C-peptide promotes lesion development in a mouse model of arteriosclerosis.

Patients with insulin resistance and early type 2 diabetes exhibit an increased propensity to develop a diffuse and extensive pattern of arteriosclerosis. Typically, these patients show elevated serum levels of the proinsulin cleavage product C-peptide and immunohistochemical data from our group revealed C-peptide deposition in early lesions of these individuals. Moreover, in vitro studies suggest that C-peptide could promote atherogenesis. This study examined whether C-peptide promotes vascular inflammation and lesion development in a mouse model of arteriosclerosis. ApoE-deficient mice on a high fat diet were treated with C-peptide or control injections for 12 weeks and the effect on lesion size and plaque composition was analysed. C-peptide treatment significantly increased C-peptide blood levels by 4.8-fold without having an effect on glucose or insulin levels, nor on the lipid profile. In these mice, C-peptide deposition in atherosclerotic plaques was significantly increased compared with controls. Moreover, lesions of C-peptide-treated mice contained significantly more macrophages (1.6 ± 0.3% versus 0.7 ± 0.2% positive area; P < 0.01) and more vascular smooth muscle cells (4.8 ± 0.6% versus 2.4 ± 0.3% positive area; P < 0.01). Finally, lipid deposition measured by Oil-red-O staining in the aortic arch was significantly higher in the C-peptide group compared with controls. Our results demonstrate that elevated C-peptide levels promote inflammatory cell infiltration and lesion development in ApoE-deficient mice without having metabolic effects. These data obtained in a mouse model of arteriosclerosis support the hypothesis that C-peptide may have an active role in atherogenesis in patients with diabetes and insulin resistance.

Glucagon-like peptide-1(1-37) inhibits chemokine-induced migration of human CD4-positive lymphocytes.

The present study examined the effect of GLP-1(1-37) on chemokine-induced CD4-positive lymphocyte migration as an early and critical step in atherogenesis. Pretreatment with GLP-1(1-37) reduced the SDF-induced migration of isolated human CD4-positive lymphocytes in a concentration-dependent manner. Similar effects were seen when RANTES was used as a chemokine. GLP-1(1-37)'s effect on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity. Downstream, GLP-1(1-37) inhibited SDF-induced phosphorylation of MLC and cofilin and limited f-actin formation as well as ICAM3 translocation. Furthermore, exendin-4 inhibited SDF-induced migration of CD4-positive lymphocytes similarly to GLP-1(1-37), and transfection of these cells with GLP-1 receptor siRNA abolished GLP-1(1-37)'s action on chemokine-induced ICAM3 translocation, suggesting an effect mediated via the GLP-1 receptor. Thus, GLP-1(1-37) inhibits chemokine-induced CD4-positive lymphocyte migration by inhibition of the PI3-kinase pathway and via the GLP-1 receptor. This effect provides a potential novel mechanism for how GLP-1(1-37) may modulate vascular disease.

LXR activation inhibits chemokine-induced CD4-positive lymphocyte migration.

Migration of CD4-positive lymphocytes into the vessel wall is a critical step in atherogenesis. Recent data suggest that CD4-positive lymphocytes express the nuclear transcription factors Liver-X-Receptor (LXR) alpha and beta with an effect of LXR activators on TH1-cytokine release from these cells. However, the role of LXR in lymphocyte migration remains currently unexplored. Therefore, the present study investigated whether LXR activation might modulate chemokine-induced migration of these cells. Stimulation of CD4-positive lymphocytes with SDF-1 leads to a 2.5 +/- 0.8-fold increase in cell migration (P < 0.05; n = 12). Pretreatment of cells with the LXR activator T0901317 reduces this effect in a concentration-dependent manner to a maximal 0.9 +/- 0.4-fold induction at 1 micromol/L T0901317 (P < 0.05 compared to SDF-1-treated cells; n = 12). Similar results were obtained with the LXR activator GW3965. The effect of LXR activators on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity as determined by PI-3 kinase activity assays. Downstream, T0901317 inhibited activation of the small GTPase Rac and phosphorylation of the myosin light chain (MLC). Moreover, LXR activator treatment reduced f-actin formation as well as ICAM3 translocation to the uropod of the cell, thus interfering with two important steps in T cell migration. Transfection of CD4-positive lymphocytes with LXRalpha/beta siRNA abolished T0901317 inhibitory effect on MLC phosphorylation and ICAM3 translocation. LXR activation by T0901317 or GW3965 inhibits chemokine-induced migration of CD4-positive lymphocytes. Given the crucial importance of chemokine-induced T cell migration in early atherogenesis, LXR activators may be promising tools to modulate this effect.

Ivabradine reduces chemokine-induced CD4-positive lymphocyte migration.

AIMS: Migration of CD4-positive lymphocytes into the vessel wall is a critical step in atherogenesis. Recent data suggest that ivabradine, a selective I(f)-channel blocker, reduces atherosclerotic plaque formation in apolipoprotein E-deficient mice, hitherto nothing is known about the mechanism by which ivabradine modulates plaque formation. Therefore, the present study investigated whether ivabradine regulates chemokine-induced migration of lymphocytes. METHODS AND RESULTS: Stimulation of CD4-positive lymphocytes with SDF-1 leads to a 2.0 ± 0.1 fold increase in cell migration (P < .01; n = 7). Pretreatment of cells with ivabradine reduces this effect to a maximal 1.2 ± 0.1 fold induction at 0.1 µmol/L ivabradine (P < .01 compared to SDF-1-treated cells, n = 7). The effect of ivabradine on CD4-positive lymphocyte migration was mediated through an early inhibition of chemokine-induced PI-3 kinase activity as determined by PI-3 kinase activity assays. Downstream, ivabradine inhibits activation of the small GTPase Rac and phosphorylation of the Myosin Light Chain (MLC). Moreover, ivabradine treatment reduces f-actin formation as well as ICAM3 translocation to the uropod of the cell, thus interfering with two important steps in T cell migration. CONCLUSION: Ivabradine inhibits chemokine-induced migration of CD4-positive lymphocytes. Given the crucial importance of chemokine-induced T-cell migration in early atherogenesis, ivabradine may be a promising tool to modulate this effect.

