Alice Récalde

Microparticles From Ischemic Muscle Promotes Postnatal Vasculogenesis

Background— We hypothesized that microparticles (MPs) released after ischemia are endogenous signals leading to postischemic vasculogenesis. Methods and Results— MPs from mice ischemic hind-limb muscle were detected by electron microscopy 48 hours after unilateral femoral artery ligation as vesicles of 0.1- to 1-&mgr;m diameter. After isolation by sequential centrifugation, flow cytometry analyses showed that the annexin V+ MP concentration was 3.5-fold higher in ischemic calves than control muscles (1392±406 versus 394±180 annexin V+ MPs per 1 mg; P<0.001) and came mainly from endothelial cells (71% of MPs are CD144+). MPs isolated from ischemic muscles induced more potent in vitro bone marrow–mononuclear cell (BM-MNC) differentiation into cells with endothelial phenotype than those isolated from control muscles. MPs isolated from atherosclerotic plaques were ineffective, whereas those isolated from apoptotic or interleukin-1&bgr;–activated endothelial cells 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<0.05) and p67 subunits (16-fold; P<0.001) than controls, whereas gp91 subunit expression was unchanged. BM-MNC differentiation was reduced by 2-fold with MPs isolated from gp91-deficient animals compared with wild-type mice (P<0.05). MP effects on postischemic revascularization were then examined in an ischemic hind-limb model. MPs isolated from ischemic muscles were injected into ischemic legs in parallel with venous injection of BM-MNCs. MPs increased the proangiogenic effect of BM-MNC transplantation, and this effect was blunted by gp91 deficiency. In parallel, BM-MNC proangiogenic potential also was reduced in ABCA1 knockout mice with impaired vesiculation. Conclusion— MPs produced during tissue ischemia stimulate progenitor cell differentiation and subsequently promote postnatal neovascularization.

Inhibition of Prolyl Hydroxylase Domain Proteins Promotes Therapeutic Revascularization

Background— The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O2-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization. Methods and Results— 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-1&agr; (shHIF-1&agr;), 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-1&agr;. 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-1&agr;–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-1&agr; with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1–dependent pathways mediated the proangiogenic effects of PHD silencing. Conclusions— 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.

Regulation of monocyte subset systemic levels by distinct chemokine receptors controls post-ischaemic neovascularization

AIMS Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets-Ly6C(hi) and Ly6C(lo)-to the blood and their respective role in post-ischaemic neovascularization are not clearly understood. Here, we hypothesized that distinct chemokine/chemokine receptor pathways, namely CCL2/CCR2, CX3CL1/CX3CR1, and CCL5/CCR5, differentially control monocyte subset systemic levels, and might thus impact post-ischaemic vessel growth. METHODS AND RESULTS In a model of murine hindlimb ischaemia, both Ly6C(hi) and Ly6C(lo) monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6C(hi) and Ly6C(lo) monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6C(lo) monocyte levels, whereas CCL5/CCR5 had no role. Only CCL2/CCR2 signalling influenced neovascularization, which was increased in mice overexpressing CCL2, whereas it markedly decreased in CCL2-/- mice. Moreover, adoptive transfer of Ly6C(hi)-but not Ly6C(lo)-monocytes enhanced vessel growth and blood flow recovery. CONCLUSION Altogether, our data demonstrate that regulation of proangiogenic Ly6C(hi) monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6C(lo) monocytes have no major role in this setting.

