiovascular disease has been suggested, it is not clear yet whether stress-inducing factors damage mitochondria and insulin signaling pathway in cardiovascular tissues.

Methods

We investigated whether stress-induced mitochondrial dysfunction might alter the insulin/Akt signaling pathway in A10 rat vascular smooth muscle cells (VSMC).

Results

The treatment of oxidized low density lipoprotein (oxLDL) decreased ATP contents, mitochondrial respiration activity, mRNA expressions of OXPHOS subunits and IRS-1/2 and insulin-mediated phosphorylations of Akt and AMP-activated protein kinase (AMPK). Similarly, dideoxycytidine (ddC), the mtDNA replication inhibitor, or rotenone, OXPHOS complex I inhibitor, inhibited the insulin-mediated pAkt while increased pAMPK regardless of insulin. Reciprocally, an inhibitor of Akt, triciribine (TCN), decreased cellular ATP contents. Overexpression of Akt dominant positive reversed the oxLDL- or ddC-mediated ATP decrease but AMPK activator did not. Akt activation also normalized the aberrant VSMC migration induced by ddC.

Conclusions

Defective insulin signaling and mitochondrial function may collectively contribute to developing cardiovascular disease.

General significance

Akt may be a possible therapeutic target for treating insulin resistance-associated atherosclerosis.     

Characterization of phospholipase A1, A2, C activity in Ureaplasma urealyticum membranes

Neointimal thickening following catheter injury is characterized, in part, by growth factor-induced vascular smooth muscle cell (VSMC) proliferation. It was hypothesized that a reduction in serum insulin-like growth factor-1 (IGF-1), characteristic of chemically-induced diabetes, would result in decreased VSMC proliferation and attenuate neointimal thickening. It was found that alloxan-treated New Zealand White rabbits exhibit varying degrees of glycemia. Rabbits classified as diabetic (glucose = 400 mg/dL) had significantly decreased serum concentration of IGF-1 (87.4 ± 14 nmol/L vs. 170 ± 14 nmol/L) and significantly decreased intimal/medial (I/M) ratios 2, 4, and 8 weeks after aortic injury compared to euglycemic rabbits (13.7 ± 2, 21.1 + mn; 2, 32.4 ± 3 in euglycemics and 6.6 ± 1, 14 ± 2, 19 ± 5 in diabetics, respectively). The I/M for high hyperglycemic animals (glucose 286-399 mg/dL) was comparable to diabetic animals yet their serum IGF-1 levels were normal rather than depressed. Vascular IGF-1 content similarly increased upon injury in both diabetic and euglycemic animals. In diabetic animals, proliferating cell nuclear antigen (PCNA) immunostaining was present by day 1 peaked by day 5 and returned to control by day 14. In euglycemic animals, staining by day 1 continued to increase through day 14. A similar increase in mitogen-activated protein kinase (MAPK) activity occurred from day 1 through day 5 in both diabetic and euglycemic animals. This is the first demonstration of an association between MAPK activity and VSMC proliferation following vascular injury in diabetic animals as previously reported in euglycemic animals. In conclusion, this study provides evidence against a direct effect of IGF-1 in the reduction in neointimal thickening, VSMC proliferation, and MAPK activity upon catheter injury in chemically-induced diabetic rabbits.     

Krüppel样因子5介导血管平滑肌细胞的增殖和迁移

Krüppel样因子5（krüppel-like factor 5,KLF5）是KLF家族中与胚胎发育、细胞增殖和肿瘤发生密切相关的转录调节因子。为观察KLF5在体外培养的大鼠血管平滑肌细胞（vascularsmooth muscle cells,VSMCs）增殖和迁移活性中的作用,通过构建KLF5腺病毒表达载体并感染细胞以过表达KLF5或用特异性siRNA敲低KLF5,用MTT、流式细胞术以及免疫细胞化学染色和伤口愈合实验检测其对VSMCs增殖和迁移活性的影响。结果发现,KLF5过表达可加速细胞由G0/G1期向S期转变,促进细胞增殖和迁移;反之,敲低KLF5后细胞增殖活性明显低于转染无关序列NS-siRNA对照组细胞,G0/G1期细胞数所占比例增多,S期细胞数所占比例减少,VSMCs迁移活性也明显降低。结果表明KLF5可参与介导VSMCs的增殖和迁移。     

Fingolimod inhibits PDGF-B-induced migration of vascular smooth muscle cell by down-regulating the S1PR1/S1PR3 pathway

