Atorvastatin inhibits myocardin expression in vascular smooth muscle cells

Atorvastatin (ATV), an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is widely prescribed as a lipid-lowering drug. It also inhibits the RhoA-Rho-associated kinase pathway in vascular smooth muscle (SM) cells and critically inhibits SM function. Myocardin is a coactivator of serum response factor, which upregulates SM contractile proteins. The RhoA-Rho-associated kinase pathway, which directly triggers SM contraction, also increases myocardin gene expression. Therefore, we investigated whether ATV inhibits myocardin gene expression in SM cells. In mice injected with ATV (IP 20 μg/g per day) for 5 days, myocardin gene expression was significantly downregulated in aortic and carotid arterial tissues with decreased expression of myocardin target genes SM α-actin and SM22. Correspondingly, the contractility of aortic rings in mice treated with ATV or the Rho-associated kinase inhibitor Y-27632 was reduced in response to treatment with either KCl or phenylephrine. In cultured mouse and human aortic SM cells, KCl treatment stimulated the expression of myocardin, SM α-actin, and SM22. These stimulatory effects were prevented by ATV treatment. ATV-induced inhibition of myocardin expression was prevented by pretreatment with either mevalonate or geranylgeranylpyrophosphate but not farnesylpyrophosphate. Treatment with Y-27632 mimicked ATV effects on the gene expression of myocardin, SM α-actin, and SM22, further suggesting a role for the RhoA-Rho-associated kinase pathway in ATV effects. Furthermore, ATV treatment inhibited RhoA membrane translocation and activation; these effects were prevented by pretreatment with mevalonate. We conclude that ATV inhibits myocardin gene expression in vivo and in vitro, suggesting a novel mechanism for ATV inhibition of vascular contraction.     

Angiotensin II, focal adhesion kinase, and PRX1 enhance smooth muscle expression of lipoma preferred partner and its newly identified binding partner palladin to promote cell migration

Lipoma preferred partner (LPP) is a proline rich LIM domain family protein highly expressed at plasma membrane dense bodies and focal adhesions in smooth muscle cells.(1) Using the C-terminus of LPP as bait in a yeast two hybrid system, palladin, an actin-associated protein was identified. The palladin interacting region of LPP was mapped to the first and second LIM domains. The N-terminus of palladin interacted with LPP both in vitro and in vivo, but not solely through its FPLPPP and FPPPP motifs. Like LPP, palladin, is highly expressed in differentiated smooth muscle, colocalized at focal adhesions, at isolated lamellipodia and at dense bodies in smooth muscle tissue. Both LPP and palladin enhanced cell migration and spreading. LPP and palladin expression was markedly decreased, in contrast to vinculin or paxillin, in migration defective focal adhesion kinase null cells, but was restored by expression of the paired-related homeobox gene-1 protein. We have previously shown in focal adhesion kinase null cells, that tetracycline induced expression of focal adhesion kinase upregulated expression of LPP(2) and now show upregulation of palladin, and paired-related homeobox gene-1 protein. The expression of both LPP and palladin, like smooth muscle alpha-actin, was increased by angiotensin II, regulated by actin dynamics, upregulated by myocardin and expressed in the neointima of injured aorta. Overall, the data suggest that the function of LPP and palladin is context dependent, that they play a critical role in cytoskeletal remodeling, respond to signals induced by vascular injury as well as signals that induce smooth muscle cell hypertrophy, such as angiotension II.     

Similarities and differences in smooth muscle alpha-actin induction by TGF-beta in smooth muscle versus non-smooth muscle cells.

Transforming growth factor-beta (TGF-beta) has been shown to stimulate smooth muscle (SM) alpha-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-beta control element (TCE) located within the first 125 bp of the SM alpha-actin promoter were required for TGF-beta inducibility of SM alpha-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-beta induction of SM alpha-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-beta can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22alpha, h(1) calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-beta inducibility of SM alpha-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from (-155) completely repressed TGF-beta responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-beta enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-beta also stimulated expression of the SMC differentiation marker SM22alpha in non-SMCs. However, in contrast to SMCs, TGF-beta did not induce expression of h(1) calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-beta-induced expression of SM alpha-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-beta stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-beta alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.     

Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions

The aims of the present studies were to define sufficient promoter sequences required to drive endogenous expression of smooth muscle (SM) alpha-actin and to determine whether regulation of SM alpha-actin expression in vivo is dependent on CArG (CC(A/T)6GG) cis elements. Promoter deletions and site directed mutagenesis techniques were used to study gene regulation in transgenic mice as well as in smooth muscle cell (SMC) cultures. Results demonstrated that a Lac Z transgene that contained 547 bp of the 5' rat SM alpha-actin promoter was sufficient to drive embryonic expression of SM alpha-actin in the heart and in skeletal muscle but not in SMCs. Transient transfections into SMC cultures demonstrated that the conserved CArG element in the first intron had significant positive activity, and gel shift analyses demonstrated that the intronic CArG bound serum response factor. A transgene construct from -2600 through the first intron (p2600Int/Lac Z) was expressed in embryos and adults in a pattern that closely mimicked endogenous SM alpha-actin expression. Expression in adult mice was completely restricted to SMCs and was detected in esophagus, stomach, intestine, lung, and nearly all blood vessels, including coronary, mesenteric, and renal vascular beds. Mutation of CArG B completely inhibited expression in all cell types, whereas mutation of the intronic CArG selectively abolished expression in SMCs, which suggests that it may act as an SMC-specific enhancer-like element. Taken together, these results provide the first in vivo evidence for the importance of multiple CArG cis elements in the regulation of SM alpha-actin expression.     

Functional tissue-engineered blood vessels from bone marrow progenitor cells

OBJECTIVE: Stem cells have significant potential for development of cell-based therapeutics for cardiovascular tissue regeneration. METHODS: We developed a novel method for isolating smooth muscle cells (SMC) from ovine bone marrow using a tissue-specific promoter and fluorescence-activated cell sorting. RESULTS: As compared to vascular SMC, bone marrow-derived smooth muscle progenitor cells (BM-SMPC) exhibited similar morphology, showed higher proliferation potential and expressed several SMC markers including alpha-actin, calponin, myosin heavy chain, smoothelin, caldesmon and SM22. When embedded in fibrin hydrogels, BM-SMPC contracted the matrix and displayed receptor- and non-receptor-mediated contractility, indicating that BM-SMPC can generate force in response to vasoreactive agonists. We also prepared tissue-engineered blood vessels from BM-SMPC and BM-derived endothelial cells and implanted them into the jugular veins of lambs. As early as five weeks post-implantation, grafted tissues displayed a confluent endothelial layer overlaying the medial layer in which BM-SMPC were aligned circumferentially and synthesized significant amounts of collagen. In contrast to previous results with vascular SMC, BM-SMPC synthesized high amounts of elastin that was organized in a fibrillar network very similar to that of native vessels. CONCLUSIONS: Our results suggest that BM-SMPC may be useful in studying SMC differentiation and have high potential for development of cell therapies for the treatment of cardiovascular disease.     

