Céline Loinard

BAFF Receptor Deficiency Reduces the Development of Atherosclerosis in Mice—Brief Report

Objective—The goal of this study was to assess the role of B-cell activating factor (BAFF) receptor in B-cell regulation of atherosclerosis. Methods and Results—Male LDL receptor-deficient mice (Ldlr−/−) were lethally irradiated and reconstituted with either wild type or BAFF receptor (BAFF-R)–deficient bone marrow. After 4 weeks of recovery, mice were put on a high-fat diet for 6 or 8 weeks. BAFF-R deficiency in bone marrow cells led to a marked reduction of conventional mature B2 cells but did not affect the B1a cell subtype. This was associated with a significant reduction of dendritic cell activation and T-cell proliferation along with a reduction of IgG antibodies against malondialdehyde-modified low-density lipoprotein. In contrast, serum IgM type antibodies were preserved. Interestingly, BAFF-R deficiency was associated with a significant reduction in atherosclerotic lesion development and reduced numbers of plaque T cells. Selective BAFF-R deficiency on B cells led to a similar reduction in lesion size and T-cell infiltration but in contrast did not affect dendritic cell activation. Conclusion—BAFF-R deficiency in mice selectively alters mature B2 cell-dependent cellular and humoral immune responses and limits the development of atherosclerosis.

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.

Hypertension Impairs Postnatal Vasculogenesis: Role of Antihypertensive Agents

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 &mgr;mol/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.

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.

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.

HIF‐Prolyl Hydroxylase 2 Inhibition Enhances the Efficiency of Mesenchymal Stem Cell‐Based Therapies for the Treatment of Critical Limb Ischemia

Upregulation of hypoxia‐inducible transcription factor‐1α (HIF‐1α), through prolyl‐hydroxylase domain protein (PHD) inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. We aimed to unravel the effect of specific PHD2 isoform silencing in cell‐based strategies designed to promote therapeutic revascularization in patients with critical limb ischemia (CLI). PHD2 mRNA levels were upregulated whereas that of HIF‐1α were downregulated in blood cells from patients with CLI. We therefore assessed the putative beneficial effects of PHD2 silencing on human bone marrow‐derived mesenchymal stem cells (hBM‐MSC)‐based therapy. PHD2 silencing enhanced hBM‐MSC therapeutic effect in an experimental model of CLI in Nude mice, through an upregulation of HIF‐1α and its target gene, VEGF‐A. In addition, PHD2‐transfected hBM‐MSC displayed higher protection against apoptosis in vitro and increased rate of survival in the ischemic tissue, as assessed by Fluorescence Molecular Tomography. Cotransfection with HIF‐1α or VEGF‐A short interfering RNAs fully abrogated the beneficial effect of PHD2 silencing on the proangiogenic capacity of hBM‐MSC. We finally investigated the effect of PHD2 inhibition on the revascularization potential of ischemic targeted tissues in the diabetic pathological context. Inhibition of PHD‐2 with shRNAs increased postischemic neovascularization in diabetic mice with CLI. This increase was associated with an upregulation of proangiogenic and proarteriogenic factors and was blunted by concomitant silencing of HIF‐1α. In conclusion, silencing of PHD2, by the transient upregulation of HIF‐1α and its target gene VEGF‐A, might improve the efficiency of hBM‐MSC‐based therapies. Stem Cells 2014;32:231–243

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.

Deletion of Chromosome 9p21 Noncoding Cardiovascular Risk Interval in Mice Alters Smad2 Signaling and Promotes Vascular Aneurysm

Background—Vascular aneurysm is an abnormal local dilatation of an artery that can lead to vessel rupture and sudden death. The only treatment involves surgical or endovascular repair or exclusion. There is currently no approved medical therapy for this condition. Recent data established a strong association between genetic variants in the 9p21 chromosomal region in humans and the presence of cardiovascular diseases, including aneurysms. However, the mechanisms linking this 9p21 DNA variant to cardiovascular risk are still unknown. Methods and Results—Here, we show that deletion of the orthologous 70-kb noncoding interval on mouse chromosome 4 (chr4&Dgr;70kb/&Dgr;70kb mice) is associated with reduced aortic expression of cyclin-dependent kinase inhibitor genes p19Arf and p15Inkb. Vascular smooth muscle cells from chr4&Dgr;70kb/&Dgr;70kb mice show reduced transforming growth factor-&bgr;–dependent canonical Smad2 signaling but increased cyclin-dependent kinase–dependent Smad2 phosphorylation at linker sites, a phenotype previously associated with tumor growth and consistent with the mechanistic link between reduced canonical transforming growth factor-&bgr; signaling and susceptibility to vascular diseases. We also show that targeted deletion of the 9p21 risk interval promotes susceptibility to aneurysm development and rupture when mice are subjected to a validated model of aneurysm formation. The vascular disease of chr4&Dgr;70kb/&Dgr;70kb mice is prevented by treatment with a cyclin-dependent kinase inhibitor. Conclusions—The results establish a direct mechanistic link between 9p21 noncoding risk interval and susceptibility to aneurysm and may have important implications for the understanding and treatment of vascular diseases.

Monocytes/Macrophages Mobilization Orchestrate Neovascularization after Localized Colorectal Irradiation

In patients undergoing radiotherapy for cancer, radiation dose to healthy tissue can occur, causing microvascular damage. Monocytes that have been shown to promote tissue revascularization comprise the subsets: CD11b+Ly6G–7/4hi/monocyteshi and CD11b+Ly6G–7/4lo/monocyteslo. We hypothesized that monocytes were implicated in postirradiation blood vessel formation. C57Bl6 mice underwent localized colorectal irradiation and were sacrificed at different times after exposure. Bone marrow, spleen, blood and colon were collected. Fourteen days postirradiation, colons expressed proangiogenic actors and adhesion molecules. Monocyteshi, which were the main subset of infiltrating monocytes, mobilized to the blood from spleen and bone marrow, peaking at day 14 postirradiation, and were associated with lymphocyte Th1 polarization. At day 28 postirradiation, angiographic score and capillary density increased by ∼1.8-fold, and then returned to nonirradiated levels at day 60. Clodronate-mediated depletion of circulating monocytes prior to irradiation resulted in a ∼1.4-fold decrease in angiographic score and capillary density compared to the nontreated control. Histological analysis of the colon in clodronate-treated mice revealed a massive decrease of macrophage and lymphocyte infiltration as well as reduced collagen deposition in crypt area at day 21. However, late depletion of monocytes from day 25 postirradiation had no effect on fibrotic process. These findings demonstrate a central role for monocyte/macrophage activation in the orchestration of a neovascularization mechanism after localized colorectal irradiation.