Effect of gliclazide on monocyte-endothelium interactions in diabetes.

Enhanced monocyte-endothelial cell interactions have been documented in diabetes. Because adherence of monocytes to the endothelium is one of the earliest events in the development of atherosclerosis, its alteration may represent one of the mechanisms leading to accelerated atherosclerosis in diabetic patients. Previous studies have suggested that lipoprotein oxidation and protein glycation may contribute to the increased monocyte binding to the diabetic vasculature. Based on the recent finding that gliclazide has free-radical scavenging activity, we examined the ex vivo and in vitro effects of this drug on human monocyte binding to endothelial cells. Our results demonstrate that short-term administration of gliclazide to patients with type 2 diabetes lowers the enhanced adhesion of diabetic monocytes observed before gliclazide treatment (163+/-24% over control values, p<0.005) to levels similar to those observed in controls. They also show that gliclazide (10 microg/ml) reduces in vitro by approximately 35% both oxidized low-density lipoprotein (LDL)- and glycated albumin-induced monocyte adhesion to endothelial cells. Based on these results, we next investigated the molecular mechanisms responsible for the inhibitory effect of gliclazide on glycated albumin-induced monocyte adhesion to endothelium. In glycated albumin-treated endothelial cells, we observed induction of cell-associated expression of E-selectin (ELAM-1; 170+/-10% over control values, p<0.005), intercellular cell adhesion molecule-1 (ICAM-1; 131+/-8% over control values, p<0.005) and vascular cell adhesion molecule-1 (VCAM-1; 134+/-8% over control values, p<0.005), augmentation in the levels of the transcripts of these molecules, and an increase in the DNA binding of NF-kappaB in the promoters of these antigens. Gliclazide markedly inhibited the induction of all these parameters. Because the oxidative stress-sensitive transcription factor NF-kappaB is implicated in endothelial cell activation, the observed inhibitory effect of gliclazide on NF-kappaB activation and glycated albumin-induced expression of DNA binding activity for the NF-kappaB site in the ELAM-1, ICAM-1 and VCAM-1 promoters seems to be due to its antioxidant properties. These results suggest that gliclazide, by its ability to reduce endothelial activation, may exert potential beneficial effects in the prevention of atherosclerosis associated with type 2 diabetes.

Gliclazide decreases low-density lipoprotein oxidation and monocyte adhesion to the endothelium.

Increasing evidence implicates oxidized low-density lipoprotein (LDL) and advanced glycation end products (AGE) in the atherogenesis associated with diabetes mellitus. In the present study, we examined the in vitro effects of gliclazide on LDL oxidation and monocyte adhesion to endothelial cells induced by oxidized LDL and glycated albumin. To assess the clinical relevance of our in vitro findings, we also measured the effect on monocyte adhesion of gliclazide administration to type 2 diabetic patients. Incubation of human monocytes and endothelial cells with increasing concentrations of gliclazide (0 to 10 microg/mL) and native LDL (100 microg/mL) induced a dose-dependent diminution of cell-mediated LDL oxidation. Pretreatment of endothelial cells with gliclazide (0 to 10 microg/mL) before addition of native LDL (100 microg/mL) or glycated albumin (100 microg/mL) resulted in a dose-dependent diminution of oxidized LDL- and glycated albumin-induced monocyte adhesion to endothelial cells. In type 2 diabetic patients, administration of gliclazide inhibits the increased adhesiveness of monocytes to levels similar to those observed in control subjects. These results indicate that gliclazide is an antioxidant and suggest a beneficial effect of this drug in the prevention of atherosclerosis associated with type 2 diabetes.

Growth factors stimulate neointimal cells in vitro and increase the thickness of the neointima formed at the neck of porcine aneurysms treated by embolization.

