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Objective— Hypercholesterolemia is an atherosclerotic condition that is associated with impaired neovascularization in response to ischemia. This study sought to define the role of microRNAs in that pathophysiology. Approach and Results— Next-generation sequencing and quantitative reverse transcription polymerase chain reaction analyses identified miR-150 as a proangiogenic microRNA, which expression is significantly reduced in the ischemic muscles of hypercholesterolemic apolipoprotein E–deficient (ApoE −/− ) mice, and in human umbilical vein endothelial cells exposed to oxidized low-density lipoprotein. Forced expression of miR-150 using a miR mimic could rescue oxidized low-density lipoprotein–mediated impairment of endothelial cell migration and tubule formation in vitro. In a mouse model of hindlimb ischemia, intramuscular injection of miR-150 mimic restored blood flow recuperation, vascular densities in ischemic muscles, and functional mobility in ApoE −/− mice. Treatment of ApoE −/− mice with miR-150 also increased the number and the activities of proangiogenic cells. miR-150 targets SRC kinase signaling inhibitor 1, an important regulator of Src (proto-oncogene tyrosine-protein kinase Src) activity. Here we found that hypercholesterolemia and oxidized low-density lipoprotein exposure are associated with increased SRC kinase signaling inhibitor 1 expression and decreased Src activity. However, treatment with miR-150 mimic reduces SRC kinase signaling inhibitor 1 expression and restores Src and downstream endothelial nitric oxide synthase and Akt (protein kinase B) activities both in vitro and in vivo. We also demonstrate the interrelation between miR-150 and SRC kinase signaling inhibitor 1 and their importance for endothelial cell angiogenic activities. Conclusions— Hypercholesterolemia is associated with reduced expression of miR-150, impaired Src signaling, and inefficient neovascularization in response to ischemia. Forced expression of miR-150 using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Aging is associated with impaired neovascularization in response to ischemia. MicroRNAs are small noncoding RNAs emerging as key regulators of physiological and pathological processes. Here we investigated the potential role of microRNAs in endothelial cell senescence and age-dependent impairment of neovascularization. Next generation sequencing and qRT-PCR analyses identified miR-130a as a pro-angiogenic microRNA which expression is significantly reduced in old mouse aortic endothelial cells (ECs). Transfection of young ECs with a miR-130a inhibitor leads to accelerated senescence and reduced angiogenic functions. Conversely, forced expression of miR-130a in old ECs reduces senescence and improves angiogenesis. In a mouse model of hindlimb ischemia, intramuscular injection of miR-130a mimic in older mice restores blood flow recovery and vascular densities in ischemic muscles, improves mobility and reduces tissue damage. miR-130a directly targets antiangiogenic homeobox genes MEOX2 and HOXA5. MEOX2 and HOXA5 are significantly increased in the ischemic muscles of aging mice, but forced expression of miR-130a reduces the expression of these factors. miR-130a treatment after ischemia is also associated with increased number and improved functional activities of pro-angiogenic cells (PACs). Forced expression of miR-130a could constitute a novel strategy to improve blood flow recovery and reduce ischemia in older patients with ischemic vascular diseases.

Adverse perinatal conditions can lead to developmental programming of cardiovascular diseases. Prematurely born infants are often exposed to high oxygen levels, which in animal models has been associated with endothelial dysfunction, hypertension, and cardiac remodeling during adulthood. Here we found that adult mice that have been transiently exposed to O2 after birth show defective neovasculariation after hindlimb ischemia, as demonstrated by impaired blood flow recovery, reduced vascular density in ischemic muscles and increased tissue damages. Ischemic muscles isolated from mice exposed to O2 after birth exhibit increased oxidative stress levels and reduced expression of superoxide dismutase 1 (SOD1) and vascular endothelial growth factor (VEGF). Pro-angiogenic cells (PACs) have been shown to have an important role for postnatal neovascularisation. We found that neonatal exposure to O2 is associated with reduced number of PACs in adults. Moreover, the angiogenic activities of both PACs and mature mouse aortic endothelial cells (MAECs) are significantly impaired in mice exposed to hyperoxia after birth. Our results indicate that neonatal exposure to high oxygen levels leads to impaired ischemia-induced neovascularization during adulthood. The mechanism involves deleterious effects on oxidative stress levels and angiogenic signals in ischemic muscles, together with dysfunctional activities of PACs and mature endothelial cells.