Regulatory T Cells Modulate Postischemic Neovascularization

Background— CD4+ and CD8+ T lymphocytes are key regulators of postischemic neovascularization. T-cell activation is promoted by 2 major costimulatory signalings, the B7/CD28 and 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 cells), 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 immunoinflammatory response to ischemic injury, leading to alteration of postischemic vessel growth. Methods and Results— Ischemia was induced by right femoral artery ligation in CD28-, B7-1/2–, or CD40-deficient mice (n=10 per group). CD40 deficiency led to a significant reduction in the postischemic 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 showed a profound reduction in the number of Treg cells compared with controls. Similarly, disruption of B7-1/2 signaling or anti-CD25 treatment and subsequent Treg deletion significantly enhanced postischemic neovascularization. These effects were associated with enhanced accumulation of CD3-positive T cells and Mac-3–positive macrophages in the ischemic leg. Conversely, treatment of CD28−/− mice with the nonmitogenic anti-CD3 monoclonal antibody enhanced the number of endogenous Treg cells and led to a significant reduction of the postischemic inflammatory response and neovascularization. Finally, coadministration of Treg cells and CD28−/− splenocytes in Rag1−/− mice with hindlimb ischemia abrogated the CD28−/− splenocyte-induced activation of the inflammatory response and neovascularization. Conclusion— Treg cell response modulates postischemic neovascularization.

The Chemokine Decoy Receptor D6 Prevents Excessive Inflammation and Adverse Ventricular Remodeling After Myocardial Infarction

Objective—Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and overwhelming infiltration of innate immune cells has been shown to promote adverse remodeling and cardiac rupture. Recruitment of inflammatory cells in the ischemic heart depends highly on the family of CC-chemokines and their receptors. Here, we hypothesized that the chemokine decoy receptor D6, which specifically binds and scavenges inflammatory CC-chemokines, might limit inflammation and adverse cardiac remodeling after infarction. Methods and Results—D6 was expressed in human and murine infarcted myocardium. In a murine model of myocardial infarction, D6 deficiency led to increased chemokine (C-C motif) ligand 2 and chemokine (C-C motif) ligand 3 levels in the ischemic heart. D6-deficient (D6−/−) infarcts displayed increased infiltration of pathogenic neutrophils and Ly6Chi monocytes, associated with strong matrix metalloproteinase-9 and matrix metalloproteinase-2 activities in the ischemic heart. D6−/− mice were cardiac rupture prone after myocardial infarction, and functional analysis revealed that D6−/− hearts had features of adverse remodeling with left ventricle dilation and reduced ejection fraction. Bone marrow chimera experiments showed that leukocyte-borne D6 had no role in this setting, and that leukocyte-specific chemokine (C-C motif) receptor 2 deficiency rescued the adverse phenotype observed in D6−/− mice. Conclusion—We show for the first time that the chemokine decoy receptor D6 limits CC-chemokine–dependent pathogenic inflammation and is required for adequate cardiac remodeling after myocardial infarction.

Homeostatic and Tissue Reparation Defaults in Mice Carrying Selective Genetic Invalidation of CXCL12/Proteoglycan Interactions

Background— Interaction with heparan sulfate proteoglycans is supposed to provide chemokines with the capacity to immobilize on cell surface and extracellular matrix for accomplishing both tissue homing and signaling of attracted cells. However, the consequences of the exclusive invalidation of such interaction on the roles played by endogenous chemokines in vivo remain unascertained. Methods and Results— We engineered a mouse carrying a Cxcl12 gene (Cxcl12Gagtm) mutation that precludes interactions with heparan sulfate structures while not affecting CXCR4-dependent cell signaling of CXCL12 isoforms (&agr;, &bgr;, &ggr;). Cxcl12Gagtm/Gagtm mice develop normally, express normal levels of total and isoform-specific Cxcl12 mRNA, and show increased counting of circulating CD34+ hematopoietic precursor cells. After induced acute ischemia, a marked impaired capacity to support revascularization was observed in Cxcl12Gagtm/Gagtm animals associated with a reduced number of infiltrating cells in the ischemic tissue despite the massive expression of CXCL12 isoforms. Importantly, exogenous administration of CXCL12&ggr;, which binds heparan sulfate with the highest affinity ever reported for a cytokine, fully restores vascular growth, whereas heparan sulfate–binding CXCL12&ggr; mutants failed to promote revascularization in Cxcl12Gagtm/Gagtm animals. Conclusion— These findings prove the role played by heparan sulfate interactions in the functions of CXCL12 in both homeostasis and physiopathological settings and document for the first time the paradigm of chemokine immobilization in vivo.

