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The protein tyrosine phosphatase PTPRJ/DEP-1 has been implicated in negative growth regulation in endothelial cells, where its expression varies at transitions between proliferation and contact inhibition. However, in the same cells, DEP-1 has also been implicated in VEGF-dependent Src activation, permeability, and capillary formation, suggesting a positive role in regulating these functions. To resolve this dichotomy in vivo, we investigated postnatal angiogenesis and vascular permeability in a DEP-1–deficient mouse. In this study, we report that DEP-1 is required for Src activation and phosphorylation of its endothelial cell–specific substrate, VE-cadherin, after systemic injection of VEGF. Accordingly, VEGF-induced vascular leakage was abrogated in the DEP-1–deficient mice. Furthermore, capillary formation was impaired in murine aortic tissue rings or Matrigel plugs infused with VEGF. In the absence of DEP-1, angiogenesis triggered by ischemia or during tumor formation was defective, which in the latter case was associated with reduced tumor cell proliferation and increased apoptosis. Macrophage infiltration was also impaired, reflecting reduced vascular permeability in the tumors or a possible cell autonomous effect of DEP-1. Consequently, the formation of spontaneous and experimental lung metastases was strongly decreased in DEP-1–deficient mice. In clinical specimens of cancer, less vascularized tumors exhibited lower microvascular expression of DEP-1. Altogether, our results established DEP-1 as an essential driver of VEGF-dependent permeability, angiogenesis, and metastasis, suggesting a novel therapeutic route to cancer treatment. Cancer Res; 76(17); 5080–91. ©2016 AACR.

Legend to Supp. Fig. S1

Fig. S1. VEGF expression in tumors collected from WT or DEP-1 KO mice.

Background and aims MicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions. Methods Next generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE−/−) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro. Results We found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE−/− mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE−/− mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs). Conclusions Hypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.

Objective Adaptive thermogenesis, which is partly mediated by sympathetic input on brown adipose tissue (BAT), is a mechanism of heat production that confers protection against prolonged cold exposure. Various endogenous stimuli, for example, norepinephrine and FGF-21, can also promote the conversion of inguinal white adipocytes to beige adipocytes, which may represent a secondary cell type that contributes to adaptive thermogenesis. We previously identified an essential role of the molecular scaffold 14-3-3ζ in adipogenesis, but one of the earliest, identified functions of 14-3-3ζ is its regulatory effects on the activity of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of norepinephrine. Herein, we examined whether 14-3-3ζ could influence adaptive thermogenesis via actions on BAT activation or the beiging of white adipocytes. Methods Transgenic mice over-expressing a TAP-tagged human 14-3-3ζ molecule or heterozygous mice without one allele of Ywhaz, the gene encoding 14-3-3ζ, were used to explore the contribution of 14-3-3ζ to acute (3 h) and prolonged (3 days) cold (4 °C) exposure. Metabolic caging experiments, PET-CT imaging, and laser Doppler imaging were used to determine the effect of 14-3-3ζ over-expression on thermogenic and vasoconstrictive mechanisms in response to cold. Results Transgenic over-expression of 14-3-3ζ (TAP) in male mice significantly improved tolerance to acute and prolonged cold. In response to cold, body temperatures in TAP mice did not decrease to the same extent when compared to wildtype (WT) mice, and this was associated with increased UCP1 expression in beige inguinal white tissue (iWAT) and BAT. Of note was the paradoxical finding that cold-induced changes in body temperatures of TAP mice were associated with significantly decreased energy expenditure. The marked improvements in tolerance to prolonged cold were not due to changes in sensitivity to β-adrenergic stimulation or BAT or iWAT oxidative metabolism; instead, over-expression of 14-3-3ζ significantly decreased thermal conductance and heat loss in mice via increased peripheral vasoconstriction. Conclusions Despite being associated with elevations in cold-induced UCP1 expression in brown or beige adipocytes, these findings suggest that 14-3-3ζ regulates an alternative, non-thermogenic mechanism via vasoconstriction to minimize heat loss during cold exposure., Graphical abstract Image 1, Highlights • 14-3-3ζ over-expression in male mice improves tolerance to acute and prolonged cold. • Increasing 14-3-3ζ expression promotes beiging of inguinal white adipose tissue. • Cold-induced changes in body temperature can be dissociated from energy expenditure. • 14-3-3ζ-dependent decreases in heat loss are associated with vasoconstriction.

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.

