Postischemic Neovascularization Research Articles

Magnetic Tagging Increases Delivery of Circulating Progenitors in Vascular Injury

We sought to magnetically tag endothelial progenitor cells (EPCs) with a clinical agent and target them to a site of arterial injury using a magnetic device positioned outside the body. Circulating EPCs are involved in physiological processes such as vascular re-endothelialization and post-ischemic neovascularization. However, the success of cell therapies depends on the ability to deliver the cells to the site of injury. Human EPCs were labeled with iron oxide superparamagnetic nanoparticles. Cell viability and differentiation were tested using flow cytometry. Following finite element modeling computer simulations and flow testing in vitro, angioplasty was performed on rat common carotid arteries to denude the endothelium and EPCs were administered with and without the presence of an external magnetic device for 12 min. Computer simulations indicated successful external magnetic cell targeting from a vessel with flow rate similar to a rat common carotid artery; correspondingly there was a 6-fold increase in cell capture in an in vitro flow system. Targeting enhanced cell retention at the site of injury by 5-fold at 24 h after implantation in vivo. Using an externally applied magnetic device, we have been able to enhance EPC localization at a site of common carotid artery injury. This technology could be more widely adapted to localize cells in other organs and may provide a useful tool for the systemic injection of cell therapies.

Inhibition of Prolyl Hydroxylase Domain Proteins Promotes Therapeutic Revascularization

The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O(2)-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization. Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1alpha (shHIF-1alpha), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1alpha. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1alpha-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2- and 2.6-fold, capillary density by 1.8- and 2.1-fold, and foot perfusion by 1.2- and 1.4-fold, respectively, compared with shCON (P<0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1alpha with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing. We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization.

D022 Natural CD4/CD25/Foxp3 regulatory t cells modulate post-ischemic inflammatory response: role in neovascularization

CD4+ and CD8+ T lymphocytes control revascularization after vascular occlusion. T cell activation is mediated by two major costimulatory signalings: the B7/CD28 and the CD40-CD40 ligand pathways. Interestingly, CD28 interactions with the structurally related ligands B7-1 and B7-2 are also required for the generation and homeostasis of CD4+CD25+ regulatory T cells (Treg), whichactively maintain immunological tolerance to self and nonself antigens. We hypothesized that naturally arising Treg modulate the immuno-inflammatory response to ischemic injury, and subsequently vessel growth. Ischemia was induced by right femoral artery ligation in CD28-deficient mice (n = 10 per group). After 21 days of ischemia, CD28 deficiency showed a profound reduction in Treg number and upregulated post-ischemic inflammatory response and neovascularization. Similarly, injection of splenocytes isolated from CD28-/- mice in Rag1-/- mice with hindlimb ischemia increased angiographic score, foot perfusion, and capillary density by 2.2-, 2.3- and 1.1-fold, respectively, compared to PBS-injected Rag1-/- mice. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3 positive macrophages in the ischemic leg of Rag1-/- mice treated with CD28-/- splenocytes. Interestingly, cotransfer of Treg with CD28-/- splenocytes in Rag1- /- mice abrogated activation of neovascularization induced by CD28-/- splenocytes. Inflammatory cells accumulation was also decreased in Rag1-/- transplanted with both Treg and CD28-/- splenocytes compared to mice receiving CD28-/- splenocytes only. In contrast, treatment of C57Bl/6 Wild-Type mice with an anti- CD25 antibody (PC61) markedly reduced endogenous Treg levels in blood and spleen. At day 14 of ischemia, inflammatory response and neovascularization were markedly increased in anti-CD25 treated Wild-Type mice compared to untreated mice. These results provide new insights into the immunoregulation of post-ischemic neovascularization.

F016 Angiopoietin-like 4 protects tissues following myocardial infarction through modulation of post-ischaemic neovascularization and inflammation