BACKGROUND AND PURPOSE: Growth factors (GFs) may favor the healing of aneurysms treated with endovascular techniques by stimulating neointima formation. METHODS: Bilateral carotid aneurysms were constructed with venous pouches in 50 pigs and embolized intraoperatively with collagen sponges with and without GFs (platelet-derived growth factor-BB [PDGF-BB] 0.15 or 1.5 microg or transforming growth factor-beta(1) [TGF-beta(1)] 60 or 600 ng) in each animal. DNA synthesis, cell proliferation, and collagen secretion assays were performed to assess the in vitro effects of GFs on neointimal cells harvested from the treated aneurysms. (125)I-PDGF-BB was used to study in vivo GF release from sponges. The thickness of the neointima at the surface of the sponges was measured 2 weeks after surgery. Since porcine aneurysms tend to heal after collagen sponge embolization, this experiment was repeated in dogs, which have shown a propensity for recurrence with the same technique, with 600 ng TGF-beta(1) or platelet extracts. RESULTS: PDGF-BB stimulated DNA synthesis and cell proliferation, while TGF-beta(1) strongly increased collagen synthesis of neointimal cells in vitro. Clearance of (125)I-PDGF-BB from the sponges followed a biphasic curve, with 1.5% of exogenous PDGF-BB remaining at 1 week. The local delivery of PDGF-BB (0.15 or 1.5 microg) and TGF-beta(1) (600 ng) significantly increased neointimal thickness at the neck of porcine aneurysms, while 60 ng of TGF-beta(1) had no demonstrable effect. TGF-beta(1) (600 ng) or platelet extracts had no influence on canine aneurysms. CONCLUSIONS: PDGF-BB and TGF-beta(1) can stimulate neointimal cells in vitro and neointima formation in vivo, but TGF-beta(1) and platelet extracts do not compensate for deficient thrombosis in canine aneurysms. Effects on the long-term results of embolization remain speculative.

Fibrinogen and vascular smooth muscle cell grafts promote healing of experimental aneurysms treated by embolization.

BACKGROUND AND PURPOSE: Residual necks and recurrences frequently occur after endovascular treatment of cerebral aneurysms. Addition of fibrinogen and vascular smooth muscle cells (VSMCs) to the embolic material may promote healing of embolized aneurysms by increasing neointima formation at the neck. METHODS: Bilateral carotid aneurysms were constructed with venous pouches in 31 dogs. Aneurysms were packed intraoperatively with bare Gelfoam sponges, sponges treated with fibrinogen, or fibrinogen sponges seeded with the animal's own VSMCs or peripheral blood mononuclear cells. Animals were killed after angiography at 3 weeks, and morphometric studies were performed to measure the thickness of the neointima at the neck of treated lesions. Angiographic results and mean thickness of neointimas were compared using ANOVA. In 8 animals, 1 aneurysm was embolized with sponge seeded with VSMCs transduced by adenoviral infection to express a fluorescent protein (green fluorescent protein), and gene expression was monitored for 4, 7, 14, and 21 days by fluorescent microscopy. RESULTS: Aneurysms treated with sponges seeded with VSMCs had significantly thicker neointimas and were more completely obliterated at 3 weeks than control aneurysms treated with fibrinogen sponges. Peripheral blood mononuclear cells could not reproduce these findings. Sponges treated with fibrinogen alone promoted formation of a thicker neointima than bare sponges. Transduced cells transplanted into in vivo aneurysms still expressed green fluorescent protein at 3 weeks. CONCLUSIONS: VMSC grafts can improve healing of experimental aneurysms treated by embolization. Transplantation of cells transduced to express a foreign gene opens the way for in situ gene therapy for cerebral aneurysms.

Gliclazide reduces the induction of human monocyte adhesion to endothelial cells by glycated albumin.