SummaryFollowing prolonged cold exposure, adaptive thermogenic pathways are activated to maintain homeothermy, and elevations in body temperature are generally associated with UCP1-dependent and -independent increases in energy expenditure. One of the earliest, identified functions of the molecular scaffold, 14-3-3ζ, was its role in the synthesis of norepinephrine, a key endogenous factor that stimulates thermogenesis. This suggests that 14-3-3ζ may have critical roles in cold-induced thermogenesis. Herein, we report that transgenic over-expression of TAP-14-3-3ζ in mice significantly improved tolerance to prolonged cold. When compared to wildtype controls, TAP mice displayed significantly elevated body temperatures and paradoxical decreases in energy expenditure. No changes in β-adrenergic sensitivity or oxidative metabolism were observed; instead, 14-3-3ζ over-expression significantly decreased thermal conductance via increased peripheral vasoconstriction. These findings suggest 14-3-3ζ mediates alternative, non-thermogenic mechanisms to mitigate heat loss for homeothermy. Our results point to an unexpected role of 14-3-3ζ in the regulation of body temperature.Graphical abstract

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.

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.

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.

The protein tyrosine phosphatase PTPRJ/DEP-1 has been implicated in negative growth regulation in endothelial cells, where its expression varies at transitions between proliferation and contact inhibition. However, in the same cells, DEP-1 has also been implicated in VEGF-dependent Src activation, permeability, and capillary formation, suggesting a positive role in regulating these functions. To resolve this dichotomy in vivo, we investigated postnatal angiogenesis and vascular permeability in a DEP-1–deficient mouse. In this study, we report that DEP-1 is required for Src activation and phosphorylation of its endothelial cell–specific substrate, VE-cadherin, after systemic injection of VEGF. Accordingly, VEGF-induced vascular leakage was abrogated in the DEP-1–deficient mice. Furthermore, capillary formation was impaired in murine aortic tissue rings or Matrigel plugs infused with VEGF. In the absence of DEP-1, angiogenesis triggered by ischemia or during tumor formation was defective, which in the latter case was associated with reduced tumor cell proliferation and increased apoptosis. Macrophage infiltration was also impaired, reflecting reduced vascular permeability in the tumors or a possible cell autonomous effect of DEP-1. Consequently, the formation of spontaneous and experimental lung metastases was strongly decreased in DEP-1–deficient mice. In clinical specimens of cancer, less vascularized tumors exhibited lower microvascular expression of DEP-1. Altogether, our results established DEP-1 as an essential driver of VEGF-dependent permeability, angiogenesis, and metastasis, suggesting a novel therapeutic route to cancer treatment. Cancer Res; 76(17); 5080–91. ©2016 AACR.

Background: Exposure to cigarette smoke is associated with impaired formation of new blood vessels (neovascularization) in response to ischemia. Here we investigated the potential role of miRs in that physiopathology. Methods and Results: Using Affimetrix GeneChip miRNA array analysis, we identified let-7f as a pro-angiogenic miR which expression is reduced following cigarette smoke exposure. qRT-PCR analyses confirmed that the expression of let-7f 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 ischemia, intramuscular injection of let-7f mimic restored ischemia-induced neovascularisation in MES. At day 21 after ischemia, Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let-7f mimic compared to those treated with a mimic control. Clinically, mice treated with let-7f mimic also exhibited reduced ambulatory impairment and hindlimb ischemic damages. Interestingly, we found that treatment with let-7f mimic can rescue endothelial progenitor cell (EPC) number and function (migration, integration into tubules) in MES. Let-7f has previously been shown to target ALK5 (TGF-βRI), an important modulator of angiogenesis. We found that ALK5 is significantly increased in HUVECs exposed to CSE and in the ischemic muscles of MES. This is associated with a downstream activation of anti-angiogenic factors such as SMAD2 and PAI-1. Importantly, treatment with let-7f mimic reduces the expression of ALK5, SMAD2 and PAI-1 both in vitro and in vivo. Moreover, let-7f mimic can also rescue angiogenesis in HUVECs exposed to CSE. Conclusion: Cigarette smoke exposure is associated with reduced expression of let-7f, which leads to impaired neovascularization following ischemia. Our results suggest that the mechanism involves increased activation of ALK5, together with a downstream stimulation of the anti-angiogenic SMAD2/PAI-1 pathway. Overexpression of let-7f using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in pathological conditions.

