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International audience; 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. © 2017 by Radiation Research Society.

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

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.

Auteur(s) : Celine Loinard Paris Centre Cardiovasculaire @ HEGP, Inserm U970, 56 rue Leblanc, 75015 Paris En 1981, Robert Furchgott, qui etait un brillant biochimiste, a ete le premier a demontrer, sur des arteres isolees in vitro, le role essentiel des cellules endotheliales dans la vasorelaxation induite par l’acetylcholine via l’activation des recepteurs muscariniques. La comprehension des mecanismes qui determinent l’effet vasodilatateur de l’acetylcholine [...]

We hypothesized that activation of angiogenesis by chronic hypoxia may affect vascular resistance and, subsequently, blood pressure levels in spontaneously hypertensive rats (SHRs). Five-week-old prehypertensive SHRs and age-matched normotensive Wistar–Kyoto (WKY) rats (n=8 per group) were maintained under normobaric normoxic or hypoxic (10% O 2 ) conditions for 8 weeks. Three weeks later, the systolic blood pressure was lower by 26% in hypoxic SHRs compared to normoxic SHRs ( P P P P P P

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.

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 Δ70kb/Δ70kb mice) is associated with reduced aortic expression of cyclin-dependent kinase inhibitor genes p19Arf and p15Inkb . Vascular smooth muscle cells from chr4 Δ70kb/Δ70kb mice show reduced transforming growth factor-β–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-β 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 Δ70kb/Δ70kb 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.

Therapeutic neovascularization represents an alternative treatment modality for patients with advanced ischemic coronary disease or critical limb ischemia (CLI). Upregulation of HIF-1α, through specific PHDs 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. The aim of this study was to unravel the efficiency of specific prolyl-hydroxylase domain protein (PHDs) silencing in both cell and non cell-based therapeutic strategies in CLI. We first showed that PHD2 and PHD3 mRNA levels were up-regulated whereas that of HIF-1α were down-regulated in mononuclear circulating cells and in ischemic legs of diabetic and non diabetic patients with CLI. We then used short hairpin RNAs (shRNAs) to downregulate murine PHD1, PHD2 or PHD3 levels and analyzed their efficiency as a therapeutic strategy in diabetic mice with hindlimb ischemia. PHDs silencing increased post-ischemic neovascularization. This increase was associated with an up-regulation of two key pro-angiogenic factors, VEGF-A and endothelial NOS and with that of pro-arteriogenic factors including fractalkine, MCP-1, and SDF-1 leading to activation of postischemic immunoinflammatory response. In addition, concomitant silencing of PHD2 and HIF-1α abrogated the shPHD2-related effects, demonstrating that activation of HIF-1α signaling mediated the pro-angiogenic and pro-arteriogenic effects of shPHD2. Finally, we assessed the putative beneficial effects of PHDs silencing on human endothelial progenitor cells (hEPC) or bone-marrow derived mesenchymal stem cells (hMSC)-based therapies. Cells were transfected with siRNA directed against PHDs and injected in Nude mice with hind-limb ischemia. We showed that PHDs silencing enhanced human cells therapeutic effect in the ischemic leg of Nude mice compared to animals receiving untreated cells. In conclusion, our study reveals, for the first time, that silencing of PHDs by the transient and local upregulation of endogenous HIF-1α improves therapeutic angiogenesis. This study also paves the way for future strategy based on administration of siRNAs directed against PHDs to enhance the efficiency of cell-based strategy.

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.

Aims Monocyte systemic levels are known to be a major determinant of ischaemic tissue revascularization, but the mechanisms mediating mobilization of different monocyte subsets—Ly6Chi and Ly6Clo—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 Ly6Chi and Ly6Clo monocyte circulating levels were increased after femoral artery ligation. CCL2/CCR2 activation enhanced blood Ly6Chi and Ly6Clo monocyte counts, although the opposite effect was seen in mice with CCL2 or CCR2 deficiency. CX3CL1/CX3CR1 strongly impacted Ly6Clo 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 Ly6Chi—but not Ly6Clo—monocytes enhanced vessel growth and blood flow recovery. Conclusion Altogether, our data demonstrate that regulation of proangiogenic Ly6Chi monocytes systemic levels by CCL2/CCR2 controls post-ischaemic vessel growth, whereas Ly6Clo monocytes have no major role in this setting.

Background— 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. 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-1α (shHIF-1α), 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-1α. 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-1α-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 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.

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

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.

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.

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

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 P P P P P

Diabetes-induced reactive oxygen species overproduction impairs neovascularization. CHOP 10 is a novel developmentally regulated nuclear protein that emerges as critical transcriptional integrator among pathways regulating differentiation, proliferation and survival. Of interest, CHOP-10 has been shown to trigger oxidative stress-induced β cells apoptosis in the setting of diabetes. Here, we analyzed the role of CHOP-10 in postnatal neovascularization and bone-marrow-derived mononuclear cells (BMC) pro-angiogenic potential in type I diabetic mice with hindlimb ischemia.Ischemia was induced by right femoral artery ligation in C57/ Bl6 animals (WT, n=8), diabetic C57/Bl6 animals (diab WT, n=8, Streptozotocin 40mg/kg) and diabetic CHOP-10-deficient animals (diab CHOP-10 KO, n=8). Two days after ischemia, CHOP-10 mRNA and protein levels were increased by 7- (p

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.

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, 100mg/kg, i.p., 2 days), clenbuterol (2mg/kg, i.p., 5 days), dopamine (DA, 50mg/kg, i.p., 5 days), NA (2.5mg/kg, i.p., 5 days) and eticlopride (10mg/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