Jessika Groleau

Moderate consumption of red wine (cabernet sauvignon) improves ischemia-induced neovascularization in ApoE-deficient mice: effect on endothelial progenitor cells and nitric oxide

Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hind‐limb ischemia in hypercholesterolemic ApoE‐deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascu‐larization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine‐drinking mice. The wine used in our study is a cabernet sauvignon from Langue‐doc‐Roussillon, France, which contains a relatively high concentration (4–6 mg/L) of the polyphenolic antiox‐idant resveratrol. We demonstrate that resveratrol can rescue oxidized low‐density lipoprotein (oxLDL)‐in‐duced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Res‐veratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia‐induced neovascularization in high‐cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt‐eNOS‐NO pathway.— Lefevre J., Michaud, S.‐E., Haddad, P., Dussault, S., Ménard C., Groleau, J., Turgeon, J., Rivard A. Moderate consumption of red wine (cabernet sauvignon) improves ischemia‐induced neovascularization in ApoE‐deficient mice: effect on endothelial progenitor cells and nitric oxide. FASEB J. 21, 3845–3852 (2007)

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.

Caspase activation regulates the extracellular export of autophagic vacuoles.

The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.

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.

MFG-E8 Released by Apoptotic Endothelial Cells Triggers Anti-Inflammatory Macrophage Reprogramming

Apoptotic endothelial cells are an important component of the “response to injury” process. Several atherosclerosis risk factors such as hyperglycemia and oxidized low-density lipoproteins, and immune injuries, such as antibodies and complement, induce endothelial cell apoptosis. While endothelial cell apoptosis is known to affect neighboring vascular wall cell biology, its consequences on macrophage reprogramming are ill defined. In this study, we report that apoptosis of human and mouse endothelial cells triggers the release of milk fat globule-epidermal growth factor 8 (MFG-E8) and reprograms macrophages into an anti-inflammatory cells. We demonstrated that MFG-E8 is released by apoptotic endothelial cells in a caspase-3-dependent manner. When macrophages were exposed to conditioned media from serum-starved apoptotic endothelial cells, they adopt a high anti-inflammatory, low pro-inflammatory cytokine/chemokine secreting phenotype that is lost if MFG-E8 is absent from the media. Macrophage treatment with recombinant MFG-E8 recapitulates the effect of conditioned media. Finally, we showed that MFG-E8-mediated reprogramming of macrophages occurs through increased phosphorylation of signal transducer and activator of transcription-3 (STAT-3). Taken together, our study suggests a key role of MFG-E8 release from apoptotic endothelial cells in macrophage reprogramming and demonstrates the importance of the apoptotic microenvironment in anti-inflammatory macrophage responses.

Essential Role of Copper-Zinc Superoxide Dismutase for Ischemia-Induced Neovascularization Via Modulation of Bone Marrow–Derived Endothelial Progenitor Cells

Objective—To investigate the effect of oxidative stress on ischemia-induced neovascularization in copper-zinc (CuZn) superoxide dismutase (SOD)–deficient mice. Methods and Results—In the vascular wall, CuZnSOD is essential for protecting against excessive oxidative stress and maintaining endothelial function. However, its specific role for the development of new vessels in response to ischemia is unknown. After surgically induced hind limb ischemia, CuZnSOD-deficient mice showed impaired neovascularization, as assessed by blood flow recuperation (laser Doppler) and capillary density in the ischemic muscles. This was associated with increased levels of oxidative stress in ischemic tissues and peripheral blood, together with reduced plasmatic NO production. CuZnSOD-deficient mice demonstrated an important reduction in the number of endothelial progenitor cells (EPCs) in the bone marrow and spleen. Moreover, EPCs isolated from CuZnSOD-deficient mice showed increased oxidative stress levels, decreased NO production, and a reduced ability to migrate and integrate into capillary-like networks. Importantly, the functional activities of CuZnSOD-deficient EPCs were rescued after treatment with the SOD-mimetic Tempol (a membrane-permeable radical scavenger) or the NO donor sodium nitroprusside (SNP). Moreover, the neovascularization defect in CuZnSOD-deficient mice could be rescued by wild-type (but not CuZnSOD-deficient) EPC supplementation. Conclusion—Protection against oxidative stress by CuZnSOD may be essential for EPC function and reparative neovascularization after ischemia.

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.

Accelerated Vascular Aging in CuZnSOD-Deficient Mice: Impact on EPC Function and Reparative Neovascularization

Objective Aging is associated with increased oxidative stress levels and impaired neovascularization following ischemia. CuZnSOD has an important role to limit oxidative stress in the vasculature. Here we investigated the role of CuZnSOD for the modulation of ischemia-induced neovascularisation during aging. Methods and Results Hindlimb ischemia was surgically induced in young (2- month-old) or older (8-month-old) wild type (WT) and CuZnSOD−/− mice. We found that blood flow recovery after ischemia and vascular density in ischemic muscles were significantly reduced in older compared to young WT mice. Both in young and older mice, CuZnSOD deficiency led to a further reduction of neovascularization. Accordingly, the resulting neovascularisation potential in a young CuZnSOD−/− mouse was similar to that of an older WT mouse. Oxidative stress levels were also increased to similar levels in the ischemic muscles of young CuZnSOD−/− and older WT mice. To identify potential mechanisms involved, we investigated the effect of aging and CuZnSOD deficiency on the number and the function of endothelial progenitor cells (EPCs). Both aging and CuZnSOD deficiency were associated with reduced number of bone marrow and peripheral EPCs. The effect of moderate aging alone on specific functional activities of EPCs (migration, integration into tubules) was modest. However, CuZnSOD deficiency was associated with severe age-dependent defects in EPC functional activities. Conclusions CuZnSOD deficiency is associated with accelerated vascular aging and impaired ischemia-induced neovascularization. Our results suggest that in the context of aging, CuZnSOD has an essential role to protect against excessive oxidative stress in ischemic tissues and preserve the function of EPCs.