桡动脉、乳内动脉、大隐静脉中RhoA/ROK mRNA 的表达与心血管危险因素的相关性

目的 探讨桡动脉（RA）、乳内动脉(IMA)、大隐静脉(GSV) 中RhoA/Rho 激酶（ROK）mRNA表达的差异，以及心血管危险因素对其表达的影响。方法 取14例CABG术中废弃的配对RA、IMA、GSV各5～10 mm，用RT-PCR检测RhoA/ROK mRNA的水平。采用多元逐步回归分析心血管危险因素对3种桥血管中RhoA/ROK表达的影响。结果 ①RT-PCR:IMA与GSV比较，RhoA和ROK mRNA表达的水平无显著差异；但RA中RhoA 和ROK mRNA表达的水平显著高于IMA（P<0.01，P<0.05）。②多元逐步回归分析显示，GSV中RhoA和ROK mRNA表达的水平与糖化血红蛋白呈显著正相关（r=0.642、r=0.692,P<0.05）。3种血管中RhoA、ROK mRNA的表达与年龄、总胆固醇、三酰甘油、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、载脂蛋白A1、载脂蛋白B、C反应蛋白、胰岛素、胰岛素抵抗及体质量指数均无显著相关。结论 ①RA、IMA、GSV管壁中RhoA/ROK mRNA的表达有差异，RA中Rho/ROK mRNA的表达明显强于IMA。②糖化血红蛋白可影响GSV 中RhoA/ROK mRNA的表达。     

Contractile smooth muscle cells derived from hair-follicle stem cells

AIMS: We hypothesized that hair-follicle stem cells can differentiate toward smooth contractile muscle cells, providing an autologous cell source for cardiovascular tissue regeneration. METHODS AND RESULTS: Smooth muscle progenitor cells (SMPCs) were obtained from ovine hair follicles using a tissue-specific promoter and fluorescence-activated cell sorting. Hair-follicle smooth muscle progenitor cells (HF-SMPCs) expressed several markers of vascular smooth muscle including alpha-actin, calponin, myosin heavy chain (MHC), caldesmon, smoothelin, and SM22. HF-SMPCs were highly proliferative and showed high clonogenic potential without any signs of chromosomal abnormalities as evidenced by karyotype analysis. HF-SMPCs compacted fibrin hydrogels to a similar extent as vascular smooth muscle cells from ovine umbilical veins (V-SMCs), indicating the development of the force-generating machinery. In addition, cylindrical tissue equivalents prepared with HF-SMPCs displayed significant contractility in response to vasoactive agonists including KCl and the thromboxane A2 mimetic U46619, suggesting that these cells had developed receptor and non-receptor-mediated pathways of contractility. Finally, transforming growth factor-beta1 promoted differentiation of HF-SMPCs toward a mature SMC phenotype as suggested by increased expression of MHC and enhanced matrix compaction. CONCLUSION: Our results suggest that hair follicles may be an easily accessible, autologous, and rich source of functional SMPC for cardiovascular tissue engineering and regenerative medicine.     

黏着斑激酶对血小板源性生长因子刺激的血管平滑肌细胞迁移和黏附的调控

目的探讨黏着斑激酶(FAK)信号分子在雷帕霉素抑制血小板源性生长因子(PDGF)诱导血管平滑肌细胞(VSMC)迁移、黏附中的调控作用。方法将培养的大鼠VSMC分为对照组、PDGF组、雷帕霉素+PDGF组(雷帕霉素组)和FAK反义寡核苷酸+PDGF组(FAK组)。用PDGF诱导VSMC的迁移和黏附,计数贴壁细胞;采用Boyden检测细胞迁移;采用RT-PCR、Western blot、免疫沉淀方法分别检测FAK基因、蛋白及蛋白磷酸化表达量。将FAK反义寡核苷酸经脂质体转染VSMC,观察FAK mRNA及蛋白磷酸化、细胞迁移和黏附的变化。结果与对照组比较,PDGF组明显诱导细胞迁移和黏附,上调FAK mRNA的表达,提高FAK蛋白和磷酸化FAK表达,差异有统计学意义(P0.05),与PDGF组比较,FAK组和雷帕霉素组细胞迁移、黏附能力减弱,FAK mRNA、FAK蛋白和磷酸化FAK表达水平明显降低,差异有统计学意义(P0.05)。结论 PDGF诱导细胞迁移和黏附可能是FAK介导的,雷帕霉素可能是通过抑制FAK蛋白和磷酸化FAK来抑制VSMC的迁移和黏附。