Background: Exposure to cigarette smoke is associated with impaired neovascularization in response to ischemia. The precise mechanisms involved in that process remain to be determined. Micro RNA (miR) are emerging as key regulators of several physiological processes, including angiogenesis. Here we investigated the potential role of miRs for the modulation of neovascularization in the context of cigarette smoking. Methods and Results: Human Umbilical Vascular Endothelial Cells (HUVECs) were exposed or not to cigarette smoke extracts (CSE). Using Affimetrix GeneChip miRNA array analysis, we found that the pro-angiogenic miR let-7f was downregulated by 40% in HUVECs exposed to CSE. Using an inhibitor of let-7f, we demonstrated reduced migration and tube formation in HUVECs, reproducing the phenotype induced by CSE. A let-7f mimic could rescue cellular migration and tube formation in HUVECs exposed to CSE. Moreover, the expression of let-7f is significantly reduced in the ischemic muscles of mice exposed to cigarette smoke (CS). In vivo, hindlimb ischemia was surgically provoked by femoral artery removal to mice exposed (SMK) or not to CS for two weeks with a local injection of a control or a let-7f mimic. Let-7f mimic could rescue blood flow recuperation and capillary density in ischemic muscles 21 days post-ischemia associated with improved mobility. We found that CS was associated with reduced number of endothelial progenitor cells (EPCs) and impairment of angiogenic activities. Importantly, let-7f mimic rescued EPC number and EPC functional activities in SMK group. TGF-β-RI and HIF1AN are predicted to be targeted by let-7f and both are increased in SMK mice, whereas the expression of HIF-1a and VEGF are reduced in these mice. Interestingly, SMK mice injected with a let-7f mimic have decreased muscle expression of TGF-β-RI and HIF1AN associated with normalized HIF-1 and VEGF expression. Conclusion: Our results suggest that a reduction in the expression of let-7f could be involved in the cigarette smoke-induced inhibition of angiogenesis through modulation of TGF-β-RI and HIF1AN. Overexpression of let-7f using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in pathological conditions.

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.

Endothelial dysfunction is one of the earliest pathological effects of cigarette smoking. Vascular endothelial growth factor (VEGF) has been shown to be an important regulator of endothelial healing and growth. Accordingly, we tested the hypothesis that cigarette smoke exposure impairs VEGF actions in endothelial cells. In human umbilical vein endothelial cells (HUVECs), cigarette smoke extracts (CSE) inhibited VEGF-induced tube formation in the matrigel assay. CSE did not affect HUVECs proliferation, but significantly reduced cellular migration in response to VEGF. This impaired migratory activity was associated with a reduced expression of alpha(v)beta(3), alpha(v)beta(5), alpha(5)beta(1) and alpha(2)beta(1) integrins. The Akt/eNOS/NO pathway has been shown to be important for VEGF-induced endothelial cell migration. We found that CSE inhibited Akt/eNOS phosphorylation and NO release in VEGF-stimulated HUVECs. This was associated with an increased generation of reactive oxygen species (ROS). Importantly, in HUVECs exposed to CSE, treatment with antioxidants (NAC, vitamin C) reduced ROS formation and rescued VEGF-induced NO release, cellular migration and tube formation. Moreover, treatment with NO donors (SNAP, SNP) or a cGMP analog (8-Br-cGMP) rescued integrin expression, cellular migration and tube formation in endothelial cells exposed to CSE. (1) Cigarette smoke exposure impairs VEGF-induced endothelial cell migration and tube formation. (2) The mechanism involves increased generation of ROS, decreased expression of surface integrins together with a blockade of the Akt/eNOS/NO pathway. (3) These findings could contribute to explain the negative effect of cigarette smoking on endothelial function and vessel growth.

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 : 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

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.

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.

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.

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.

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.

Background: Sildenafil is widely used to treat erectile dysfunction. Sildenafil inhibits phospho-diesterase 5 (PDE5), thereby increasing cGMP levels in target tissues. The NO-cGMP pathway has been shown to be essential for angiogenesis, vasculogenesis and postnatal neovascularization. Therefore, here we tested the hypothesis that sildenafil might improve neovascularization following ischemia. Methods and results: Hypercholesterolemic ApoE−/− mice, which are characterised by reduced neovascularization in response to ischemia, were treated or not with sildenafil (40 mg/kg/day in water) for the whole duration of the study. Sildenafil was well tolerated by ApoE−/− mice and led to a significant increase of plasmatic cGMP levels compared to controls (13.4±1.9 vs. 4.6±2.6 pmol/mL, p Conclusions: Sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic conditions. The mechanisms involve beneficial effects of sildenafil on angiogenic pathways in ischemic tissues together with an increase in the number and the functional activity of EPCs.

