Proteomic profiling of cellular responses to Carvedilol enantiomers in vascular smooth muscle cells by iTRAQ-coupled 2-D LC–MS/MS

Carvedilol is a third-generation β-blocker, with the S-enantiomer being more active than the R-enantiomer. Clinically, it has been used in the treatment of hypertension, congestive heart failure and angina pectoris. Each enantiomer of Carvedilol exhibits differential pharmacological effects. However, the cellular effects of individual enantiomer are not well understood. To gain insights into how each enantiomer affects cells, we analysed differential protein expression levels in vascular smooth muscle cells (A7r5) incubated separately with S- and R-Carvedilol by iTRAQ-coupled 2-D LC–MS/MS approach. Thirteen proteins were identified with statistically significant changes in cells incubated with S-Carvedilol, while the changes of most proteins incubated with R-Carvedilol were less significant. Among these proteins, actin in aortic smooth muscle (ACTA2), calmodulin, S100-A6, S100-A10, S100-A11, thioredoxin, lactadherin and heat-shock protein 105 kDa were found to be closely relevant with the clinical effects of Carvedilol. Furthermore, the changes in protein levels were validated by Western blot. Our findings thus provided molecular evidence on a comprehensive protein profile on Carvedilol–cell interaction, which may shed new light in molecular events underlying Carvedilol treatment.     

Heparin induces specific protein release from human intestinal smooth muscle cells

Human intestinal smooth muscle cells have recently been identified as the major cell type responsible for stricture formation in Crohn's disease. Heparin, a sulfated glycosaminoglycan, has been shown to be a key modulator of vascular smooth muscle cell growth both in vivo and in vitro and to affect the release of proteins from these cells. Heparin has also been shown to affect the growth of human intestinal smooth muscle cells. In this report we demonstrate that heparin, in addition to its effects on proliferation, also has very specific effects on proteins released by these cells in vitro. Examination of the culture medium proteins of heparin-treated human intestinal cells revealed an increase in three proteins of molecular weight between 150-250 kd, an increase in a 37 kd protein and a decrease in synthesis of lower molecular weight (less than 20 kd) proteins. In substrate-attached material a transient effect on a 48 kd protein was observed. No effects on intracellular labeled proteins could be demonstrated. The 35S-methionine labeled protein profile of human intestinal smooth muscle cells exposed to heparin is similar to that observed in rat vascular smooth muscle cells yet distinct differences do exist. Extracellular processing does not account for the released proteins nor is de novo protein synthesis required suggesting that altered intracellular protein processing is the mechanism for the heparin-induced protein pattern. The release of specific proteins following exposure to heparin may reflect a significant influence of this glycosaminoglycan on the metabolism of smooth muscle cells in general and particularly in the human intestine.     

OxLDL-induced gene expression patterns in CASMC are mimicked in apoE-/- mice aortas

Oxidized low density lipoprotein (oxLDL) contributes to the pathophysiology of atherosclerosis, partly by altering gene expression in vascular cells. Here, we show 221 genes differentially regulated by oxLDL in coronary artery smooth muscle cells (CASMC), using oligonucleotide microarrays. These genes were classified into 14 functional groups. A comparable gene expression pattern was detected in apoE(-/-) mice. OxLDL induced an oxidative stress response in CASMC, but not the unfolded protein response. OxLDL also caused CASMC death which was accompanied by increased expression of FasL, Bax, and p53 but was caspase-independent. This approach provides further insight into disease pathology and prognosis.     

Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells

Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration. To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library. The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen. FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression. Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells. FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells. In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI. Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP. Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region. PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1. These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction. Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.     

RGC-32 increases p34CDC2 kinase activity and entry of aortic smooth muscle cells into S-phase.

Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34CDC2-cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34CDC2-mediated phosphorylation and resulted in loss of p34CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34CDC2.     

Activation-dependent surface expression of LOX-1 in human platelets

Lectin-like oxidized LDL receptor-1 (LOX-1) was initially identified as an oxidized LDL receptor in aortic endothelial cells. Here we identified LOX-1 mRNA and protein in human platelets in addition to recent findings on the expression in macrophages and smooth muscle cells. The presence of LOX-1 was further confirmed in the megakaryocytic cell lines. Flow cytometric analyses revealed that LOX-1 was exposed on the surface of platelets in an activation-dependent manner. Consistently, the activation-dependent binding of OxLDL to platelets was mostly inhibited by anti-LOX-1 antibody. Immunohistochemistry of the atherosclerotic plaque from a patient with unstable angina pectoris (UAP) revealed accumulation of LOX-1 protein at the site of thrombus. As LOX-1 recognizes and binds activated platelets, exposure of LOX-1 on activated platelets surface might assist thrombosis formation.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.     