Angiopoietin-like 4 (ANGPTL4) is a secreted protein of the Angiopoietins family involved in angiogenesis and vessel maintenance. We identified angptl4 as a hypoxia induced gene in endothelial cells in vitro. In humans, ANGPTL4 is expressed in hypoxic areas such as vascular cells of human critical leg ischemia samples. In the heart, focal expression in myocytes, vessels and infiltrating monocytes was also evidenced in infarcted areas. We used C57/Bl6 angptl4 knock-out mice (Regeneron pharmaceuticals, Tarrytown). The pattern of expression of angptl4 was determined during development using Lac Z staining allowed by the insertion of a beta-galactosidase cassette. At the adult stage, myocardial infarction was induced by ligation of the left coronary artery for 45mn. The hearts were analyzed 24 h, 48 h and 2 weeks after reperfusion. During embryonic development, angptl4 is expressed in endothelial cells from intersomitic, limb bud (E10,5-E11,5) and heart vessels (E12,5-E14,5). At later stages (from E15,5), angptl4 is also expressed in skin vessels. Angptl4 knock-out mice showed an increased infarcted area compared to wild type littermates mice (+31 %, p=0.01) 48h after reperfusion, suggesting a cardioprotective role of ANGPTL4. Immunohistological analyzes revealed dramatic tissue damages in the angptl4 knock-out mice in term of oedema, hemorragies and necrosis correlated with an increased circulating monocytes infiltration (+36 %, p=0.004). PECAM-1 stainings revealed morphologically modified angiogenic capillaries in angptl4 KO mice compared to controls suggesting a modulated vascular permeability. No difference was observed between isolated cardiomyocytes from both groups submitted to a survival assay under hypoxia. Angptl4 is expressed by endothelial cells during embryonic and during adult stage following hypoxic stress. Experiments performed using the ischemia-reperfusion model show that ANGPTL4 has a cardioprotective role modulating both endothelium integrity and post-ischaemic inflammation.

D026 Catecholamines regulate vascular progenitor cells mobilization from bone marrow

Endogenous catecholamine stimulation of adrenergic receptors contributes to artery growth and remodeling in pathological and adaptive physiological settings. Recently, it has been shown that enforced stem/progenitor cells egress from bone marrow (BM) niches depends critically on the nervous system. In particular, pharmacological or genetic ablation of adrenergic neurotransmission indicates that noradrenalin (NA) signaling controls bone SDF-1 downregulation, and hematopoietic stem cells mobilization. We hypothesized that catecholamines may control vascular progenitor cells mobilization from bone marrow and subsequently trigger post-ischemic neovascularization. Ischemia was induced by right femoral artery ligation in C57Bl6 mice (n = 7 per group) treated with or without 6-hydroxydopamin (6-OHDA, 100 mg/kg, i.p., 2 days), clenbuterol (2 mg/kg, i.p., 5 days), dopamine (DA, 50 mg/kg, i.p., 5 days), NA (2.5 mg/kg, i.p., 5 days) and eticlopride (10 mg/kg, i.p., 5 days). Sympathectomy induced by 6-OHDA led to a decrease in foot perfusion, angiographic score and capillary density by 41.4 % (p < 0.001), 20.2 % (p < 0.01) and 21.6 % (p < 0.05) respectively, compared to controls, 21 days after ischemia. In contrast, administration of DA and NA increased angiographic score by 57.4 % (p = 0.05) and 80.3 % (p < 0.05), respectively and raised capillary density by around 15 % (p < 0.01). Treatment with the β2-receptor agonist, clenbuterol, increased vessel and capillary densities by 30 % and 13 %, respectively (p < 0.01 versus untreated control). Injection of eticlopride, a specific DA D2 receptor antagonist, also raised vessel density compared to controls. We next assessed the mobilization of stem/progenitor cells from bone marrow and their recruitment to ischemic tissue using chimeric mice lethally irradiated and transplanted with BM mononuclear cells isolated from GFP mice. Three days after ischemia, the number of GFP/ BS-1 lectin positive cells was reduced by 53.7 % (p < 0.05) in the ischemic muscle of mice treated with 6-OHDA compared with controls. In contrast, clenbuterol increased by 46.4 % (p < 0.05) the number of double positive cells suggesting that β2-receptor activation mediates BM stem cells mobilization and incorporation into vascular structure. Therefore, activation of the β2-receptor promotes postnatal vessel growth in response to ischemia. This effect is likely mediated by activation of stem/progenitor cells migration out of the bone marrow.

