Psychological stress impairs ischemia-induced neovascularization: Protective effect of fluoxetine.

BACKGROUND: Psychological stress (PS) has been associated with the development of cardiovascular diseases and adverse long-term outcomes after ischemic events. However, the precise mechanisms involved are not completely understood. Here we investigated the effect of PS on ischemia-induced neovascularization, and the potential therapeutic effect of fluoxetine in this condition. METHODS AND RESULTS: Balb/c mice were subjected or not to chronic restraint stress. After 3 weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that blood flow recovery was significantly impaired in mice exposed to PS compared to controls (Doppler flow ratio (DFR) 0.61 ± 0.07 vs. 0.80 ± 0.07, p < 0.05). At the microvascular level, capillary density was significantly reduced in ischemic muscles of mice exposed to PS (38 ± 1 vs. 74 ± 3 capillaries per field, p < 0.001). This correlated with increased oxidative stress levels and reduced expression of VEGF and VEGF signalling molecules (p44/p42 MAPK, Akt) in ischemic muscles. We found that the number of pro-angiogenic cells (PACs) was significantly reduced in mice exposed to PS. In addition, oxidative stress levels (DCF-DA, DHE) were increased in PACs isolated from mice exposed to PS, and this was associated with impaired PAC functional activities (migration, adhesion, and integration into tubules). Importantly, treatment of mice exposed to PS with the selective serotonin reuptake inhibitor (SSRI) fluoxetine improved all the angiogenic parameters, and completely rescued PS-induced impairment of neovascularization. CONCLUSION: PS impairs ischemia-induced neovascularization. Potential mechanisms involved include reduced activation of the VEGF pathway in ischemic tissues, increased oxidative stress levels and reduced number and functional activities of PACs. Our results suggest that fluoxetine may represent a novel therapeutic strategy to improve neovascularization and reduce ischemia in patients suffering from cardiovascular diseases and exposed to PS.

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.

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.

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.

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.

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.

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.

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.

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.

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. DESIGN: Descriptive clinical trial. METHODS: Serum glucose, insulin, insulin resistance, lipids, high sensitivity C-reactive protein (hsCRP), interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and soluble E-selectin (sELAM-1) serum levels were determined in 15 patients with hyperprolactinaemia at baseline (compared with 20 healthy subjects) and after 12 weeks of cabergoline therapy (0.5-1 mg twice per week). We also measured mononuclear cell NF-kappaB activation and TNF-alpha production in a subset of subjects. RESULTS: Serum levels of prolactin (PRL), insulin, insulin resistance (HOMA-IR) index and hsCRP were significantly higher in patients than in control subjects. Markers of mononuclear cell activation did not differ between the groups. Hyperprolactinaemia, BMI and age were predictors of hsCRP. BMI was the only predictor of HOMA-IR. Cabergoline therapy significantly reduced serum PRL, insulin, hsCRP and sELAM-1 levels. CONCLUSIONS: These data suggest that hyperprolactinaemia is associated with insulin resistance related to increased BMI and low-grade inflammation independently of BMI. Short-term cabergoline therapy can reduce the inflammatory markers.

Linoleic acid increases lectin-like oxidized LDL receptor-1 (LOX-1) expression in human aortic endothelial cells.

Results from in vitro studies suggest that selected fatty acids, and especially linoleic acid (LA), can elicit endothelial dysfunction (ED). Because LA is increased in all LDL subfractions in patients with type 2 diabetes, this alteration may contribute to ED associated with diabetes. Lectin-like oxidized LDL receptor-1 (LOX-1) is the major endothelial receptor for oxidized LDL (oxLDL), and uptake of oxLDL through LOX-1 induces ED. To evaluate whether LA may contribute to the upregulation of endothelial LOX-1 in diabetes, we studied the effect of LA on LOX-1 expression in cultured human aortic endothelial cells (HAECs). Treatment of HAECs with LA increased, in a time- and dose-dependent manner, endothelial LOX-1 protein expression. Pretreatment of HAECs with antioxidants and inhibitors of NADPH oxidase, protein kinase C (PKC), and nuclear factor-kappaB (NF-kappaB) inhibited the stimulatory effect of LA on LOX-1 protein expression. Furthermore, in LA-treated HAECs, increased expression of classic PKC isoforms was observed. LA also led to a significant increase in LOX-1 gene expression and enhanced the binding of nuclear proteins extracted from HAECs to the NF-kappaB regulatory element of the LOX-1 gene promoter. Finally, LA enhanced, through LOX-1, oxLDL uptake by endothelial cells. Overall, these results demonstrate that LA enhances endothelial LOX-1 expression through oxidative stress-sensitive and PKC-dependent pathways. This effect seems to be exerted at the transcriptional level and to involve the activation of NF-kappaB. Upregulation of LOX-1 by LA may contribute to ED associated with type 2 diabetes.

Enhanced lipoprotein lipase secretion and foam cell formation by macrophages of patients with growth hormone deficiency: possible contribution to increased risk of atherogenesis?

GH deficiency is associated with increased prevalence of atherosclerosis, and recent data indicate a proatherogenic role for macrophage lipoprotein lipase (LPL) in the arterial wall. In this pilot study, we determined LPL expression and foam cell formation in monocyte-derived macrophages of 12 control subjects and nine patients with GH deficiency without GH replacement therapy. LPL mRNA levels, mass, and activity were increased in macrophages of patients with GH deficiency. In these subjects, macrophage LPL activity correlated with body mass index and fat mass. Incubation of patient macrophages with IGF-I for 24 h or differentiation of monocytes isolated from GH-deficient patients into macrophages in the presence of this growth factor decreased the amount of LPL mass. Compared with control cells, macrophages derived from GH-deficient patients took up and stored increased amounts of proatherogenic lipoproteins and were more easily converted to foam cells. In the supernatants of these cells, increased levels of free fatty acids and TNFalpha were also documented. These results demonstrate that macrophages of patients with GH deficiency secrete increased amounts of proatherogenic cytokines and are more susceptible to foam cell formation. These alterations may contribute to the increased cardiovascular risk in patients with GH deficiency.

Leptin increases lipoprotein lipase secretion by macrophages: involvement of oxidative stress and protein kinase C.

Recent data suggest that plasma leptin may represent a cardiovascular risk factor in diabetic patients. To gain further insight into the role of leptin in atherogenesis associated with diabetes, we investigated in the present study the role of this hormone in the regulation of macrophage lipoprotein lipase (LPL), a proatherogenic cytokine overexpressed in patients with type 2 diabetes. Treatment of human macrophages with leptin (1-10 nmol/l) increased LPL expression, at both the mRNA and protein levels. Pretreatment of these cells with anti-leptin receptor (Ob-R) antibody, protein kinase C (PKC) inhibitors, calphostin C, and GF109203X, or the antioxidant N-acetylcysteine (NAC) blocked the effects of leptin. Similar results were observed in leptin-treated J774 macrophages. In these cells, leptin increased the membrane expression of conventional PKC isoforms and downregulation of endogenous PKC expression abolished the effects of leptin on macrophage LPL expression. In leptin-treated J774 cells, enhanced LPL synthetic rate and increased binding of nuclear proteins to the activated protein-1 (AP-1) consensus sequence of the LPL gene promoter were also observed. This latter effect was abrogated by GF109203X. Overall, these data demonstrate that binding of leptin at the macrophage cell surface increases, through oxidative stress- and PKC-dependent pathways, LPL expression. This effect appears to be exerted at the transcriptional level and to involve AP-1 activation.