In vitro prevention of cyclosporin-induced cell contraction by mycophenolic acid

Nephrotoxicity is a major side-effect of cyclosporin A (CsA), which induces a vasoconstrictive response in vascular smooth muscle and mesangial cells. Mycophenolic acid (MPA) is used in combination with low-dose CsA to reduce nephrotoxicity. We previously demonstrated that MPA affected mesangial cell contractile response to angiotensin II or KCl. Aims of the present study were to evaluate if MPA can prevent CsA-induced contraction of human mesangial and aortic smooth muscle cells (ASMC). Using a morphoquantitative approach, we evidenced that pretreatment with MPA (1 microM) prevented the reduction of cell area induced by CsA within 30 min in both cell types. We then compared the expression of three main cytoskeleton proteins: tubulin, alpha-smooth actin (SMA) and basic calponin, in ASMC and in mesangial cells treated with MPA and/or CsA. CsA alone did not significantly change the expression level of these proteins neither in mesangial cells nor in ASMC. MPA decreased the expression level of tubulin in both mesangial cells and ASMC. Surprisingly, MPA, which stimulated SMA and calponin expression in mesangial cells, exerted an inhibitory effect on both contractile protein expression in ASMC. In conclusion, our results evidenced opposite effects of MPA on calponin and SMA protein expression in ASMC and in mesangial cells, despite similar antiproliferative properties, suggesting that sarcomeric protein expression is controlled by different intracellular mechanisms in mesangial and smooth muscle cells. However, MPA interferes in both cell types with the constrictive properties CsA, which may partially explain the protective effects of MPA against CsA nephrotoxicity.     

OxLDL stimulates cell proliferation through a general induction of cell cycle proteins

Oxidized low-density lipoprotein (oxLDL) may be involved in atherosclerosis by stimulating proliferation of cells in the vessel wall. The purpose of this study was to identify the mechanism by which oxLDL induces proliferation. Quiescent human fibroblasts and rabbit smooth muscle cells were treated with 0, 10, or 50 microg/ml oxLDL for 24-48 h. This resulted in significant increases in total cell counts at both concentrations of oxLDL, at both time points, for both types of cells. Western blot analysis revealed that oxLDL-stimulated cell proliferation was associated with significant increases in the expression of proteins that regulate entry into and progression through the cell cycle [cell division cycle 2, cyclin-dependent kinase (cdk) 2, cdk 4, cyclin B1, cyclin D1, and PCNA]. Surprisingly, the expression of cell cycle inhibitors (p21 and p27) was stimulated by oxLDL as well, but this was to a lesser extent than the effects on cell cycle-activating proteins. OxLDL also induced nuclear localization of all cell cycle proteins examined. The similar effects of oxLDL on the translocation and expression of both cell cycle-activating and -inhibiting proteins may explain the controlled proliferative phenomenon observed in atherosclerosis as opposed to the more rapid proliferative event characteristic of cancer.     

Functional Effects of FGF-13 on Human Lung Fibroblasts,Dermal Microvascular Endothelial Cells,and Aortic Smooth Muscle Cells

We studied the effects of FGF-13 and FGF-2 on human lung fibroblasts, dermal microvascular endothelial cells, and aortic smooth muscle cells. FGF-13 induced cell growth of lung fibroblasts and aortic smooth muscle cells but had no effect on dermal vascular endothelial cells. FGF-2 induced cell growth in all the three cell types. FGF-13 and FGF-2 had little effect on IL-6 production by lung fibroblasts and aortic smooth muscle cells and substantially enhanced that induced by IL-1α. In contrast, FGF-13 and FGF-2 had little effect on IL-6 production by dermal vascular endothelial cells, either alone or in synergy with IL-1α.     

Internalization and processing of human angiogenin by cultured aortic smooth muscle cells

Human angiogenin is a 14-kDa plasma protein with angiogenic and ribonucleolytic activities. Angiogenin binds specifically to aortic smooth muscle cells, activates second messenger pathways, and inhibits their proliferation. Human and bovine aortic smooth muscle cells were used to study the internalization and intracellular fate of human angiogenin at 37 degrees C. Using a specific antibody against angiogenin, we found that the internalized native protein was localized in the perinuclear region at 30 min and then dispersed throughout the cytoplasm. In conditions favoring receptor-mediated endocytosis, internalization of iodinated angiogenin showed a first peak at 5 min and then further increased for up to 24 h. The half-life of the molecule, calculated as 12 h in chase experiments, could contribute to its intracellular accumulation. In cell extracts, in addition to the 14-kDa protein, a 8.7-kDa fragment was observed at 24 h, and three fragments with molecular mass of 10.5, 8.7, and 6. 1 kDa were detected at 48 h. Our data point to a specific internalization and processing of human angiogenin by aortic smooth muscle cells.     

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.     

Expression of receptors for human angiogenin in vascular smooth muscle cells.