Abstract 5448: Regulatory T Cells Control Post-Ischemic Neovascularization

CD4+ and CD8+ T lymphocytes are key regulators of post-ischemic neovascularization. T cell activation is promoted by two major costimulatory signalings: the CD28/B7 and the CD40ligand-CD40 pathways. Interestingly, CD28 interactions with the structurally related ligands B7–1 and B7–2 are also required for the generation and homeostasis of CD4+CD25+ regulatory T cells (Treg), which play a critical role in the suppression of immune responses and the control of T-cell homeostasis. We hypothesized that Treg cell activation may modulate the immuno-inflammatory response to ischemic injury, leading to alteration of post-ischemic vessel growth. Ischemia was induced by right femoral artery ligation in CD28-, B7–1/2- or CD40-deficient mice (n=10 per group). CD40 deficiency did not affect Treg levels and led to significant reduction in post-ischemic inflammatory response and vessel growth. In contrast, at day 21 after ischemia, angiographic score, foot perfusion, and capillary density were increased by 2.0-, 1.2-, and 1.8-fold respectively in CD28-deficient mice, which show profound reduction in Treg, compared to controls. Similarly, disruption of B7–1/2 signaling and subsequent Treg deletion significantly enhanced post-ischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3 positive macrophages in the ischemic leg of CD28−/− or B7−/− mice compared with controls. Proinflammatory cytokines, IL-1β, TNF-α and IL-6, were also upregulated and antiinflammatory mediators, IL-10 and TGF-β1 downregulated in the ischemic legs of CD28−/− or B7−/− mice. Finally, treatment of CD28−/− mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced expression of the regulatory T-cell marker Foxp3 in blood and ischemic tissue of CD28−/− mice, led to reduction in post-ischemic inflammatory response and neovascularization in CD28-deficient mice. In conclusion, endogenous Treg cell response controls post-ischemic neovascularization.

Abstract 3666: Major Role of Ly6chi/7/4hi/ccr2+ Monocytes in Post-ischemic Neovascularization

Circulating mouse monocytes consist of two main subsets, Ly6CHi7/ 4HiCCR2+CX3CR1loCCR5+ and Ly6Clo7/4loCCR2-CX3CR1HiCCR5+ monocytes.We sought to investigate their respective role in postischemic neovascularization and unravel the mechanisms involved in their recruitment to ischemic tissues. Postischemic neovascularization was impaired in MCP-1−/− or CCR2−/− but not in CCR5−/− or CX3CR1−/− mice with operatively-induced hindlimb ischemia (n=10 per groups). Overexpression of MCP-1 in the ischemic muscle by plasmid electrotransfer improved foot perfusion, angiographic score and capillary density by 1.4-, 1.8- and 1.4-fold, respectively (p&lt;0 .01 versus control). In contrast, overexpression of Fractalkine or Rantes, the ligands of CX3CR1 and CCR5 respectively, had no significant effects (n=10 per groups) suggesting that recruitment of 7/4HiCCR2+ monocytes play a significant role in this setting. In this line, flow cytometry analysis revealed that monocytes infiltration in ischemic muscle peaked at day 3 and dropped to levels comparable to those in non ischemic muscle on day 7. 7/4Hi monocytes predominated from day 1 to day 3 (52% of infiltrated Cd11b+ cells) whereas 7/4lo prevailed from day 7 onward. MCP-1 or CCR2 deficiency fully abrogated infiltration of both monocytes subsets, while overexpression of MCP-1 in the ischemic muscle selectively increased 7/4Hi monocytes infiltration (287% of wild-type, p&lt;0.05). In contrast, monocytes subsets infiltration was slightly reduced in CX3CR1−/− mice (p&lt;0.05 versus MCP-1−/−) and unchanged in CCR5−/− mice. Interestingly, MCP-1 deficiency or upregulation also led to changes in monocyte subtypes levels in both bone marrow and blood. MCP-1/CCR2 signaling plays a major role in monocytes mobilization from the bone marrow and recruitment to the ischemic tissue. We also suggest that inflammatory 7/4Hi/CCR2 monocytes represent a major component of the inflammatory response in post-ischemic neovascularization.