OBJECTIVE: To examine the kinetic of human monocyte adhesion to endothelial cells stimulated by glycated albumin, the contributive role of cell adhesion molecules to this effect, and the effect of gliclazide--an hypoglycemic drug with antioxidant properties--on these parameters. METHODS: In-vitro experiments performed in the presence and absence of gliclazide consisted of: (1) time-dependent determination of human monocyte adhesion to human endothelial cells (ECs) pre-exposed to glycated albumin; (2) evaluation of adhesion after incubation of ECs with antibodies against cell surface adhesion molecules; and (3) determination of EC surface adhesion molecules and of the activity of the transcription factor NF-kappaB. RESULTS: Exposition of human ECs for 1-48 h to 100 microg/ml glycated albumin led to a time-dependent increase in human monocyte adhesion to endothelium. Pretreatment of ECs with 10 microg/ml gliclazide significantly decreased the glycated albumin-stimulated monocyte adhesion to these cells. Anti-antibodies against E-selectin (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) also reduced the stimulatory effect of glycated albumin on monocyte adhesion. In glycated albumin-treated ECs, an induction of both soluble and cell associated expression of ELAM-1, VCAM-1 and ICAM-1, an augmentation in the levels of these molecule transcripts and an increase in the DNA binding activity for NF-kappaB in the promoters of these antigens were observed. Gliclazide markedly inhibited the induction of all these parameters. CONCLUSIONS: Glycated albumin stimulates human monocyte adhesion to ECs by inducing cell associated ELAM-1, ICAM-1 and VCAM-1. Gliclazide effectively inhibits monocyte adhesion to ECs by reducing glycated albumin induction of EC adhesion molecules and NF-kappaB activation. These results suggest that gliclazide may be beneficial in the prevention of endothelial disturbances associated with hyperglycemia in diabetic patients.

Normalization of plasma lipid peroxides, monocyte adhesion, and tumor necrosis factor-alpha production in NIDDM patients after gliclazide treatment.

OBJECTIVE: To evaluate the effect of gliclazide administration to NIDDM patients on 1) monocyte adhesion to cultured endothelial cells, 2) plasma cytokine and lipid peroxide levels, and 3) monocyte cytokine production. RESEARCH DESIGN AND METHODS: Poorly controlled glyburide-treated diabetic patients (n = 8) and healthy control subjects (n = 8) were recruited. At the beginning of the study, glyburide was replaced by an equivalent hypoglycemic dose of gliclazide. Serum and monocytes were isolated from blood obtained from control and diabetic subjects before and after 3 months of treatment with gliclazide. RESULTS: Plasma lipid peroxide levels and monocyte adhesion to endothelial cells are enhanced in NIDDM patients, and gliclazide administration totally reverses these abnormalities. Before gliclazide treatment, serum levels of cytokines did not differ in the control and the diabetic groups, with the exception of an enhancement of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL)-6 in NIDDM subjects. Basal and lipopolysaccharide (LPS)-stimulated monocyte production of interleukin-1 beta, IL-6, and IL-8 did not differ between the two groups. Furthermore, basal monocyte production of TNF-alpha was similar in the control and the diabetic groups, whereas a marked increase in the LPS-stimulated monocyte production of TNF-alpha was observed in the NIDDM group. Gliclazide treatment lowered LPS-stimulated TNF-alpha production by diabetic monocytes to levels similar to those observed in control subjects. CONCLUSIONS: Gliclazide administration to NIDDM patients inhibits the increased adhesiveness of diabetic monocytes to endothelial cells and reduces the production of TNF-alpha by these cells. These results suggest that treatment of NIDDM subjects with gliclazide may be beneficial in the prevention of atherosclerosis associated with NIDDM.

Lipoprotein lipase enhances human monocyte adhesion to aortic endothelial cells.