Human angiogenin is a plasma protein with angiogenic and ribonucleolytic activities. Angiogenin inhibited both DNA replication and proliferation of aortic smooth muscle cells. Binding of 125I-angiogenin to bovine aortic smooth muscle cells at 4 degrees C was specific, saturable, reversible and involved two families of interactions. High-affinity binding sites with an apparent dissociation constant of 0.2 nm bound 1 x 104 molecules per cell grown at a density of 3 x 104.cm-2. Low-affinity binding sites with an apparent dissociation constant of 0.1 micrometer bound 4 x 106 molecules.cell-1. High-affinity binding sites decreased as cell density increased and were not detected at confluence. 125I-angiogenin bound specifically to cells routinely grown in serum-free conditions, indicating that the angiogenin-binding components were cell-derived. Affinity labelling of sparse bovine smooth muscle cells yielded seven major specific complexes of 45, 52, 70, 87, 98, 210 and 250-260 kDa. The same pattern was obtained with human cells. Potential modulators of angiogenesis such as protamine, heparin and the placental ribonuclease inhibitor competed for angiogenin binding to the cells. Together these data suggest that cultured bovine and human aortic smooth muscle cells express specific receptors for human angiogenin.     

Integrated membrane protein analysis of mature and embryonic stem cell-derived smooth muscle cells using a novel combination of CyDye/biotin labeling

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.     

Cyclic stretch upregulates SDF-1α/CXCR4 axis in human saphenous vein smooth muscle cells

Cyclic stretch (CS) mediates different cellular functions in vascular smooth muscle cells and involves in neointimal hyperplasia and subsequent atherosclerosis of vein grafts. Here, we investigated whether CS can modulate stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in human saphenous vein smooth muscle cells. We found CS induced the upregulation of SDF-1α and CXCR4 in human saphenous vein smooth muscle cells in vitro, which was dependent on PI3K/Akt/mTOR pathway. Furthermore, CS augmented human saphenous vein smooth muscle migration and focal adhesion kinase (FAK) activation by PI3K/Akt/mTOR pathway. Interestingly, the upregulation of SDF-1α/CXCR4 axis was instrumental in CS-induced saphenous vein smooth muscle cell migration and FAK activation, as showed by AMD3100, an inhibitor of SDF-1α/CXCR4 axis, partially but significantly blocked the CS-induced cellular effects. Thus, those data suggested SDF-1α/CXCR4 axis involves in CS-mediated cellular functions in human saphenous vein smooth muscle cells.     

Distinct apolipoprotein E isoform preference for inhibition of smooth muscle cell migration and proliferation

The current study compared the effectiveness of the various human apolipoprotein E (apoE) isoforms in inhibiting platelet-derived growth factor- (PDGF-) stimulated smooth muscle cell proliferation and migration. The incubation of primary mouse aortic smooth muscle cells with apoE3 resulted in dose-dependent inhibition of smooth muscle cells stimulated by 10 ng/mL PDGF. Greater than 50% inhibition of smooth muscle cell proliferation was observed at 15 microg/mL of human apoE3. Human apoE2 was less effective, requiring a higher concentration to achieve inhibition comparable to that of apoE3. Human apoE4 was the least effective of the apoE isoforms with no significant inhibition of cell proliferation observed at concentrations up to 15 microg/mL. Interestingly, apoE inhibition of PDGF-directed smooth muscle cell migration did not show preference for any apoE isoforms. Human apoE2, apoE3, and apoE4 were equally effective in inhibiting smooth muscle cell migration toward PDGF. These results are consistent with previous data showing that apoE inhibition of smooth muscle cell proliferation is mediated through its binding to heparan sulfate proteoglycans, whereas its inhibition of cell migration is mediated via binding to the low-density lipoprotein receptor related protein. The low efficiency of apoE4 to inhibit smooth muscle cell proliferation also suggested another mechanism to explain the association between the apolipoprotein epsilon4 allele with increased risk of coronary artery disease.     

Proteomic profiling and identification of cofilin responding to oxidative stress in vascular smooth muscle

We used 2-DE and MALDI-TOF/TOF to identify proteins of vascular smooth muscle cells whose expression was or was not altered by exposure to 500 microM H2O2 for 30 min. We detected more than 800 proteins on silver-stained gels of whole protein extracts from rat aortic smooth muscle strips. Of these proteins, 135 clearly unaffected and 19 having levels altered by exposure to H2O2 were identified. Protein characterization revealed that the most prominent vascular smooth muscle proteins were those with antioxidant, cytoskeletal structure, or muscle contraction. In addition, cofilin, an isoform of the actin depolymerizing factor family, shifted to its basic site on the 2-DE gel as a result of H2O2 treatment. In Western blot analysis of proteins from A7r5 aortic smooth muscle cells, the phosphorylation, but not the expression, of cofilin was decreased by H2O2 in a dose-dependent manner. The H2O2-induced dephosphorylation of cofilin and apoptosis was inhibited by Na3VO4, an inhibitor of protein tyrosine phosphatase (PTP). These results suggest that cofilin is one of the proteins regulated by H2O2 treatment in vascular smooth muscle, and has an important role in the induction of vascular apoptosis through PTP-dependent mechanisms.