Abstract 3390: Impairment of Post-ischemic Neovascularization in Mice Lacking IQGAP1, an Actin-binding Scaffold Protein: Role in Macrophage Infiltration and ROS production

Neovascularization is important repair mechanism in response to ischemic injury and its process is in part dependent on reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is key regulator for actin cytoskeleton and motility. We demonstrated that IQGAP1 is expressed in endothelial cells (ECs) and mediates VEGF-induced ROS production and migration of cultured ECs. However, its role in postnatal neovascularization is unknown. Here we show that neovascularization induced by hindlimb ischemia is significantly inhibited in IQGAP1-deficient mice as evaluated by laser Doppler blood flow (38%), capillary density (48%) and alpha-actin positive arterioles (68%). In wild-type (WT) mice, western and immunofluorescence analysis reveal that IQGAP1 protein expression in ischemic tissues is significantly increased at Mac3+ macrophages and lectin+ capillary-like ECs at 3 days and 7 days after ischemia, respectively, which is associated with increased superoxide production. Of note, macrophage infiltration is important for post-ischemic revascularization. Mice lacking IQGAP1 show significant decrease in the number of infiltrated Mac3-positive macrophage and superoxide production in ischemic muscles (30% and 52%, respectively) as compared to WT mice. Numbers of inflammatory cells and cKit+/Flk1+ endothelial progenitor cells in peripheral blood are not affected in these knockout mice. Moreover, thioglycollate-induced peritoneal leukocyte recruitment and accumulation are significantly inhibited in IQGAP1-deficient mice (53%), whereas white blood cell number is not changed. In vitro, peritonitis-elicited (activated) macrophages obtained from IQGAP1-deficient mice show impaired polarization of actin, as visualized by phalloidin staining as well as complete inhibition of migration towards stromal derived factor-1 and VEGF, as measured by modified Boyden-chambered method. In summary, IQGAP1 plays a key role in reparative neovascularization in response to tissue ischemia by regulating not only ECs but also macrophage migration/infiltration as well as ROS production in the vessel growth area. Thus, IQGAP1 is a potential therapeutic target to promote neovascularization in ischemic cardiovascular diseases. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Neurotrophin p75 Receptor (p75 NTR ) Promotes Endothelial Cell Apoptosis and Inhibits Angiogenesis

Diabetes impairs endothelial function and reparative neovascularization. The p75 receptor of neurotrophins (p75 NTR ), which is scarcely present in healthy endothelial cells (ECs), becomes strongly expressed by capillary ECs after induction of peripheral ischemia in type-1 diabetic mice. Here, we show that gene transfer-induced p75 NTR expression impairs the survival, proliferation, migration, and adhesion capacities of cultured ECs and endothelial progenitor cells (EPCs) and inhibits angiogenesis in vitro. Moreover, intramuscular p75 NTR gene delivery impairs neovascularization and blood flow recovery in a mouse model of limb ischemia. These disturbed functions are associated with suppression of signaling mechanisms implicated in EC survival and angiogenesis. In fact, p75 NTR depresses the VEGF-A/Akt/eNOS/NO pathway and additionally reduces the mRNA levels of ITGB1 [beta (1) integrin] , BIRC5 (survivin ), PTTG 1 ( securin ) and VEZF1 . Diabetic mice, which typically show impaired postischemic muscular neovascularization and blood perfusion recovery, have these defects corrected by intramuscular gene transfer of a dominant negative mutant form of p75 NTR . Collectively, our data newly demonstrate the antiangiogenic action of p75 NTR and open new avenues for the therapeutic use of p75 NTR inhibition to combat diabetes-induced microvascular liabilities.

Hypertension Impairs Postnatal Vasculogenesis

We analyzed the effect of hypertension on postischemic vasculogenesis. Ischemia was induced by right femoral artery ligature in Wistar Kyoto rats (WKY) or spontaneously hypertensive rats (SHR) treated with or without angiotensin-converting enzyme inhibitor (Perindopril, 0.76 mg/kg/d) and angiotensin type 1 receptor blocker (losartan, 30 mg/kg/d). Basal postischemic neovascularization was reduced in SHR compared to WKY (P<0.05, n=8). Treatment with ACE inhibitor or angiotensin type 1 receptor blocker decreased blood pressure levels by 1.4- and 1.3-fold (P<0.001), respectively and restored vessel growth in SHR to WKY levels. Interestingly, 14 days after bone-marrow mononuclear cell (BM-MNC) transfusion, angiographic scores, capillary density, and foot perfusion were decreased by 1.4-, 1.5-, and 1.2-fold, respectively in SHR transfused with BM-MNCs isolated from SHR compared to those receiving BM-MNCs of WKY (P<0.05, n=6). Alteration in BM-MNCs proangiogenic potential was likely related to the reduction in their ability to mobilize into peripheral circulation, as revealed by the 2.9-fold decrease in number of circulating CD34+/CD117+ cells (P<0.001) and to differentiate into cells with endothelial phenotype, as revealed by the 2.1-fold reduction in percentages of DilLDL/BS-1 lectin positive cells (P<0.001). In addition, reactive oxygen species (ROS) levels were increased by 2.2-fold in SHR BM-MNCs compared to WKY BM-MNCs (P<0.01), as assessed by L-012 luminescence. Cotreatment with ACE inhibitor, angiotensin type 1 receptor blocker, or antioxidants (NAC 3 mmol/L, Apocynin 200 micromol/L) reduced ROS levels, improved the number of DilLDL/BS-1 lectin-positive cells by around 1.5-fold, and restored BM-MNCs proangiogenic effects in ischemic hindlimb. In conclusion, alteration in progenitor cell proangiogenic function may participate to the hypertension-induced impairment in postischemic revascularization.