This study sought to determine the potential role of microRNAs (miRNAs) in the detrimental effects of cigarette smoke on angiogenesis and neovascularization. Using large‐scale miRNA profiling and qRT‐PCR analyses, we identified let‐7f as a pro‐angiogenic miRNA which expression is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischaemia, intramuscular injection of let‐7f mimic restored ischaemia‐induced neovascularization in MES. Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let‐7f mimic. Clinically, this was associated with reduced ambulatory impairment and hindlimb ischaemic damage. Treatment with let‐7f mimic could also rescue pro‐angiogenic cell (PAC) number and function (attachment, proliferation, migration) in MES. ALK5 (TGF‐βR1), an important modulator of angiogenesis, is a target of let‐7f. Here we show that ALK5 is increased in HUVECs exposed to CSE and in the ischaemic muscles of MES. This is associated with a downstream activation of the anti‐angiogenic factors SMAD2/3 and PAI‐1. Importantly, treatment with let‐7f mimic reduces the expression of ALK5, SMAD2/3 and PAI‐1 both in vitro and in vivo. Moreover, let‐7f overexpression or ALK5 inhibition can rescue angiogenesis in HUVECs exposed to CSE. Cigarette smoke exposure is associated with reduced expression of let‐7f and activation of the anti‐angiogenic TGF‐β/ALK5 pathway. Overexpression of let‐7f using a miRNA mimic could constitute a novel therapeutic strategy to improve ischaemia‐induced neovascularization in pathological conditions.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naive mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20 S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Renin is the rate limiting step for the activation of the renin-angiotensin-aldosterone system, which is linked to the development of endothelial dysfunction, hypertension and atherosclerosis. However, the specific role of renin during physiological responses to tissue ischemia is currently unknown. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization in response to ischemia.At a dose that did not modulate blood pressure (10 mg/kg), aliskiren led to improved blood flow recovery after hindlimb ischemia in C57BL/6 mice (Doppler flow ratios 0.71 ± 0.07 vs. 0.55 ± 0.03; P0.05). In ischemic muscles, treatment with aliskiren was associated with a significant increase of vascular density, reduced oxidative stress levels and increased expression of VEGF and eNOS. Aliskiren treatment also significantly increased the number of bone marrow-derived endothelial progenitor cells (EPCs) after hindlimb ischemia. Moreover, the angiogenic properties of EPCs (migration, adhesion, integration into tubules) were significantly improved in mice treated with aliskiren. In vitro, aliskiren improves cellular migration and tubule formation in HUVECs. This is associated with an increased expression of nitric oxide (NO), and a significant reduction of oxidative stress levels. Importantly, the angiogenic properties of aliskiren in vitro and in vivo are completely abolished following treatment with the NOS inhibitor l-NAME.Direct renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanism involves beneficial effects of aliskiren on oxidative stress and NO angiogenic pathway, together with an increase in the number and the functional activities of EPCs.

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.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.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.

Background: The activation of the renin-angiotensin system is associated with impaired formation of new blood vessels (neovascularization) in response to ischemia. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization following ischemia. Methods and Results: C57BL/6 mice were treated with a high dose of aliskiren (50 mg/kg), a low dose of aliskiren (10 mg/kg), or drinking water only. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. Treatment with aliskiren led to a significantly faster rate of blood flow recovery after hindlimb ischemia (Laser Doppler). Interestingly the lower dose of aliskiren, which did not reduce blood pressure, provided similar improvement of blood flow recuperation compared to the higher dose of aliskiren. At day 21 after surgery, Doppler flow ratios were significantly improved in mice treated with aliskiren (0.69+/-0.07 vs. 0.52+/-0.03; p Conclusions: Direct renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanisms involve beneficial effects of aliskiren on NO and angiogenic pathways in ischemic tissues, together with an increase in the number and the functional activity of EPCs.

