cyclin-d1 and Arteriosclerosis

Increased levels of C-peptide, a cleavage product of proinsulin, circulate in patients with insulin resistance and early type 2 diabetes mellitus. Recent data suggest a potential causal role of C-peptide in atherogenesis by promoting monocyte and T-lymphocyte recruitment into the vessel wall. The present study examined the effect of C-peptide on vascular smooth muscle cells (VSMCs) proliferation and evaluated intracellular signaling pathways involved. In early arteriosclerotic lesions of diabetic subjects, C-peptide colocalized with VSMCs in the media. In vitro, stimulation of human or rat VSMCs with C-peptide induced cell proliferation in a concentration-dependent manner with a maximal 2.6+/-0.8-fold induction at 10 nmol/L human C-peptide (P<0.05 compared with unstimulated cells; n=9) and a 1.8+/-0.2-fold induction at 0.5 nmol/L rat C-peptide (P<0.05 compared with unstimulated cells; n=7), respectively, as shown by [H3]-thymidin incorporation. The proliferative effect of C-peptide on VSMCs was inhibited by Src short interference RNA transfection, PP2, an inhibitor of Src-kinase, LY294002, an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059. Moreover, C-peptide induced phosphorylation of Src, as well as activation of PI-3 kinase and ERK1/2, suggesting that these signaling molecules are involved in C-peptide-induced VSMC proliferation. Finally, C-peptide induced cyclin D1 expression as well as phosphorylation of Rb in VSMCs. Our results demonstrate that C-peptide induces VSMC proliferation through activation of Src- and PI-3 kinase as well as ERK1/2. These data suggest a novel mechanism how C-peptide may contribute to plaque development and restenosis formation in patients with insulin resistance and early type 2 diabetes mellitus.

Topics: Animals; Arteriosclerosis; C-Peptide; Cell Proliferation; Cells, Cultured; Cyclin D1; Diabetic Angiopathies; Enzyme Activation; Humans; Intracellular Membranes; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases; Proto-Oncogene Proteins c-akt; Rats; Retinoblastoma Protein; Signal Transduction; src-Family Kinases

Current theories suggest that atherosclerosis, plaque rupture, stroke, and restenosis after angioplasty may involve defective apoptotic mechanisms in vascular cells. Prior work has demonstrated that cells from human atherosclerotic lesions, and cells from the aorta of aged rats, exhibit functional resistance to apoptosis induced by TGF-beta and glucocorticoids. The present studies demonstrate that human lesion-derived cells (LDC) are also resistant to apoptosis induced by fas ligation compared to cells derived from the adjacent media, and that in vitro expansion of LDC causes acquired resistance to apoptosis. Microarray profiling of fas-resistant versus sensitive cells identified a set of genes including STATs, caspase 1, cyclin D1, Bcl-xL, VDAC2, and BAD. The STAT proteins have been implicated in resistance to apoptosis, potentially via their ability to modulate caspase 1 (ICE), Bcl-xL, and cyclin D1 expression. Western blot analysis of sensitive and resistant LDC clonal lines confirmed increases in cyclin D1, STAT6, Bcl-xL, and BAD, with decreased expression of caspase 1. Thus, transcript profiling has identified a potential pathway of apoptotic regulation in subsets of lesion cells. The resistant phenotype may contribute to plaque stability and excessive vascular repair, while sensitive cells may be involved in plaque rupture and infarction. The data suggests both genetic interventions and novel small-molecule inhibitors that may be effective modulators of apoptosis in atherosclerosis, angina, and in-stent restenosis.

Topics: Apoptosis; Arteriosclerosis; bcl-Associated Death Protein; bcl-X Protein; Blotting, Western; Carrier Proteins; Cell Survival; Cyclin D1; DNA-Binding Proteins; fas Receptor; Gene Expression Profiling; Genome, Human; Humans; Models, Anatomic; Models, Biological; Oligonucleotide Array Sequence Analysis; Porins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT6 Transcription Factor; Trans-Activators; Voltage-Dependent Anion Channel 2; Voltage-Dependent Anion Channels

Transplant arteriosclerosis is the result of intima proliferation in large vessels upon organ transplantation. Obliteration of the vascular lumen will ultimately lead to ischemia and late graft failure. Gene array analysis was performed to identify factors involved in the pathogenesis of transplant arteriosclerosis. Aortic transplants from Dark Agouti to Wistar Furth rats were performed to identify potential target genes. Hierarchical clustering of genes specifically upregulated in allogeneic but not in syngeneic aortas revealed 19 genes. A gene that fulfilled these criteria is prothymosin alpha (PTMA), a regulator of estrogen receptor transcriptional activity. PTMA gene and protein expression levels were confirmed by PCR and immunohistochemistry. Estrogen receptor staining was increased in allogeneic aortas. Furthermore, cyclin D1 a downstream target of PTMA, was also up regulated in allogeneic aortas. In conclusion, PTMA was identified as potential candidate gene involved in transplant arteriosclerosis.