Combination of the Angiotensin-Converting Enzyme Inhibitor Perindopril and the Diuretic Indapamide Activate Postnatal Vasculogenesis in Spontaneously Hypertensive Rats

Cardiovascular risk factors are associated with reduction in both the number and function of vascular progenitor cells. We hypothesized that 1) hypertension abrogates postnatal vasculogenesis, and 2) antihypertensive treatment based on the combination of perindopril (angiotensin-converting enzyme inhibitor) and indapamide (diuretic) may counteract hypertension-induced alteration in progenitor cell-related effects. Postischemic neovascularization was significantly lower in untreated spontaneously hypertensive rats (SHRs) compared with Wistar Kyoto (WKY) rats (p < 0.05). Treatment of SHRs with perindopril and the combination of perindopril/indapamide reduced the blood pressure levels and normalized vessel growth in ischemic area. Cotreatment with perindopril and indapamide increased vascular endothelial growth factor and endothelial nitric-oxide synthase protein contents, two key proangiogenic factors. It is interesting to note that 14 days after bone marrow mononuclear cell (BM-MNC) transplantation, revascularization was significantly lower in ischemic SHRs receiving BM-MNCs isolated from SHRs compared with those receiving BM-MNCs isolated from WKY rats (p < 0.05). Alteration in proangiogenic potential of SHR BM-MNCs was probably related to the reduction in their ability to differentiate into endothelial progenitor cells in vitro. Furthermore, the number of circulating endothelial progenitor cells (EPCs) was reduced by 3.1-fold in SHRs compared with WKY rats (p < 0.001). Treatments with perindopril or perindopril/indapamide restored the ability of BM-MNCs to differentiate in vitro into EPCs, increased the number of circulating EPCs, and re-established BM-MNC proangiogenic effects. Therefore, hypertension is associated with a decrease in the number of circulating progenitor cells and in the BM-MNC proangiogenic potential, probably leading to vascular complications in this setting. The combination of perindopril and indapamide counteracts hypertension-induced alterations in progenitor cell-related effects and restores blood vessel growth.

Post-ischaemic neovascularization and inflammation

Four principal processes-vasculogenesis, angiogenesis, arteriogenesis, and collateral growth-characterize tissue repair and remodelling in acute and chronic ischaemic vascular diseases. The relative importance of each process remains unclear, but it is likely that they may complement each other. In addition, these processes are driven by distinct, but partially overlapping, cellular and molecular pathways. In particular, inflammation might be one of the most important stimuli for initiation of vessel growth in the setting of ischaemia. Here, we summarize the current knowledge on the inflammatory response in the context of ischaemia and review the major factors that may be involved in inflammation-induced, post-ischaemic neovascularization.

Abstract 1444: Overexpression Of Endothelial Nitric Oxide Synthase Restores Post-natal Neovascularization In Atherosclerosis.

