Fritz Maingrette

The influences of hyperprolactinemia and obesity on cardiovascular risk markers: effects of cabergoline therapy

Objective  In view of the association of hyperprolactinaemia with insulin resistance, we hypothesized that patients with hyperprolactinaemia may present increased cardiovascular risk markers.

Nox2-Containing NADPH Oxidase Deficiency Confers Protection From Hindlimb Ischemia in Conditions of Increased Oxidative Stress

Objective—Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. Methods and Results—To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2−/−). Nox2−/− mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2+/+ mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2−/− endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2+/+ endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. Conclusions—Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.

Nox2-derived reactive oxygen species contribute to hypercholesterolemia-induced inhibition of neovascularization: Effects on endothelial progenitor cells and mature endothelial cells

BACKGROUND Hypercholesterolemia has been associated with impaired angiogenesis and reduced blood flow recuperation after ischemia. However, the precise mechanisms involved are unknown. Here we investigated the role of Nox2-derived reactive oxygen species (ROS) in the modulation of neovascularization by hypercholesterolemia. METHODS AND RESULTS Mice deficient for the Nox2-containing NADPH oxidase (Nox2(-/-)) and control mice (Nox2(+/+)) were put on a high cholesterol diet (HCD) for a total of 15 weeks. After three months, hindlimb ischemia was surgically induced by femoral artery removal. Nox2 expression and oxidative stress levels in ischemic tissues were significantly increased by HCD in control mice, but not in Nox2(-/-) mice. Nox2(-/-) mice were also protected against hypercholesterolemia-induced impairment of neovascularization, as demonstrated by faster blood flow recovery after ischemia and increased capillary density in ischemic muscles. Nox2 deficiency was associated with preserved activity of eNOS in ischemic tissues, and improved activity of endothelial progenitor cells (EPCs). In vitro, HUVECs treated with the NADPH oxidase inhibitor apocynin or endothelial cells isolated from the aorta of Nox2(-/-) mice exhibited reduced ROS formation following exposure to oxLDL. This was associated with improved nitric oxide (NO) bioavailability and protection against oxLDL-induced inhibition of angiogenic activities. CONCLUSIONS Nox2-containing NADPH oxidase deficiency protects against hypercholesterolemia-induced impairment of neovascularization. The potential mechanisms involved include reduced ROS formation, preserved activation of angiogenic signals, and improved functional activities of EPCs and mature endothelial cells.

Sildenafil Increases Endothelial Progenitor Cell Function and Improves Ischemia-Induced Neovascularization in Hypercholesterolemic Apolipoprotein E–Deficient Mice

Hypercholesterolemia is associated with impaired neovascularization in response to ischemia. Potential mechanisms include defective NO bioactivity and a reduction in the number/function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that sildenafil, a phosphodiesterase 5 inhibitor that increases NO-driven cGMP levels, could stimulate EPC function and improve ischemia-induced neovascularization in hypercholesterolemic conditions. Apolipoprotein E–deficient (ApoE−/−) mice were treated (or not treated) with sildenafil (40 mg/kg per day in water), and hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to an improved blood flow recovery, an increased capillary density, and a reduction of oxidative stress levels in ischemic muscles at day 7 after surgery. Sildenafil therapy is associated with an increased activation of angiogenic transduction pathways, including Akt, p44/42 mitogen-activated protein kinase, and p38. In vitro, sildenafil increases cellular migration and tubule formation of mature endothelial cells (human umbilical vascular endothelial cells) in a cGMP-dependent manner. In vivo, ApoE−/− mice treated with sildenafil exhibit a significant increase in the number of bone marrow–derived EPCs. Moreover, the angiogenic activities of EPCs (migration and adhesion) are significantly improved in ApoE−/− mice treated with sildenafil. In summary, this study demonstrates that sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic ApoE−/− mice. The mechanisms involve beneficial effects on angiogenic transduction pathways together with an increase in the number and the functional activity of EPCs. Sildenafil could constitute a novel therapeutic strategy to reduce tissue ischemia in atherosclerotic diseases.

Protection against vascular aging in Nox2-deficient mice: Impact on endothelial progenitor cells and reparative neovascularization

BACKGROUND Aging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in the context of aging. METHODS AND RESULTS Hindlimb ischemia was surgically induced by femoral artery removal in young (2 months) and old (10 months) Nox2-deficient (Nox2(-/-)) and wild type mice. We found that Nox2 expression is increased by aging in ischemic muscles of wild type mice. This is associated with a significant reduction of blood flow recovery after ischemia in old compared to young mice at day 21 after surgery (Doppler flow ratios: 0.51 ± 0.05 vs. 0.72 ± 0.05; p < 0.05). We also demonstrate that capillary and arteriolar densities are significantly reduced in ischemic muscles of old animals, while oxidative stress levels are increased (nitrotyrosine immunostaining). Importantly, Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation and vascular densities in old animals. Endothelial progenitor cells (EPCs) have an important role for postnatal neovascularization. Here we show that the functional activities of EPCs (migration, adhesion to mature endothelial cells) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPC functional activities in old animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels in EPCs (DHE, DCF-DA) that is not present in Nox2-deficient animals. CONCLUSION Nox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.

