Helga Bach

C-Peptide Colocalizes with Macrophages in Early Arteriosclerotic Lesions of Diabetic Subjects and Induces Monocyte Chemotaxis In Vitro

Objective—Increased levels of C-peptide, a cleavage product of proinsulin, 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. This study tested the hypothesis that C-peptide might participate in atherogenesis in these patients. Method and Results—We demonstrate significantly higher intimal C-peptide deposition in thoracic artery specimens from young diabetic subjects compared with matched nondiabetic controls as determined by immunohistochemical staining. C-peptide colocalized with monocytes/macrophages in the arterial intima of artery specimen from diabetic subjects. In vitro, C-peptide stimulated monocyte chemotaxis in a concentration-dependent manner with a maximal 2.3±0.4-fold increase at 1 nmol/L C-peptide. Pertussis toxin, wortmannin, and LY294002 inhibited C-peptide–induced monocyte chemotaxis, suggesting the involvement of pertussis toxin-sensitive G-proteins as well as a phosphoinositide 3-kinase (PI3K)-dependent mechanism. In addition, C-peptide treatment activated PI3K in human monocytes, as demonstrated by PI3K activity assays. Conclusion—C-peptide accumulated in the vessel wall in early atherogenesis in diabetic subjects and may promote monocyte migration into developing lesions. These data support the hypothesis that C-peptide may play an active role in atherogenesis in diabetic patients and suggest a new mechanism for accelerated arterial disease in diabetes.

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)γ, tumor necrosis factor (TNF)α, and interleukin (IL)-2, thus promoting atherogenesis. Recent data suggest that the nuclear transcription factors liver X receptor-alpha and liver X receptor-beta (LXRα and LXRβ) 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 LXRα and LXRβ 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 IFNγ, TNFα, and IL-2 in a concentration-dependent manner with a maximum at 1 &mgr;mol/L T0901317. Transient transfection assays revealed an inhibition of IFNγ 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 LXRα and LXRβ, 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 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.

Effect of rosiglitazone treatment on plaque inflammation and collagen content in nondiabetic patients: data from a randomized placebo-controlled trial.

Background—Therapeutic strategies to stabilize advanced arteriosclerotic lesions may prevent plaque rupture and reduce the incidence of acute coronary syndromes. Thiazolidinediones (TZDs), like rosiglitazone, are oral antidiabetic drugs with additional antiinflammatory and potential antiatherogenic properties. In a randomized, placebo-controlled, single-blind trial, we examined the effect of 4 weeks of rosiglitazone therapy on histomorphological characteristics of plaque stability in artery specimen of nondiabetic patients scheduled for elective carotid endarterectomy. Methods and Results—A total of 24 nondiabetic patients with symptomatic carotid artery stenosis were randomly assigned to rosiglitazone (4 mg BID) or placebo in addition to standard therapy. In this population of nondiabetic patients, rosiglitazone treatment did not significantly change fasting blood glucose, fasting insulin, or lipid parameters. In contrast, rosiglitazone significantly reduced CD4-lymphocyte content as well as macrophage HLA-DR expression in the shoulder region, reflecting less inflammatory activation of these cells by lymphocyte interferon-&ggr;. Moreover, rosiglitazone significantly increased plaque collagen content (7.7±1.6% versus 3.7±0.7% of plaque area; P=0.036) compared with placebo, suggesting that TZD treatment may stabilize arteriosclerotic lesions. In addition, rosiglitazone reduced serum levels of 2 inflammatory arteriosclerosis markers: C-reactive protein and serum amyloid A. Conclusions—Four weeks of treatment with rosiglitazone significantly reduces vascular inflammation in nondiabetic patients, leading to a more stable type of arteriosclerotic lesion.

Inhibition of MMP-9 expression by PPARγ activators in human bronchial epithelial cells

Background: The release of matrix degrading enzymes such as matrix metalloproteinase 9 (MMP-9) from bronchial epithelial cells is critically involved in airway wall remodelling in chronic inflammatory processes of the respiratory system. MMP-9 expression is induced by inflammatory mediators such as tumour necrosis factor (TNF)-α, but to date nothing is known about the mechanisms of inhibition of MMP-9 expression in these cells. Methods: A study was undertaken to examine whether activators of the nuclear transcription factor peroxisome proliferator activated receptor gamma (PPARγ) might modulate MMP-9 expression in two different bronchial epithelial cell lines. Results: PPARγ was expressed and was functionally active in NL20 and BEAS cells. Activation of PPARγ by rosiglitazone or pioglitazone significantly reduced TNF-α and PMA induced MMP-9 gelatinolytic activity in a concentration dependent manner in both cell lines, but did not alter the expression of tissue inhibitor of MMPs type 1 (TIMP-1), the local inhibitor of MMP-9. Northern blot analysis revealed a decrease in MMP-9 mRNA expression following treatment with PPARγ which resulted from the inhibition of NF-κB activation in these cells, as determined by transient transfection assays and electromobility shift assays. Conclusion: Activation of PPARγ in human bronchial epithelial cells limits the expression of matrix degrading MMP-9. This might have therapeutic applications in chronic inflammatory processes of the respiratory system.

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.

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.

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 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.

Signalling processes involved in C-peptide-induced chemotaxis of CD4-positive lymphocytes

Abstract.Previous data from our group demonstrated that C-peptide induces chemotaxis of CD4-positive lymphocytes in-vitro, mediated by activation of G-protein and PI 3-kinase γ, but additional signalling pathways involved in this process remained unexplored. In the present study we further analyze intracellular signalling pathways which lead to C-peptide-induced CD4-positive lymphocyte migration. We provide evidence that C-peptide-induced chemotaxis of CD4-positive lymphocytes is critically dependent on activation of Src-kinase and RhoA, Rac-1 and Cdc42 GTPases. Furthermore, C-peptide stimulates phosphorylation of PAK, LIMK and cofilin downstream of Rac-1 and Cdc42, leading to cofilin inactivation and actin filament stabilization. In addition, C-peptide induces ROCK kinase activity and MLC phosphorylation downstream of RhoA, thereby stimulating myosin mediated cell contraction. In contrast, C-peptide does not activate ERK1/2, p38 or Akt in CD4-positive lymphocytes. Our data support an active role of C-peptide in CD4-positive lymphocyte chemotaxis and elucidate molecular mechanisms in C-peptide-induced cell migration.