ALBERT

Beschreibung: Ischemia of the heart, brain, and limbs is a leading cause of morbidity and mortality worldwide. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases. But the underlying mechanism by which tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)–derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool and mobilizes CD45+CD11b+ proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b+ cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b+ cells and VEGFR-1+ cells, but not carrier-mobilized cells or CD11b− cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb ischemia. Thus, tPA mobilizes CD11b+ cells from the BM and increases systemic and local (cellular) VEGF-A, which can locally promote angiogenesis during ischemic recovery. tPA might be useful to induce therapeutic revascularization in the growing field of regenerative medicine.

Beschreibung: Atherosclerosis develops as a result of multiple inflammatory-fibroproliferative responses and is the primary cause of heart disease and stroke in Western countries. Circulating BM-derived progenitor cells (CFU-Cs or EPCs) can regenerate injured vasculature by accelerating reendothelialization and limiting atherosclerotic lesion formation. Risk factors for coronary artery disease like age and diabetes reduce the number and functional activity of these cells, thus limiting the regenerative capacity. The impairment of stem/progenitor cells by risk factors may contribute to atherosclerotic disease progression. High leukocyte counts, and especially neutrophils, a marker of inflammation have been shown to be an independent risk factor and prognostic indicator of future cardiovascular outcomes. Cyclophosphamide (CY) is a synthetic antineoplastic drug, which reduces white blood cell counts (WBC), especially neutrophils, and under certain circumstances can promote hematopoietic progenitor (colony forming unit-cells, CFU-C) mobilization from the bone marrow (BM) into the circulation. We hypothesized that administration of CY limits atherosclerotic lesion formation by decreasing inflammation-associated cells, and by increasing circulating BM-derived progenitors cells. Apolipoprotein E knockout (ApoE−/ −) mice were fed a high cholesterol diet and received different doses of CY in their drinking water (37.5mg/kg/day, 18.75mg/kg/day, 9.375mg/kg/day or nothing). To control for possible effects on progenitor release a control experiment was set up in which C57/B6 received the highest CY in their drinking water (37.5mg/kg/day) or remained untreated. Peripheral blood was drawn from mice every other week. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to cultures to detect EPCs and CFU-Cs. In CY-treated mice, WBC and neutrophil counts decreased within the first 10 days after the start of the treatment as compared to non-treated controls, and stayed low over the course of the experiment (until day 70). When apoE mice receiving a high-cholesterol diet were treated with CY, the number of circulating CFU-Cs doubled, whereas no major change was found in the c57Bl/6 control group receiving only Cy. EPCs were not detectable at any time point in the CY or control apoE group. Aortic atherosclerotic tree lesion areas were approximately 50% smaller in apoE-KO mice receiving CY in their drinking water after 12 weeks on a high-cholesterol diet than control animals. Most strikingly, apoE-KO mice treated with CY showed a prolonged survival rate as compared to untreated controls. The presence of macrophage-derived foam cells is a hallmark of the atherosclerotic lesion, and an increase in their content promotes plaque instability. Immunohistochemical analysis revealed that the number of macrophages was reduced in plaques of CY-treated animals, indicating that plaques in these might be more stable. Vascular endothelial growth factor (VEGF) is expressed in atherosclerotic plaques from local macrophages and can induce metalloproteinase-9 (MMP-9). Plasma VEGF levels were lower in the CY-treatment group, coinciding with a decrease in MMP-9 plasma levels. This study demonstrates the potential clinical use of a myelosuppressive agent for the treatment of a benign, but not less “malignant” deadly disease like atherosclerosis.