Background The composition of a diet can influence myocardial metabolism and development of left ventricular hypertrophy (LVH). The impact of a high-fat diet in chronic left ventricular volume overload (VO) causing eccentric LVH is unknown. This study examined the effects of chronic ingestion of a high-fat diet in rats with chronic VO caused by severe aortic valve regurgitation (AR) on LVH, function and on myocardial energetics and survival. Methods Male Wistar rats were divided in four groups: Shams on control or high-fat (HF) diet (15 rats/group) and AR rats fed with the same diets (ARC (n = 56) and ARHF (n = 32)). HF diet was started one week before AR induction and the protocol was stopped 30 weeks later. Results As expected, AR caused significant LV dilation and hypertrophy and this was exacerbated in the ARHF group. Moreover, survival in the ARHF group was significantly decreased compared the ARC group. Although the sham animals on HF also developed significant obesity compared to those on control diet, this was not associated with heart hypertrophy. The HF diet in AR rats partially countered the expected shift in myocardial energy substrate preference usually observed in heart hypertrophy (from fatty acids towards glucose). Systolic function was decreased in AR rats but HF diet had no impact on this parameter. The response to HF diet of different fatty acid oxidation markers as well as the increase in glucose transporter-4 translocation to the plasma membrane compared to ARC was blunted in AR animals compared to those on control diet. Conclusions HF diet for 30 weeks decreased survival of AR rats and worsened eccentric hypertrophy without affecting systolic function. The expected adaptation of myocardial energetics to volume-overload left ventricle hypertrophy in AR animals seemed to be impaired by the high-fat diet suggesting less metabolic flexibility. Electronic supplementary material The online version of this article (doi:10.1186/1471-2261-14-123) contains supplementary material, which is available to authorized users.

Background : Elsibucol is a metabolically stable derivative of probucol with antioxidant, anti-inflammatory and antiproliferative properties. Here we investigated the effect of elsibucol on the development of atherosclerosis following arterial injury in hypercholesterolemic rabbits. Methods and results : New Zealand White rabbits were fed a high cholesterol diet that was supplemented or not with 0.5% elsibucol, 1% elsibucol or 1% probucol. An angioplasty of the iliac artery was performed after 3 weeks of diet. We found that treatment with elsibucol significantly decreases blood total cholesterol, LDLc and triglyceride levels. This is associated with a significant 46% reduction of neointimal hyperplasia following arterial injury. Interestingly, the effect of elsibucol on cholesterol levels and neointimal formation appears to be more pronounced than that of probucol. In vitro, elsibucol reduces vascular smooth muscle cell proliferation without affecting cell viability. In vivo, treatment with elsibucol is associated with a significant reduction of cellular proliferation (PCNA immunostaining), oxidative stress (nitrotyrosine immunostaining), VCAM-1 expression and macrophage infiltration in injured arteries. Despite its potent effect on neointimal hyperplasia, elsibucol does not prevent endothelial healing (Evans blue staining) following arterial injury. Conclusions : In hypercholesterolemic animals, elsibucol inhibits atherosclerosis and preserves endothelial healing following arterial injury. The mechanisms involved include lowering of blood cholesterol levels together with a reduction of oxidative stress and inflammation in injured arteries.

s S277 treatment of HC mice with miR-150 mimic can rescue the number of bone marrow and peripheral EPCs. Moreover, the angiogenic properties of EPCs (adhesion, migration and integration into tubules) are restored in HC mice treated with miR-150 mimic. In vitro, miR-150 rescues angiogenesis (migration, tube formation) in HUVECs exposed to oxLDL. miR-150 has previously been shown to target SRC kinase signaling inhibitor 1 (SRCIN1), an important regulator of SRC activity. We found that SRC activity is significantly reduced in HUVECs exposed to oxLDL. Moreover, the angiogenic effects of miR-150 mimic in HUVECs exposed to oxLDL are completely abolished when the cells are also treated with an inhibitor of SRC. CONCLUSION: Hypercholesterolemia is associated with reduced expression of miR-150, which leads to impaired neovascularization following ischemia. The mechanism involves beneficial effects on both angiogenesis and EPC activities. Our results suggest that SRC signaling is at least in part responsible for the pro-angiogenic effects of miR-150 in these conditions. IRSC 525 NEONATAL EXPOSURE TO HIGH OXYGEN LEVELS IS ASSOCIATED WITH IMPAIRED ISCHEMIA-INDUCED NEOVASCULARIZATION DURING ADULTHOOD R Mathieu, M Desjarlais, W Dhahri, A Cloutier, A Nuyt, A Rivard