Platelet Derived Growth Factor (PDGF) and sphingosine-1-phosphate (S1P) pathways play a key role in mural cell recruitment during tumor growth and angiogenesis. Fingolimod, a S1P analogue, has been shown to exert antitumor and antiangiogenic properties. However, molecular targets and modes of action of fingolimod remain unclear. In this study, we confirmed the antagonizing action of S1P and PDGF-B on rat vascular smooth muscle cell (VSMCs) growth and migration. We then compared siRNA and/or fingolimod (100 nM) treatments on PDGFR-β, S1PR1 S1PR2 and S1PR3 expression. Fingolimod induced a 50% reduction in S1PR3 protein expression which was cumulative with that obtained with anti-S1PR3 siRNA. We found that siRNA-induced inhibition of both PDGFR-β and S1PR3 was the most effective means to block VSMC migration induced by PDGF-B. Finally, we observed that fingolimod treatment associated with anti-S1PR1 siRNA principally inhibited VSMC growth while in combination with anti-S1PR3 siRNA it strongly inhibited VSMC migration. These results suggest that for rat VSMCs, the PDGFR-S1PR1 pathway is predominantly dedicated to cell growth while PDGFR-S1PR3 stimulates cell migration. As an S1P analogue, fingolimod is considered a potent activator of S1PR1 and S1PR3. However, its action on the PDGFR-S1PR platform appears to be dependent on S1PR1 and S1PR3 specific downregulation. Considering that the S1P pathway has already been shown to exert various crosstalks with tyrosine kinase pathways, it seems of great interest to evaluate fingolimod potential in combination with the numerous tyrosine kinase inhibitors used in oncology.     

The Notch pathway attenuates interleukin 1β (IL1β)-mediated induction of adenylyl cyclase 8 (AC8) expression during vascular smooth muscle cell (VSMC) trans-differentiation

Vascular smooth muscle cell (VSMC) trans-differentiation, or their switch from a contractile/quiescent to a secretory/inflammatory/migratory state, is known to play an important role in pathological vascular remodeling including atherosclerosis and postangioplasty restenosis. Several reports have established the Notch pathway as tightly regulating VSMC response to various stress factors through growth, migration, apoptosis, and de-differentiation. More recently, we showed that alterations of the Notch pathway also govern VSMC acquisition of the inflammatory state, one of the major events accelerating atherosclerosis. We also evidenced that the inflammatory context of atherosclerosis triggers a de novo expression of adenylyl cyclase isoform 8 (AC8), associated with the properties developed by trans-differentiated VSMCs. As an initial approach to understanding the regulation of AC8 expression, we examined the role of the Notch pathway. Here we show that inhibiting the Notch pathway enhances the effect of IL1β on AC8 expression, amplifies its deleterious effects on the VSMC trans-differentiated phenotype, and decreases Notch target genes Hrt1 and Hrt3. Conversely, Notch activation resulted in blocking AC8 expression and up-regulated Hrt1 and Hrt3 expression. Furthermore, overexpressing Hrt1 and Hrt3 significantly decreased IL1β-induced AC8 expression. In agreement with these in vitro findings, the in vivo rat carotid balloon-injury model of restenosis evidenced that AC8 de novo expression coincided with down-regulation of the Notch3 pathway. These results, demonstrating that the Notch pathway attenuates IL1β-mediated AC8 up-regulation in trans-differentiated VSMCs, suggest that AC8 expression, besides being induced by the proinflammatory cytokine IL1β, is also dependent on down-regulation of the Notch pathway occurring in an inflammatory context.     

血管平滑肌细胞中ERK通路与TGF-β_1/Smad通路的相互作用

目的:探讨血管平滑肌细胞(VSMC)中TGF-β/Smad与ERK信号转导通路是否存在相互调节关系。方法:原代培养的大鼠胸主动脉平滑肌细胞,分四组:①对照组,②TGF-β1组,③ERK阻断剂(PD98059)组和④TGF-β1+ERK阻断剂(PD98059)组。分别用Western blot法检测VSMC内Smad2/3、ERK1/2蛋白表达及磷酸化Smad2/3、磷酸化ERK1/2蛋白含量,RT-PCR方法测VSMC中Smad2、Smad3mRNA的表达。结果:①与对照组相比,TGF-β1组P-Smad2/3、P-ERK1/2蛋白含量增多(P0.05),ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05),TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量无差异;与TGF-β1组相比,TGF-β1+ERK阻断剂组P-Smad2/3、P-ERK1/2蛋白含量减少(P0.05)。各组间Smad2/3、ERK1/2蛋白表达无差异。②各组的Smad2、Smad3mRNA表达无差异。结论:TGF-β1诱导的Smad2/3蛋白磷酸化依赖ERK通路激活,但ERK通路对Smad2/3蛋白和mRNA表达水平无影响。     

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.