Beschreibung: Abstract 3052 Poster Board II-1028 The fibrinolytic system comprises an inactive proenzyme, plasminogen (Plg), which is converted by activators such as tPA to the active enzyme, plasmin. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases (e.g. such as pulmonary embolism, myocardial infarction and stroke). Better survival in tPA-treated vs. placebo patients have been attributed to its effect on thrombus lysis. But the underlying mechanism how tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)-derived hematopoietic cells have been shown to promote neoangiogenesis during tissue regeneration or cancer growth. Here we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin mobilizes CD45+/CD11b+ pro-angiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signalling. To study the role of tPA for cell-driven tissue regeneration, we choose a model of hindlimb ischemia. tPA improves cell incorporation of CD11b+ cells into ischemic tissues, and increases expression of neoangiogenesis-related genes including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b+ cells and VEGFR-1+ cells, but not the same number of carrier-mobilized CD11b+ cells or CD11b− cells, accelerates neovascularization and ischemic tissue regeneration, showing that tPA administration had qualitatively changed CD11b+ cells and made them more angiogenic. Inhibition of VEGF-signalling suppresses tPA-induced neovascularization in a model of hindlimb ischemia. Thus, tPA mobilizes CD11b+ cells from the BM, increases systemic and local (cellular) VEGF-A, which can promote angiogenesis locally during ischemic recovery. We provide clinically relevant evidence that administration of the single agent tPA promotes ischemic tissue regeneration by recruiting pro-angiogenic CD11b+ myeloid cells, which qualitatively are more angiogenic then their PBS stimulated counterparts, and which incorporate with high efficiency into peripheral ischemic tissues. These data introduce a new paradigm in cell biology whereby fibrinolytic enzymes mediate systemic and localized effects on hematopoietic cells thereby modulating their tissue integration potential via integrin modulation and cytokine release and thus these data establish a novel role for tPA in the growing field of regenerative medicine. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Irradiation is one of the pillars in the treatment of malignancies. The combination of radiotherapy and anti-angiogenic strategies has been shown to increase the tumor response in various tumor models. The more than additive effect of irradiation and anti-angiogenic treatment suggested that irradiation might have a pro-angiogenic effect. However, the mechanism remained unclear. Bone marrow(BM)-derived progenitor cells contribute to tissue regeneration by promoting angiogenesis/vasculogenesis. We demonstrated that chemokine/cytokine mediated progenitor mobilization is dependent on the activation of matrix metalloproteinase-9 (MMP-9). Here, we show that following irradiation hematopoietic and endothelial progenitors are released into circulation in MMP-9 wild-type, but not MMP-9 deficient (−/−) mice. We have observed that low-dose irradiation fosters vascular regeneration in a limb ischemia model. Vascular regeneration was driven by the upregulation of MMP-9 mediating the release of soluble Kit-ligand (KitL) and increasing plasma vascular endothelial growth factor (VEGF), followed by mobilization of BM-derived hematopoietic and endothelial progenitor cells. Release of sKitL and production of VEGF were impaired in MMP-9-/- mice resulting in failure of mobilization of hematopoietic and endothelial progenitors, and delayed vessel formation in the ischemic limb. The blood vessels forming in the ischemic tissue of MMP-9−/− mice lacked smooth muscle cell coverage, whereas stable vessels were formed in MMP-9 wild-type animals. But which cell type might be responsible for the observed VEGF increase following irradiation? Mast cells are known to harbor a variety of growth factors and angiogenic factors, including VEGF and have been shown to have pro-angiogenic effects. We analyzed various tissues of irradiated and non-irradiated controls. The number of mast cells was increased in the irradiated muscle tissue in MMP-9 wild-type, but not MMP-9−/− mice. VEGF was mainly produced by mast cells in a MMP-9 dependent manner as determined by in situ hybridization. Likewise, the number of mast cells was increased in the ischemic tissue of MMP-9 wild-type, but not in MMP-9 deficient mice. We could show that both VEGF and KitL can promote mast cell migration and that irradiation-induced soluble KitL in collaboration with VEGF promoted migration of mast cells in vitro. Taken together, low-dose irradiation promoted hematopoietic and endothelial progenitor cell mobilization and activation of mast cells thereby promoting vasculogenesis/angiogenesis in a hind limb model. These data not only show a novel mechanism of neovascularization and tissue regeneration but suggest that low-dose irradiation can be used for therapeutic angiogenesis augmenting collateral vessel growth in ischemic tissues.