C/EBP Homologous Protein-10 (CHOP-10) Limits Postnatal Neovascularization Through Control of Endothelial Nitric Oxide Synthase Gene Expression

Background —CHOP-10 is a novel developmentally regulated nuclear protein that emerges as critical transcriptional integrator among pathways regulating differentiation, proliferation and survival. Here, we analyzed the role of CHOP-10 in postnatal neovascularization. Methods and Results —Ischemia was induced by right femoral artery ligation in wild-type (WT) and CHOP-10-/- mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes. Angiographic score, capillary density and foot perfusion were increased in CHOP-10-/-mice compared to WT. This effect was associated with a reduction in apoptosis and an upregulation of eNOS levels in ischemic legs of CHOP-10-/-mice compared to WT. In line with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 siRNA-transfected human endothelial cells whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using chromatin immunoprecipitation assay, we also showed that CHOP-10 bound to the eNOS promoter. Interestingly, enhanced post-ischemic neovascularization in CHOP-10-/-mice was fully blunted in CHOP-10/eNOS double knock out animals. Finally, we showed that induction of diabetes is associated with a marked upregulation of CHOP-10 that substantially inhibited post-ischemic neovascularization. Conclusions —This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.Background— C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. Methods and Results— Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10−/− mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10−/− mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10−/− mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA–transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10−/− mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. Conclusions— This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.

Endothelial Nitric Oxide Synthase Overexpression Restores the Efficiency of Bone Marrow Mononuclear Cell-Based Therapy

Bone marrow-derived mononuclear cells (BMMNCs) enhance postischemic neovascularization, and their therapeutic use is currently under clinical investigation. However, cardiovascular risk factors, including diabetes mellitus and hypercholesterolemia, lead to the abrogation of BMMNCs proangiogenic potential. NO has been shown to be critical for the proangiogenic function of BMMNCs, and increased endothelial NO synthase (eNOS) activity promotes vessel growth in ischemic conditions. We therefore hypothesized that eNOS overexpression could restore both the impaired neovascularization response and decreased proangiogenic function of BMMNCs in clinically relevant models of diabetes and hypercholesterolemia. Transgenic eNOS overexpression in diabetic, atherosclerotic, and wild-type mice induced a 1.5- to 2.3-fold increase in postischemic neovascularization compared with control. eNOS overexpression in diabetic or atherosclerotic BMMNCs restored their reduced proangiogenic potential in ischemic hind limb. This effect was associated with an increase in BMMNC ability to differentiate into cells with endothelial phenotype in vitro and in vivo and an increase in BMMNCs paracrine function, including vascular endothelial growth factor A release and NO-dependent vasodilation. Moreover, although wild-type BMMNCs treatment resulted in significant progression of atherosclerotic plaque in ischemic mice, eNOS transgenic atherosclerotic BMMNCs treatment even had antiatherogenic effects. Cell-based eNOS gene therapy has both proangiogenic and antiatherogenic effects and should be further investigated for the development of efficient therapeutic neovascularization designed to treat ischemic cardiovascular disease.

Sympathetic Nervous System Regulates Bone Marrow–Derived Cell Egress Through Endothelial Nitric Oxide Synthase Activation Role in Postischemic Tissue Remodeling