Alteration in post-ischemic neovascularization is a common complication of atherosclerotic disease. This results, at least in part, from abrogation of bone-marrow mononuclear cells (BM-MNC) pro-angiogenic potential. Overexpression of eNOS has been shown to promote vessel growth in the setting of ischemia. We hypothesized that eNOS overexpression could restore impaired neovascularization in atherosclerotic (ApoE KO) mice. Hind limb ischemia was induced in mice by right femoral artery ligation. After two weeks we evaluated tissue perfusion of the foot by Laser Doppler, vessel density in the hind limb by micro-angiography and histology, and atherosclerotic plaque size. In vitro BM-MNC cell culture assays were performed. Tissue perfusion and vessel density were 1.5-fold increased in transgenic mice overexpressing eNOS (eNOStg) as compared to wild type (WT) (P&lt;0.001, n=10). Transplantation of 1x106 WT- or eNOStg BM-MNC in WT recipients caused a 1.5-fold increase in tissue perfusion and vessel density compared to injection of PBS (P&lt;0.001, n=10). Next, we used ApoE KO mice and crossbreeds of eNOStg and ApoE KO mice (eNOStg*ApoE KO). Tissue perfusion and vessel density were 1.8-fold increased in eNOStg*ApoE KO mice as compared to ApoE KO mice (P&lt;0.001, n=10). Transplantation of both WT- or eNOStg*ApoE KO BM-MNC in ApoE KO recipients caused a 1.6- to 2-fold increase in tissue perfusion and vessel density compared to PBS (P&lt;0.01, n=10), while transplantation of ApoE KO BM-MNC had no positive effect on neovascularization. Moreover, transplantation of WT BM-MNC significantly increased plaque size, while eNOStg*ApoE KO BM-MNC had no effect on plaque size. eNOS overexpression did not affect BM-MNC apoptosis and secretion of growth factors but increased their ability to differentiate in vitro into EPC. Conclusion: eNOS overexpression in the endothelium improves post-ischemic neovascularization in both physiological as atherosclerotic settings. Furthermore, eNOS overexpression in the bone marrow restores the impaired pro-angiogenic potential of atherosclerotic BM-MNC without adverse effects on plaque size. Therefore, overexpression of eNOS could play a vital part in the development of therapeutic angiogenesis for atherosclerotic disease.

Abstract 464: Membrane Microparticles From Ischemic Muscle Induce In Vitro Progenitor Cells Differentiation And In Vivo Neovascularization.

Postischemic neovascularization is mediated, at least in part, by the homing of progenitor cells to sites of injury and their differentiation into endothelial cells (EC). However, the mechanisms of in situ progenitor differentiation remain for the most part unknown. We hypothesized that miproparticles (MPs) released following ischemia-induced cell activation or apoptosis are the endogenous signal of postischemic vasculogenesis. MPs were detected by electron microscopy as vesicles of 0.1 to 1 μm in diameter in mice ischemic hindlimb muscle, 48hrs after unilateral femoral artery ligation. After isolation by sequential centrifugations, flow cytometry analyses showed that AnnexinV+ MPs concentration in ischemic calf was 3.5-fold higher than in control muscle (1392±406 vs. 394±180 AnnV+MPs/mg, p&lt;0.005) and mainly originated from endothelial cells (47% of MPs are CD144+). MPs isolated from ischemic muscles induced a more potent in vitro bone marrow-mononuclear cells (BM-MNC) differentiation into EPC, than those isolated from control muscle (6.1±1.0% vs. 3.5±0.7% Dil-LDL/BS1lectin+ cells/field, p&lt;0.05). Moreover, MPs isolated from GFP+ ischemic muscles colocalized with differentiated BM-MNC. MPs isolated from atherosclerotic plaque were uneffective whereas those isolated from apoptotic or IL-1 activated EC also promoted BM-MNC differentiation. Interestingly, MPs from ischemic muscles produced more reactive oxygen species and expressed significantly higher levels of NADPH oxidase p47 (6 fold ; p&lt;0.05) and p67 subunits (16 fold ; p&lt;0.005) than those from control, whereas gp91 subunit expression was unchanged. The MPs-induced BM-MNC differentiation was reduced by two fold with MPs isolated from gp91-deficient mice (p&lt;0.05). MPs effect on post-ischemic vasculogenesis and revascularization was examined in the ischemic hindlimb model. MPs isolated from ischemic muscles were injected into ischemic legs in parallel with venous injection of BM-MNC. MPs increased the pro-angiogenic effect of BM-MNC transplantation and this effect was blunted with MPs from gp91−/− mice. These results demonstrate that MPs produced after tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization.