Background: Several risk factors for cardiovascular diseases are associated with reduced neovascularization in response to ischemia. A common feature of all cardiovascular risk factors is increased production of reactive oxygen species (ROS) such as superoxide. We have recently demonstrated that mice that are deficient for CuZnSOD present impaired neovascularization in response to ischemia. Here we tested the hypothesis that endothelial progenitor cell (EPC) dysfunction might be involved in this neovascularization defect. Methods and results: Seven days after surgically induced hindlimb ischemia, EPCs were isolated from CuZnSOD deficient mice (CuZnSOD −/ − , n=8 –10) or control mice (CuZnSOD +/+ , n=8 –10). We found that the number of EPCs was significantly reduced in the bone marrow (40.6±3.0 vs 70.5±5.0, p−/ − mice compared to controls. Moreover, EPC functional activities were significantly impaired in CuZnSOD −/ − mice. More specifically, we found that EPC adhesion was reduced by 41%, migration by 88%, proliferation by 46% and integration into tubules by 77% in CuZnSOD −/ − mice. To confirm the role of EPCs in vivo, we isolated CD117+ cells (also acLDL and lectin positive when cultured for 4 days in vitro) from the bone marrow of CuZnSOD +/+ mice and performed an intravenous injection (5X10 5 cells) into CuZnSOD −/ − mice at 24 hours after surgically-induced hindlimb ischemia. Interestingly, EPC supplementation in CuZnSOD −/ − mice led to a complete rescue of ischemia-induced neovascularization, as assessed by blood flow recuperation in the ischemic hindlimb (Laser-Doppler analysis). Moreover, EPC therapy in CuZnSOD −/ − mice was also associated with a significant reduction of oxidative stress in ischemic tissues (nitrotyrosine and DHE staining). Conclusions: CuZnSOD deficiency is associated with a significant reduction in the number and the functional activities of EPCs. Cellular therapy with EPC can rescue the impaired neovascularization in CuZnSOD −/ − mice. Our results suggest that increased oxidative stress could contribute to explain EPC dysfunction and impaired neovascularization associated with different cardiovascular risk factors.

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 hindlimb 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 neovascularization. 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 Languedoc-Roussillon, France, which contains a relatively high concentration (4-6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol 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.

Endothelial dysfunction is one of the earliest pathological effects of cigarette smoking. It has recently been suggested that endothelial progenitor cells (EPCs) could contribute to ongoing endothelial maintenance and repair. Accordingly, we tested the hypothesis that cigarette smoking is associated with EPC dysfunction.EPCs were isolated from the peripheral venous blood of 15 healthy smokers and 11 age-matched nonsmokers. The number of EPCs was significantly reduced in smokers versus control subjects (51.6+/-1.9 versus 120.3+/-10.0 per power field, p0.001). Moreover, the functional activities of EPCs isolated from smokers were severely compromised. First, the proliferative and migratory response of EPCs isolated from smokers were reduced by 75% and 19%, respectively (p0.05). Second, EPCs from smokers showed an important decreased adherence to HUVECs that had been previously activated with tumor necrosis factor-alpha (TNF-alpha) (p0.01). Finally, the participation of EPCs to tube formation in a matrigel assay was reduced by 38% in smokers versus control subjects (p0.001). We found that EPCs from smokers had a significant reduction in the expression of the endothelial cell-specific markers (VE-cadherin, KDR, and vWF). Moreover, ROS formation was significantly increased in EPCs from smokers, whereas the serum antioxidant and nitrite levels of smokers were reduced and correlated with impaired EPC number and functional activity.Cigarette smoking is associated with a reduced number of EPCs together with an important impairment of EPC differentiation and functional activities. Our results suggest that EPC dysfunction could contribute to impair blood vessel healing and growth in smokers.