Lipoprotein lipase (LPL)-mediated lipolysis of very low density lipoprotein (VLDL) has been demonstrated to increase U937 monocyte adhesion to endothelial cells. In the present study, we evaluated the ability of LPL to enhance human monocyte adhesion to bovine aortic endothelial cells (BAEC) in the absence of exogenous lipoproteins. Exposure of BAEC to 1 microgram/ml LPL at 37 degrees C resulted in a significant increase in monocyte adhesion over control values. Addition of VLDL in the culture media further enhanced the LPL effect. A significant increase in monocyte adhesion was also observed when BAEC were incubated with LPL at 4 degrees C. Heparin or heparinase treatment of BAEC totally abolished the LPL stimulatory effect on monocyte adhesion. In addition, incubation of monocytes with heparinase suppressed the ability of LPL to stimulate monocyte adhesion to endothelial cells. These treatments also markedly decreased LPL binding to the monocyte and endothelial cell surfaces. In contrast to native LPL, heat inactivated or phenylmethylsulfonyl fluoride (PMSF)-treated LPL did not increase monocyte adhesion to BAEC. Finally, incubation of LPL in the presence of the 5D2 antibody resulted in a total suppression of the LPL-induced monocyte adhesion to BAEC. Taken together, these data demonstrate that LPL activity plays an important role in LPL-induced monocyte adhesion and that LPL binding to heparan sulfate proteoglycans expressed on both monocytes and endothelial cells surfaces is required for the enhanced monocyte adhesion. These results suggest a new mechanism by which LPL may promote the development of atherosclerosis, that of facilitating monocyte adhesion to the endothelium.

Gliclazide decreases cell-mediated low-density lipoprotein (LDL) oxidation and reduces monocyte adhesion to endothelial cells induced by oxidatively modified LDL.

Low-density lipoprotein (LDL) oxidation has been suggested to play a key role in the pathogenesis of atherosclerosis, a major complication of diabetes mellitus. Gliclazide, a second-generation sulfonylurea, is widely used in the treatment of type II diabetes mellitus. Recently, a free-radical-scavenging activity of gliclazide has been reported. In the present study, we examined the effects of gliclazide on cell-mediated LDL oxidation and monocyte adhesion to endothelial cells induced by oxidatively modified LDL. Incubation of human monocytes and bovine aortic endothelial cells (BAE cells) with increasing concentrations of gliclazide (0 to 10 micrograms/mL) and native LDL (100 micrograms/mL) resulted in a dose-dependent diminution of cell-mediated LDL oxidation as assayed by measurement of thiobarbituric acid (TBA)-reactive substances (TBARS). In addition, exposure of BAE cells to gliclazide (0 to 10 micrograms/mL) and native LDL (100 micrograms/mL) induced a dose-dependent diminution of the oxidized LDL-induced monocyte adhesion to BAE cells as measured by the myeloperoxidase (MPO) assay. The effects of glyburide, another second-generation sulfonylurea, were also tested on cell-mediated oxidation of LDL and LDL-induced monocyte adhesion to the endothelium. No significant effect of this drug was observed on these two processes. These results therefore demonstrate that gliclazide is effective in vitro in reducing both cell-mediated LDL oxidation and monocyte adhesion to the endothelium. These findings suggest a potential beneficial effect of gliclazide in the prevention of atherosclerosis in diabetic patients.

Direct stimulatory effect of insulin-like growth factor-I on monocyte and macrophage tumor necrosis factor-alpha production.