Effects of red wine polyphenols on postischemic neovascularization model in rats: low doses are proangiogenic, high doses anti‐angiogenic

Polyphenols, present in green tea, grapes, or red wine, have paradoxical properties: they protect against cardiac and cerebral ischemia but inhibit angiogenesis in vitro. So we investigated the effects of polyphenols in vivo on postischemic neovascularization. Rats treated with low (0.2 mg x kg(-1) x day(-1)) or high (20 mg x kg(-1) x day(-1)) doses of red wine polyphenolic compounds (RWPC) were submitted to femoral artery ligature on the left leg. Two wks after ligature, high doses of RWPC (i.e., 7 glasses of red wine) reduced arterial, arteriolar, and capillary densities and blood flow in association with an inhibition of a PI3 kinase-Akt-endothelial NO synthase (eNOS) pathway, decreased VEGF expression, and lower metalloproteinase (MMP) activation. Low doses of RWPC (i.e., 1/10th glass of red wine) increased the left/right (L/R) leg ratio to control level in association with an increased blood flow and microvascular density. This angiogenic effect was associated with an overexpression of PI3 kinase-Akt-eNOS pathway and an increased VEGF production without effect on MMP activation. Thus, low and high doses RWPC have respectively pro- and anti-angiogenic properties on postischemic neovascularization in vivo. This unique dual effect of RWPC offers important perspectives for the treatment and prevention of ischemic diseases (low dose) or cancer growth (high dose).

Impaired therapeutic vasculogenesis by transplantation of OxLDL-treated endothelial progenitor cells

Previous in vitro studies have revealed that oxidized low density lipoprotein (OxLDL) has negative effects on the proliferation and activity of endothelial progenitor cells (EPCs). Here, we evaluated the effect of OxLDL on the therapeutic potential of EPCs in ischemia-induced neovascularization. EPCs derived from mobilized human peripheral blood mononuclear cells were cultured without or with OxLDL before transplantation. Hindlimb ischemia models were surgically induced in athymic nude mice, which then received an intracardiac injection of 3 x 10(5) EPCs. By laser Doppler perfusion image and ischemia damage score, we found that blood perfusion and ischemia damage were less well recovered in the OxLDL-treated EPC transplantation group than in controls. Histological examination showed fewer transplanted EPCs and lower capillary density in ischemic tissue. Local delivery of Stromal cell-derived factor (SDF-1) restored this defect and improved blood perfusion by recruiting OxLDL-treated EPCs to the ischemic area and increasing host capillary density. These results provide for the first time direct evidence that OxLDL impaired the therapeutic potential of EPCs in ischemia-induced neovascularization through an inhibitory effect on the migration, adhesion, and incorporation of EPCs into vasculature and/or entrapment in the perivascular region in vivo. A therapeutic strategy based on SDF-1 administration ameliorated such defects and improved postischemic neovascularization.

NADPH Oxidase-Derived Overproduction of Reactive Oxygen Species Impairs Postischemic Neovascularization in Mice with Type 1 Diabetes

We hypothesized that diabetes-induced oxidative stress may affect postischemic neovascularization. The response to unilateral femoral artery ligation was studied in wild-type or gp91(phox)-deficient control or type 1 diabetic mice or in animals treated with the anti-oxidant N-acetyl-l-cysteine (NAC) or with in vivo electrotransfer of a plasmid encoding dominant-negative Rac1 (50 microg) for 21 days. Postischemic neovascularization was reduced in diabetic mice in association with down-regulated vascular endothelial growth factor-A protein levels. In diabetic animals vascular endothelial growth factor levels and postischemic neovascularization were restored to nondiabetic levels by the scavenging of reactive oxygen species (ROS) by NAC administration or the inhibition of ROS generation by gp91(phox) deficiency or by administration of dominant-negative Rac1. Finally, diabetes reduced the ability of adherent bone marrow-derived mononuclear cells (BM-MNCs) to differentiate into endothelial progenitor cells. Treatment with NAC (3 mmol/L), apocynin (200 micromol/L), or the p38MAPK inhibitor LY333351 (10 micromol/L) up-regulated the number of endothelial progenitor cell colonies derived from diabetic BM-MNCs by 1.5-, 1.6-, and 1.5-fold, respectively (P < 0.05). In the ischemic hindlimb model, injection of diabetic BM-MNCs isolated from NAC-treated or gp91(phox)-deficient diabetic mice increased neovascularization by approximately 1.5-fold greater than untreated diabetic BM-MNCs (P < 0.05). Thus, inhibition of NADPH oxidase-derived ROS overproduction improves the angiogenic and vasculogenic processes and restores postischemic neovascularization in type 1 diabetic mice.