Topics: Animals; Aorta; Arteriosclerosis; Cyclin D1; Gene Expression Profiling; Immunohistochemistry; Male; Polymerase Chain Reaction; Protein Precursors; Rats; Rats, Inbred WF; Thymosin; Transplantation, Homologous

Activation of EGF receptors is closely involved in vascular proliferative diseases. The signaling mechanisms of EGF ligands, including betacellulin (BTC) and amphiregulin (AR), are poorly understood. We examined how BTC and AR induced DNA synthesis activity in primary cultures of human thoracic aortic smooth muscle cells (HTASMCs). BTC induced phosphorylation of all four EGF receptors present on HTASMCs: ErbB1, ErbB2, ErbB3, and ErbB4. BTC rapidly induced the phosphorylation of Akt, GSK3alpha/beta, and two FoxO factors, FKHR and AFX, in a dose- and time-dependent manner. BTC increased nuclear beta-catenin accumulation. BTC increased cyclin D1 mRNA, cyclin D1 protein, and DNA synthesis activity. Pretreatment with the phosphatidylinositol 3'-kinase (PI 3'-kinase) inhibitor wortmannin suppressed BTC-induced cyclin D1 mRNA and protein and DNA synthesis activity. In contrast, AR, a specific ErbB1 ligand, induced sustained ERK1/2 and Elk1 phosphorylation, increased cyclin D1 mRNA and protein, and increased DNA synthesis activity. AR did not produce any changes in Akt phosphorylation. Pretreatment with PD98059 suppressed AR-induced cyclin D1 mRNA and protein. Thus, the PI 3'-kinase/Akt/GSK/FoxO/beta-catenin pathway could be the major signaling cascade for BTC-induced upregulation of cyclin D1 protein, whereas a sustained ERK/Elk1 activation could be the major signaling cascade for AR-induced upregulation of cyclin D1 protein in HTASMCs. Moreover, immunohistochemical staining revealed that that BTC, ErbB1, and ErbB4 are upregulated in the plaques of human atherosclerotic coronary arteries. Taken together, BTC and AR could be potent growth factors in proliferative vascular diseases.

Topics: Agammaglobulinaemia Tyrosine Kinase; Amphiregulin; Arteriosclerosis; beta Catenin; Betacellulin; Cell Cycle; Cell Cycle Proteins; Cell Nucleus; Cells, Cultured; Cyclin D1; Cytoskeletal Proteins; DNA; DNA-Binding Proteins; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; ets-Domain Protein Elk-1; Forkhead Box Protein O1; Forkhead Transcription Factors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; RNA, Messenger; Signal Transduction; Trans-Activators; Transcription Factors; Up-Regulation

Plasma homocysteine levels are elevated in 20-30% of all patients with premature atherosclerosis. Although elevated homocysteine levels have been recognized as an independent risk factor for myocardial infarction and stroke, the mechanism by which these elevated levels cause atherosclerosis is unknown. To understand the role of homocysteine in the pathogenesis of atherosclerosis, we examined the effect of homocysteine on the growth of both vascular smooth muscle cells and endothelial cells at concentrations similar to those observed in clinical studies. As little as 0.1 mM homocysteine caused a 25% increase in DNA synthesis, and homocysteine at 1 mM increased DNA synthesis by 4.5-fold in rat aortic smooth muscle cells (RASMC). In contrast, homocysteine caused a dose-dependent decrease in DNA synthesis in human umbilical vein endothelial cells. Homocysteine increased mRNA levels of cyclin D1 and cyclin A in RASMC by 3- and 15-fold, respectively, indicating that homocysteine induced the mRNA of cyclins important for the reentry of quiescent RASMC into the cell cycle. Furthermore, homocysteine promoted proliferation of quiescent RASMC, an effect markedly amplified by 2% serum. The growth-promoting effect of homocysteine on vascular smooth muscle cells, together with its inhibitory effect on endothelial cell growth, represents an important mechanism to explain homocysteine-induced atherosclerosis.

Topics: Animals; Aorta, Thoracic; Arteriosclerosis; Base Sequence; Biomarkers; Bromodeoxyuridine; Cell Division; Cells, Cultured; Cyclin D1; Cyclins; DNA; DNA Primers; Dose-Response Relationship, Drug; Endothelium, Vascular; Gene Expression; Homocysteine; Humans; Kinetics; Male; Molecular Sequence Data; Muscle, Smooth, Vascular; Oncogene Proteins; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; RNA, Messenger; Thymidine; Time Factors; Umbilical Veins