Toluidine blue (TBO) is a cationic thiazine dye with an affinity for neoplastic tissues in vivo. The objective of this study was to explore the in vitro photosensitizing potential of TBO and its capacity to induce apoptosis in human leukaemic T cells. Jurkat cells were incubated with TBO for one hour followed by exposure to 11 J cm(-2) of visible light from a slide projector. Cytotoxicity was assessed at 24 hours using a MTT assay. DNA fragmentation was examined at different intervals after photodynamic treatment using a DNA elution-filtration assay with [14C]-thymidine labelled cells. Caspase-3 like activation induced by photodynamic treatment was studied by measuring AC-DEVD-AMC peptide hydrolysis. The MTT assay showed a 97% decrease in optical density 24 hours following photodynamic therapy with 0.15 microg ml(-1) of TBO. Dark toxicity was absent under these conditions. DNA fragmentation was detected as early as 2 hours after photodynamic therapy and reached 68% at 6 hours. At higher TBO concentrations less DNA fragmentation and more dark toxicity was observed. An increase in caspase-3 like activity was also induced by photodynamic therapy with TBO. At the time of light exposure TBO was present in the endoplasmic reticulum and Golgi regions. In conclusion, TBO-based photodynamic therapy has a potent phototoxic effect and induces apoptosis in Jurkat cells.

Angiogenesis and increased vessel permeability play key roles during tumor growth and cancer progression. Therefore, much current effort in the field aims at understanding the molecular mechanisms underlying the formation and remodeling of blood vessels as a way to identify new approaches to harness these processes. In this context, we have been focusing our efforts on the protein tyrosine phosphatase DEP-1 (also named PTPRJ or CD148), which was initially identified as a negative regulator of cell proliferation and VEGFR2 phosphorylation when highly expressed in quiescent endothelial cells. However, using an RNAi knockdown approach, we demonstrated for the first time that through its ability to dephosphorylate the Src inhibitory Y529, DEP-1 was also a positive regulator of Src activation in VEGF-stimulated endothelial cells. As Src plays key roles in the promotion of angiogenesis and vascular permeability, we further demonstrated that DEP-1 was indeed a promoter of these vascular functions in vitro, and that the tyrosine phosphorylation of its C-terminal tail, allowing Src interaction and activation, was required for this. Interestingly, the catalytic inactivation of DEP-1 in mice led to increased endothelial cell proliferation, but also disorganized vascular structures, in contrast with the apparent absence of phenotype in DEP-1 knock-out (KO) mice models. In this study, we evaluated the biological consequences of the loss of DEP-1 expression on normal and pathological angiogenesis using a complete KO mouse model. We first confirmed that DEP-1 was required for Src activation and the phosphorylation of its substrates VE-cadherin and FAK in mice lung lysates upon systemic VEGF stimulation. Consistent with these observations, VEGF-induced vascular leakage was abrogated in the DEP-1 KO mice, as determined using a modified Miles assay. Capillary formation from mouse aortic rings ex vivo and in Matrigel plugs was also impaired, demonstrating the key positive role of DEP-1 in permeability and angiogenesis. Ischemia-induced angiogenesis was also deficient in KO mice, as demonstrated by reduced limb blood flow and capillary formation in a hind limb ischemia model. To assess tumor-associated angiogenesis, the highly metastatic Mvt-1 mouse mammary tumor cells were orthotopically injected in WT and DEP-1 KO mice. The growth of tumors in DEP-1 KO mice was reduced by about 50% compared to those grown in WT animals, due to inhibition of tumor angiogenesis. As tumor-associated and leaky blood vessels contribute to dissemination of cancer cells, a 50-60% decrease in the number and size of spontaneous and experimental metastases were also observed in the lungs of DEP-1 KO mice. Altogether, these results demonstrate for the first time that DEP-1 is an essential driver of VEGF-dependent Src activation, permeability, and angiogenesis in vivo, therefore acting as a promoter of tumor cell growth and dissemination. Citation Format: Patrick Fournier, Sylvie Dussault, Alfredo Fusco, Alain Rivard, Isabelle Royal. The protein tyrosine phosphatase DEP-1/PTPRJ is an essential promoter of vascular permeability, angiogenesis and tumor progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 8. doi:10.1158/1538-7445.AM2014-8

While it can bring several benefits, IT outsourcing entails some risks. As is the case in other types of investments or business ventures, the risk associated with an IT outsourcing project must be evaluated and managed. This paper proposes a framework for the management of IT outsourcing risk and assesses the usefulness of the framework using data gathered about two cases of system development outsourcing. After providing a conceptual definition of risk and of risk exposure, the paper presents the proposed risk management framework. The two cases are then described, along with the evaluation of the level of risk exposure of each and the risk management mechanisms that were included in the contracts. The results of the study suggest that, by charting the various items that contribute to risk exposure and by specifically applying appropriate mechanisms that can target those elements with higher levels of risk exposure, outsourcing risk can be adequately managed.