Tetrapeptide AcSDKP Induces Postischemic Neovascularization Through Monocyte Chemoattractant Protein-1 Signaling

We investigated the putative proangiogenic activity and molecular pathway(s) of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in a model of surgically induced hindlimb ischemia. Hindlimb ischemia was induced by femoral artery ligature and an osmotic minipump was implanted subcutaneously to deliver low (0.12 mg/kg per day) or high (1.2 mg/kg per day) doses of AcSDKP, for 7 or 21 days. Angiography scores, arteriole density, capillary number, and foot perfusion were increased at day 21 in the high-dose AcSDKP-treated mice (by 1.9-, 1.8-, 1.3-, and 1.6-fold, respectively) compared with control animals (P<0.05, P<0.01, P<0.01, respectively). AcSDKP treatment for 24 hours upregulated the monocyte chemoattractant protein-1 (MCP-1) mRNA and protein levels by 1.5-fold in cultured endothelial cells (P<0.01). In the ischemic hindlimb model, administration of AcSDKP also enhanced MCP-1 mRNA levels by 90-fold in ischemic leg (P<0.001) and MCP-1 plasma levels by 3-fold (P<0.001 versus untreated ischemic control mice). MCP-1 levels upregulation were associated with a 2.3-fold increase in the number of Mac3-positive cells in ischemic area of AcSDKP-treated mice (P<0.001 versus untreated animals). Interestingly, AcSDKP-induced monocyte/macrophage infiltration and postischemic neovascularization was fully blunted in MCP-1-deficient animals. AcSDKP stimulates postischemic neovascularization through activation of a proinflammatory MCP-1-related pathway.

Thromboxane A2/Prostaglandin H2 Receptor Activation Mediates Angiotensin II–Induced Postischemic Neovascularization

We analyzed the involvement of thromboxane (TX) A2/prostaglandin (PG) H2 (TP) receptor in ischemia-induced neovascularization in mice. Unilateral hindlimb ischemia was induced by right femoral artery ligature in male C57BL/6J mice (n=7 per group). Animals were then treated with or without TP receptor antagonist (S18886, 5 or 10 mg/kg per day; ramatroban, 10 mg/kg per day) or aspirin (30 mg/kg per day) in drinking water for 21 days. Hindlimb ischemia raised plasma level of TXB2, the stable metabolite of TXA2, by 4.7-fold. This increase was blocked by aspirin treatment whereas S18886 (5 or 10 mg/kg per day) had no effect. However, neither S 18886 nor aspirin affected postischemic neovascularization. We next assessed the putative involvement of TXA2 signaling in angiotensin II (Ang II) proangiogenic pathway. Ang II (0.3 mg/kg per day) enhanced TXB2 plasma levels by 2.6-fold over that of control (P<0.01). Ang II-induced TXB2 upregulation was reduced by cotreatment with Ang II type I receptor antagonist (candesartan, 20 mg/kg per day). Angiographic score, capillary number, and foot perfusion were improved by 1.7-, 1.7-, and 1.4-fold, respectively, in Ang II-treated mice compared with controls (P<0.05). Ang II proangiogenic effect was associated with a 1.6-fold increase in VEGF-A protein content (P<0.05) and a 1.4-fold increase in the number of Mac-3-positive cells (ie, macrophages) in ischemic areas (P<0.05). Interestingly, treatments with TP receptor antagonists or aspirin hampered the proangiogenic effects of Ang II. Endogenous activation of TXA2 receptor by eicosanoids did not modulate spontaneous neovascularization in the setting of ischemia. Conversely, TXA2 signaling is involved in Ang II-induced AT1-dependent vessel growth.

Lactadherin promotes VEGF-dependent neovascularization

Vascular endothelial growth factor (VEGF)-induced blood vessel growth is involved in both physiological and pathological angiogenesis and requires integrin-mediated signaling. We now show that an integrin-binding protein initially described in milk-fat globule, MFG-E8 (also known as lactadherin), is expressed in and around blood vessels and has a crucial role in VEGF-dependent neovascularization in the adult mouse. Using neutralizing antibodies and lactadherin-deficient animals, we show that lactadherin interacts with alphavbeta3 and alphavbeta5 integrins and alters both VEGF-dependent Akt phosphorylation and neovascularization. In the absence of VEGF, lactadherin administration induced alphavbeta3- and alphavbeta5-dependent Akt phosphorylation in endothelial cells in vitro and strongly improved postischemic neovascularization in vivo. These results show a crucial role for lactadherin in VEGF-dependent neovascularization and identify lactadherin as an important target for the modulation of neovascularization.