Objective—Catecholamines have been shown to control bone marrow (BM)–derived cell egress, yet the cellular and molecular mechanisms involved in this effect and their subsequent participation to postischemic vessel growth are poorly understood. Methods and Results—Tyrosine hydroxylase mRNA levels, as well as dopamine (DA) and norepinephrine (NE) contents, were increased in the ischemic BM of mice with right femoral artery ligation. Angiographic score, capillary density, and arteriole number were markedly increased by treatments with DA (IP, 50 mg/kg, 5 days) or NE (IP, 2.5 mg/kg, 5 days). Using chimeric mice lethally irradiated and transplanted with BM-derived cells from green fluorescent protein mice, we showed that DA and NE enhanced by 70% (P<0.01) and 62% (P<0.001), respectively, the number of green fluorescent protein–positive BM-derived cells in ischemic tissue and promoted their ability to differentiate into cells with endothelial and inflammatory phenotypes. Similarly, both DA and NE increased the in vitro differentiation of cultured BM-derived cells into cells with endothelial phenotype. This increase was blunted by the nitric oxide synthase inhibitor N&ohgr;-nitro-L-arginine methyl ester. DA and NE also upregulated the number of CD45-positive cells in blood 3 days after ischemia and that of macrophages in ischemic tissue 21 days after ischemia. Of interest, DA and NE increased BM endothelial nitric oxide synthase (eNOS) mRNA levels and were unable to promote BM-derived cell mobilization in chimeric eNOS-deficient mice lethally irradiated and transplanted with BM-derived cells from wild-type animals. Furthermore, administration of a &bgr;2 adrenergic agonist (clenbuterol, IP, 2 mg/kg, 5 days) and that of a dopaminergic D1/D5 receptor agonist (SKF-38393, IP, 2.5 mg/kg, 5 days) also enhanced BM-derived cell mobilization and subsequently postischemic vessel growth. Conclusion—These results unravel, for the first time, a major role for the sympathetic nervous system in BM-derived cell egress through stromal eNOS activation.

CHOP-10 Limits Postnatal Neovascularization Through the Control of eNOS Gene Expression

Background —CHOP-10 is a novel developmentally regulated nuclear protein that emerges as critical transcriptional integrator among pathways regulating differentiation, proliferation and survival. Here, we analyzed the role of CHOP-10 in postnatal neovascularization. Methods and Results —Ischemia was induced by right femoral artery ligation in wild-type (WT) and CHOP-10-/- mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes. Angiographic score, capillary density and foot perfusion were increased in CHOP-10-/-mice compared to WT. This effect was associated with a reduction in apoptosis and an upregulation of eNOS levels in ischemic legs of CHOP-10-/-mice compared to WT. In line with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 siRNA-transfected human endothelial cells whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using chromatin immunoprecipitation assay, we also showed that CHOP-10 bound to the eNOS promoter. Interestingly, enhanced post-ischemic neovascularization in CHOP-10-/-mice was fully blunted in CHOP-10/eNOS double knock out animals. Finally, we showed that induction of diabetes is associated with a marked upregulation of CHOP-10 that substantially inhibited post-ischemic neovascularization. Conclusions —This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.Background— C/EBP homologous protein-10 (CHOP-10) is a novel developmentally regulated nuclear protein that emerges as a critical transcriptional integrator among pathways regulating differentiation, proliferation, and survival. In the present study, we analyzed the role of CHOP-10 in postnatal neovascularization. Methods and Results— Ischemia was induced by right femoral artery ligation in wild-type and CHOP-10−/− mice. In capillary structure of skeletal muscle, CHOP-10 mRNA and protein levels were upregulated by ischemia and diabetes mellitus. Angiographic score, capillary density, and foot perfusion were increased in CHOP-10−/− mice compared with wild-type mice. This effect was associated with a reduction in apoptosis and an upregulation of endothelial nitric oxide synthase (eNOS) levels in ischemic legs of CHOP-10−/− mice compared with wild-type mice. In agreement with these results, eNOS mRNA and protein levels were significantly upregulated in CHOP-10 short interfering RNA–transfected human endothelial cells, whereas overexpression of CHOP-10 inhibited basal transcriptional activation of the eNOS promoter. Using a chromatin immunoprecipitation assay, we also showed that CHOP-10 was bound to the eNOS promoter. Interestingly, enhanced postischemic neovascularization in CHOP-10−/− mice was fully blunted in CHOP-10/eNOS double-knockout animals. Finally, we showed that induction of diabetes mellitus is associated with a marked upregulation of CHOP-10 that substantially inhibited postischemic neovascularization. Conclusions— This study identifies CHOP-10 as an important transcription factor modulating vessel formation and maturation.