Yasmine Zouggari

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.

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.

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.

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.

D025 Phagocytosis is pivotal in the beneficial effect of bone marrow mononuclear cellsbased therapy for myocardial infarction

Cell-based therapy is a promising option for treatment of cardiovascular diseases. Based on experimental studies demonstrating that bone marrow-derived mononuclear cells (BMMNCs) improve the functional recovery after ischemia, clinical trials were initiated to address this new therapeutic concept. BMMNCs improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. Here, we hypothesized that BMMNCs have a phagocytic ability, and switch to a proangiogenic phenotype after engulfment of apoptotic cells. Activation of such angiogenic program may be pivotal in the beneficial effect of BMMNCs-based therapy. In vitro, wildtype (WT) BMMNCs ingestion of apoptotic cells upregulated the release of proangiogenic factors VEGF and HGF by 15- and 5-fold, respectively. In contrast, BMMNCs collected from mice deficient in MFG-E8, a protein that is required for attachment and engulfment of apoptotic cells by phagocytes, displayed lower phagocytic ability, leading to decrease in VEGF and HGF release. The capacity of BMMNCs to differentiate into cells with endothelial phenotype was similar in control and MFG-E8-deficient cells. In an in vivo model of mice myocardial infaction (MI), transplantation of WT BMMNCs increased fractionnal shortening (120 % of untreated control, p