Background— Aortic valve regurgitation (AR) is a volume-overload disease causing severe eccentric left ventricular (LV) hypertrophy and eventually heart failure. There is currently no approved drug to treat patients with AR. Many vasodilators including angiotensin-converting enzyme inhibitors have been evaluated in clinical trials, but although some results were promising, others were inconclusive. Overall, no drug has yet been able to improve clinical outcome in AR and the controversy remains. We have previously shown in an animal model that captopril (Cpt) reduced LV hypertrophy and protected LV systolic function, but we had not evaluated the clinical outcome. This protocol was designed to evaluate the effects of a long-term Cpt treatment on survival in the same animal model of severe aortic valve regurgitation. Methods and Results— Forty Wistar rats with AR were treated or untreated with Cpt (1 g/L in drinking water) for a period of 7 months to evaluate survival, myocardial remodeling, and function by echocardiography as well as myocardial metabolism by µ positron emission tomography scan. Survival was significantly improved in Cpt-treated animals with a survival benefit visible as soon as after 4 months of treatment. Cpt reduced LV dilatation and LV hypertrophy. It also significantly improved the myocardial metabolic profile by restoring the level of fatty acids metabolic enzymes and use. Conclusions— In a controlled animal model of pure severe aortic valve regurgitation, Cpt treatment reduced LV remodeling and LV hypertrophy and improved myocardial metabolic profile and survival. These results support the need to reevaluate the role of angiotensin-converting enzyme inhibitors in humans with AR in a large, carefully designed prospective clinical trial.

Aims Fenofibrate is a peroxisome proliferator-associated receptor alpha agonist (PPARα) used clinically for the management of dyslipidemia and is a myocardial fatty acid oxidation stimulator. It has also been shown to have cardiac anti-hypertrophic properties but the effects of fenofibrate on the development of eccentric LVH and ventricular function in chronic left ventricular (LV) volume overload (VO) are unknown. This study was therefore designed to explore the effects of fenofibrate treatment in a VO rat model caused by severe aortic valve regurgitation (AR) with a focus on cardiac remodeling and myocardial metabolism. Main methods Male Wistar rats were divided in four groups (13–15 animals/group): Shams (S) treated with fenofibrate (F; 100 mg/kg/d PO) or not (C) and severe AR receiving or not fenofibrate. Treatment was started one week before surgery and the animals were sacrificed 9 weeks later. Key findings AR rats developed severe LVH (increased LV weight) during the course of the protocol. Fenofibrate did not reduce LV weight. However, eccentric LV remodeling was strongly reduced by fenofibrate in AR animals. Fractional shortening was significantly less affected in ARF compared to ARC group. Fenofibrate also increased the myocardial enzymatic activity of enzymes associated with fatty acid oxidation while inhibiting glycolytic enzyme phosphofructokinase. Significance Fenofibrate decreased LV eccentric remodeling associated with severe VO and helped maintain systolic function. Studies with a longer follow-up will be needed to assess the long-term effects of fenofibrate in chronic volume overload caused by aortic regurgitation.

Left ventricular hypertrophy (LVH) is often associated with a change in myocardial energy substrate preference from fatty acids to glucose. A possible anti hypertrophic treatment strategy could aim at stimulating or restoring normal myocardial energy metabolism. Metformin, an adenosine monophosphate-activated protein kinase (AMPK) activator used in the management of glucose metabolism in diabetes, is also a fatty acid oxidation stimulator. The effect of metformin on the development of eccentric LVH and ventricular function in chronic left ventricular (LV) volume overload (VO) is unknown. This study was designed to study this question in a VO rat model caused by severe aortic valve regurgitation (AR). Male Wistar rats were divided in four groups (13-15 animals / group): Shams (S) treated or not (C) with metformin (M; 100 mg/kg/d PO) and severe ARreceiving or not metformin. Treatment was started one week before surgery and the animals were sacrificed 9 weeks later. As expected AR rats developed severe eccentric LVH during the course of the protocol. Metformin treatment did not influence the total heart weight. However, LV remodeling associated with the severe VO was severe in ARM than in ARC. Fractional shortening, a marker of systolic function, was significantly higher in ARM compared to ARC group. Metformin also increased the activity of enzymes associated with fatty acid oxidation while inhibiting phosphofructokinase, a glycolytic enzyme. A 2 month treatment with metformin reduced LV eccentric remodeling associated with severe VO and helped maintain a better systolic function.

The impact of a high–fat (HF) diet on eccentric left ventricular hypertrophy (LVH) from severe volume overload is still not well understood. The purpose of this study was to investigate the long–term impact of a HF diet on survival, LVH and myocardial metabolism in a model of dilated