Fish oil-enriched diet protects against ischemia by improving angiogenesis, endothelial progenitor cell function and postnatal neovascularization

BACKGROUND Fish oil consumption has been associated with a reduced incidence of cardiovascular diseases. However, the precise mechanisms involved are not completely understood. Here we tested the hypothesis that a fish oil-enriched diet improves neovascularization in response to ischemia. METHODS AND RESULTS C57Bl/6 mice were fed a diet containing either 20% fish oil, rich in long-chain n-3 polyunsaturated fatty acids (PUFAs), or 20% corn oil, rich in n-6 PUFAs. After 4 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly improved in mice fed a fish oil diet compared to those fed a corn oil diet (Doppler flow ratio (DFR) at day 21 after surgery 78 ± 5 vs. 56 ± 4; p < 0.01). Clinically, this was associated with a significant reduction of ambulatory impairment and ischemic damage in the fish oil group. At the microvascular level, capillary density was significantly improved in ischemic muscles of mice fed a fish oil diet. This correlated with increased expression of VEGF and eNOS in ischemic muscles, and higher NO concentration in the plasma. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. We found that the number of EPCs was significantly increased in mice fed a fish oil diet. In addition, oxidative stress levels (DCF-DA, DHE) were reduced in EPCs isolated from mice exposed to fish oil, and this was associated with improved EPC functional activities (migration and integration into tubules). In vitro, treatment of EPCs with fish oil resulted in a significant increase of cellular migration. In addition, the secretion of angiogenic growth factors including IL6 and leptin was significantly increased in EPCs exposed to fish oil. CONCLUSION Fish oil-enriched diet is associated with improved neovascularization in response to ischemia. Potential mechanisms involved include activation of VEGF/NO pathway in ischemic tissues together with an increase in the number and the functional activities of EPCs.

Probucol and antioxidant vitamins rescue ischemia-induced neovascularization in mice exposed to cigarette smoke: Potential role of endothelial progenitor cells

OBJECTIVE Cigarette smoking is associated with impaired neovascularization in response to ischemia. Potential mechanisms include increased generation of reactive oxygen species (ROS) and a reduction in the function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that antioxidant therapies could stimulate EPC function and improve ischemia-induced neovascularization following cigarette smoke exposure. METHODS AND RESULTS C57Bl/6 mice exposed to cigarette smoke (MES) were fed a normal diet (controls) or a diet supplemented with probucol (0.5%) or a combination of vitamin C (25 g/l in drinking water) and vitamin E (0.1% in normal chow). After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. Exposure to cigarette smoke was associated with a significant reduction of blood flow recuperation and vessel density in ischemic muscles. However, a complete rescue of neovascularization was demonstrated in MES treated with probucol or antioxidant vitamins. We found that antioxidant therapy in MES is associated with a significant reduction of oxidative stress levels both in the plasma and in ischemic muscles. Moreover, EPCs exposed to cigarette smoke extracts in vitro showed a significant impairment of their angiogenic activities (migration, adhesion, homing into ischemic tissues) that was completely rescued by probucol and antioxidant vitamins. CONCLUSIONS Probucol and antioxidant vitamins rescue cigarette smoke-dependent impairment of ischemia-induced neovascularization. The mechanisms involve beneficial effects on oxidative stress levels in ischemic tissues together with an improvement of EPC functional activities. Antioxidant therapy could constitute a novel therapeutic strategy to promote vessel growth and reduce tissue ischemia in atherosclerotic diseases.

Diabetic vasculopathy and the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1).

Type 2 diabetes is associated with an increased incidence of coronary heart disease and cardiovascular complications. One crucial step in the initiation and progression of atherosclerosis is the unregulated uptake of oxidized low-density lipoprotein (oxLDL) by vascular wall components through scavenger receptors. Identification of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) as the major receptor for oxLDL in endothelial cells has provided a new clue to the mechanisms involved in oxLDL accumulation in the vessel wall. This receptor, by facilitating the uptake of oxLDL, induces endothelial dysfunction and mediates numerous oxLDL-induced proatherogenic effects. Besides endothelial cells, LOX-1 is also expressed by smooth muscle cells and macrophages. In these cells, LOX-1 may function as a scavenger receptor and promote foam cell formation. Notably, LOX-1 is induced by multiple stimuli relevant to atherogenesis and inflammation and is up-regulated in various proatherogenic conditions, including diabetes. As such, activation of vascular cells by oxLDL through LOX-1 may be relevant to the development and progression of human diabetic vasculopathy. This review summarizes recent advances related to the role of LOX-1 in atherosclerosis, its regulation by metabolic and inflammatory factors relevant to diabetes and the impact of these factors on LOX-1-mediated proatherogenic events linked to diabetic vasculopathy.

C-reactive protein enhances macrophage lipoprotein lipase expression

High serum levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are documented in patients with type 2 diabetes. Accumulating evidence suggests that CRP could directly promote arterial damage. To determine the role of CRP in diabetic atherosclerosis, we examined the effect of CRP on the expression of macrophage lipoprotein lipase (LPL), a proatherogenic molecule upregulated in type 2 diabetes. Treatment of human macrophages with native CRP increased, in a dose- and time-dependent manner, LPL protein expression and secretion. Modified CRP reproduced these effects. Preincubation of human macrophages with antioxidants, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK) inhibitors prevented CRP-induced LPL expression. Exposure of human macrophages to CRP further increased intracellular reactive oxygen species generation, classic PKC isozymes expression, and extracellular signal-regulated protein kinase 1/2 phosphorylation. In CRP-treated J774 macrophages, increased macrophage LPL mRNA levels and enhanced binding of nuclear proteins to the activated protein-1 (AP-1)-enhancing element were observed. These effects were prevented by antioxidants, as well as by PKC, MAPK, and AP-1 inhibitors. These data show for the first time that CRP directly increases macrophage LPL expression and secretion. Given the predominant role of macrophage LPL in atherogenesis, LPL might represent a novel factor underlying the adverse effect of CRP on the diabetic vasculature.