C-Peptide induces vascular smooth muscle cell proliferation: involvement of SRC-kinase, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinase 1/2.

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.

LXR activation reduces proinflammatory cytokine expression in human CD4-positive lymphocytes.

BACKGROUND: CD4-positive lymphocytes, the major T-cell population in human atheroma, mainly secrete Th-1-type proinflammatory cytokines, like interferon (IFN)gamma, tumor necrosis factor (TNF)alpha, and interleukin (IL)-2, thus promoting atherogenesis. Recent data suggest that the nuclear transcription factors liver X receptor-alpha and liver X receptor-beta (LXRalpha and LXRbeta) limit plaque formation in animal models by modulating macrophage function. Still, the role of LXRs in CD4-positive lymphocytes is currently unexplored. METHODS AND RESULTS: Human CD4-positive lymphocytes express LXRalpha and LXRbeta mRNA and protein. Activation of CD4-positive cells by anti-CD3 mAbs, anti-CD3/CD28 mAbs, as well as PMA/ionomycin significantly increased Th1-cytokine mRNA and protein expression. Treatment with the LXR activator T0901317 reduced this increase of IFNgamma, TNFalpha, and IL-2 in a concentration-dependent manner with a maximum at 1 micromol/L T0901317. Transient transfection assays revealed an inhibition of IFNgamma promoter activity by T0901317 as the underlying molecular mechanism. Such anti-inflammatory actions were also evident in cell-cell interactions with medium conditioned by T0901317-treated CD4-positive cells attenuating human monocyte CD64 expression. CONCLUSIONS: Human CD4-positive lymphocytes express both LXRalpha and LXRbeta, and LXR activation can reduce Th-1 cytokine expression in these cells. These data provide new insight how LXR activators might modulate the inflammatory process in atherogenesis and as such influence lesion development.

C-peptide induces human renal mesangial cell proliferation in vitro, activating Src-kinase, PI-3 kinase and ERK1/2.

BACKGROUND: Elevated levels of C-peptide have been found in patients with insulin resistance and early type 2 diabetes. These patients are at greater risk to develop micro- and macrovascular complications. Since diabetic nephropathy involves glomerular hyperproliferation, the present study evaluates the role of C-peptide on human renal mesangial cell proliferation. METHODS AND RESULTS: C-peptide induces proliferation of human renal mesangial cells in a concentration-dependent manner with a maximal 2.6±0.4-fold induction at 10 nmol/L (P<0.05 compared with unstimulated cells; n=6), as revealed by [3H]-thymidine incorporation experiments. The proliferative effect of C-peptide is prevented by Src-kinase inhibitor-PP2, PI-3 kinase inhibitor-LY294002, and the ERK1/2 inhibitor-U126. Moreover, C-peptide induces phosphorylation of Src, as well as activation of PI-3 kinase and ERK1/2. Furthermore, C-peptide induces cyclin D1 expression as well as phosphorylation of retinoblastoma protein (Rb). CONCLUSIONS: These results demonstrate an active role of C-peptide on the proliferation of human renal mesangial cells in vitro involving PI-3 kinase and MAP kinase signaling pathways, suggesting a possible role of C-peptide in glomerular hyperproliferation in patients with diabetic nephropathy.

Resistin: a newly identified chemokine for human CD4-positive lymphocytes.

AIMS: Increased levels of resistin, a peptide secreted by adipocytes and inflammatory cells, circulate in patients with insulin resistance and early type 2 diabetes, a high-risk population for the development of a diffuse and extensive pattern of arteriosclerosis. Recent data suggest that resistin may activate vascular cells such as smooth muscle cells and endothelial cells, but hitherto nothing is known about the role of resistin in CD4-positive lymphocytes. Therefore, the present study examined the effect of resistin on CD4-positive lymphocyte migration, an important process in early atherogenesis. METHODS AND RESULTS: Resistin stimulated CD4-positive cell chemotaxis in a concentration-dependent manner with a maximal induction of 2.25 +/- 0.54 at 100 ng/mL (P < 0.05, n = 7). This process involves pertussis toxin-sensitive G-proteins as well as activation of Src- and phosphoinositide 3-kinase (PI 3-K). Biochemical analysis showed that resistin induces phosphorylation of Src and PI 3-K activation in human CD4-positive cells. In addition, resistin activates RhoA, Rac-1, and Cdc42 in these cells as shown by affinity precipitation experiments. Finally, resistin-induced phosphorylation of myosin light chain was inhibited by Src short interference RNA transfection, underscoring the importance of the upstream signalling molecule Src in resistin-induced migration. CONCLUSION: These data support an active role of resistin in CD4-positive lymphocyte chemotaxis and elucidate molecular mechanisms in resistin-induced cell migration.