GH has been demonstrated to play a physiological role in the priming of macrophages for tumor necrosis factor-alpha (TNF alpha) synthesis. Although evidence has been presented that GH exerts this effect by an indirect mechanism, the mediators of GH stimulation of TNF alpha synthesis have not been identified. Because insulin-like growth factor-I (IGF-I) is a major mediator of many GH effects, in the present study we investigated the direct in vitro effect of this growth factor on macrophage TNF alpha production. Treatment of murine macrophages with physiological concentrations of IGF-I (0.13-130 nM) enhanced both basal and lipopolysaccharide-stimulated macrophage TNF alpha release and messenger RNA levels. Induction of basal TNF alpha production was also observed after treatment of the cells with supraphysiological concentrations of insulin (130-1300 nM). Exposure of human monocytes to IGF-I led to a similar increase of basal TNF alpha production and messenger RNA expression. Preexposure of macrophages with specific antibodies against IGF-I and IGF-I receptor before IGF-I addition resulted in a complete abrogation of the stimulatory effect of IGF-I on TNF alpha production, indicating that specific binding of IGF-I to its receptor is required for macrophage TNF alpha induction by IGF-I. In contrast to the stimulatory effect of IGF-I, neither GH (0.1-10 micrograms/ml) nor IGF-II (0.13-130 nM) enhanced macrophage TNF alpha release in vitro. To assess the role of the tyrosine kinase system in mediating IGF-I-induced basal TNF alpha production, macrophages were preincubated with the specific tyrosine kinase inhibitors, genistein and tyrphostin A9, before IGF-I exposure. Addition of these compounds resulted in a dose-dependent inhibition of the stimulatory effect of IGF-I on macrophage TNF alpha release, indicating that protein tyrosine kinase activation is required for TNF alpha stimulation by IGF-I. Taken together, these results demonstrate that IGF-I is a monocyte/macrophage activating factor that enhances TNF alpha production, and that such effect is mediated via the IGF-I receptor and involves tyrosine kinase activation.

Role of oxidant injury on macrophage lipoprotein lipase (LPL) production and sensitivity to LPL.

We investigated, in the present study, the role of reactive oxygen intermediates (ROI) in the control of macrophage lipoprotein lipase (LPL) secretion. Exposure of murine macrophages to increasing concentrations of hydrogen peroxide (H2O2) resulted in enhanced basal LPL production and mRNA levels. The increase of LPL production was reduced in the presence of antioxidants. Oxidant stress also modulated the regulation of macrophage LPL production by tumor necrosis factor alpha (TNF alpha). While antioxidants accentuated the inhibition of LPL by TNF alpha, addition of H2O2 significantly attenuated TNF alpha-induced LPL inhibition. As LPL has been shown to induce macrophage TNF alpha release, the effect of reactive oxygen species on LPL-induced TNF alpha production was also examined. Simultaneous treatment of macrophages with LPL and H2O2 or pretreatment of macrophages with H2O2 prior to LPL stimulation decreased the LPL-induced TNF alpha release by macrophages to the same extent. Under these experimental conditions, LPL binding to macrophages was markedly decreased. These data indicate that ROI are effective enhancers of macrophage LPL production and modulate macrophage response to LPL. These effects may represent additional mechanisms through which oxidant stress may participate to the development of atherosclerosis.

The effects of a selective alpha-1 adrenergic blockade on the activity of adipose tissue lipoprotein lipase in female hamsters.

Lipoprotein lipase, is an enzyme responsible for the hydrolysis of triacylglycerols at the surface of endothelial cells. Its regulation is not completely elucidated and seems, among other things, under the influence of the sympathetic nervous system. The adrenergic regulation of lipoprotein lipase activity is complex and the alpha 1 adrenergic pathway appears involved in this regulation. In the present study, adipose tissues of female hamsters are investigated following a single injection of doxazosin and phenylephrine and are compared to controls for the activity of lipoprotein lipase. After an acute treatment with a selective alpha 1 antagonist (doxazosin), lipoprotein lipase activity was decreased in the parametrial white adipose tissue and increased in brown adipose tissue (p < or = 0.05). Moreover, a treatment with phenylephrine, an alpha 1 adrenergic agonist, increased the activity of lipoprotein lipase, in the parametrial fat pad only. On the other hand, the activity of lipoprotein lipase in heart and in skeletal muscle was not modified by an alpha 1 stimulation or blockade. In this study, calcium and norepinephrine did not appear involved in the regulation of lipoprotein lipase activity. On the contrary, the increase of plasma glycerol after an acute treatment with doxazosin suggests that the lipolytic activity of white adipose tissue could be involved in the decrease of lipoprotein lipase activity in the parametrial white adipose tissue.