Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration.

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.

Low-dose irradiation promotes tissue revascularization through VEGF release from mast cells and MMP-9-mediated progenitor cell mobilization.

Mast cells accumulate in tissues undergoing angiogenesis during tumor growth, wound healing, and tissue repair. Mast cells can secrete angiogenic factors such as vascular endothelial growth factor (VEGF). Ionizing irradiation has also been shown to have angiogenic potential in malignant and nonmalignant diseases. We observed that low-dose irradiation fosters mast cell-dependent vascular regeneration in a limb ischemia model. Irradiation promoted VEGF production by mast cells in a matrix metalloproteinase-9 (MMP-9)-dependent manner. Irradiation, through MMP-9 up-regulated by VEGF in stromal and endothelial cells, induced the release of Kit-ligand (KitL). Irradiation-induced VEGF promoted migration of mast cells from the bone marrow to the ischemic site. Irradiation-mediated release of KitL and VEGF was impaired in MMP-9-deficient mice, resulting in a reduced number of tissue mast cells and delayed vessel formation in the ischemic limb. Irradiation-induced vasculogenesis was abrogated in mice deficient in mast cells (steel mutant, Sl/Sl(d) mice) and in mice in which the VEGF pathway was blocked. Irradiation did not induce progenitor mobilization in Sl/Sl(d) mice. We conclude that increased recruitment and activation of mast cells following irradiation alters the ischemic microenvironment and promotes vascular regeneration in an ischemia model. These data show a novel mechanism of neovascularization and suggest that low-dose irradiation may be used for therapeutic angiogenesis to augment vasculogenesis in ischemic tissues.

Granulocyte colony-stimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils.

The granulocyte colony-stimulating factor (G-CSF) promotes angiogenesis. However, the exact mechanism is not known. We demonstrate that vascular endothelial growth factor (VEGF) was released by Gr-1+CD11b- neutrophils but not Gr-1-CD11b+ monocytes prestimulated with G-CSF in vitro and in vivo. Similarly, in vivo, concomitant with an increase in neutrophil numbers in circulation, G-CSF augmented plasma VEGF level in vivo. Local G-CSF administration into ischemic tissue increased capillary density and provided a functional vasculature and contributed to neovascularization of ischemic tissue. Blockade of the VEGF pathway abrogated G-CSF-induced angiogenesis. On the other hand, as we had shown previously, VEGF can induce endothelial progenitor cell (EPC) mobilization. Here, we show that G-CSF also augmented the number of circulating VEGF receptor-2 (VEGFR2) EPCs as compared with untreated controls. Blocking the VEGF/VEGFR1, but to a much lesser extent, the VEGF/VEGFR2 pathway in G-CSF-treated animals delayed tissue revascularization in a hindlimb model. These data clearly show that G-CSF modulates angiogenesis by increasing myelomonocytic cells (VEGFR1+ neutrophils) and their release of VEGF. Our results indicated that administration of G-CSF into ischemic tissue provides a novel and safe therapeutic strategy to improve neovascularization.

Adipocyte-derived microvesicles are associated with multiple angiogenic factors and induce angiogenesis in vivo and in vitro.

We previously reported that 3T3-L1 and rat primary adipocytes secreted microvesicles, known as adipocyte-derived microvesicles (ADMs). In the present study, we further characterized the 3T3-L1 ADMs and found that they exhibited angiogenic activity in vivo. Antibody arrays and gelatin zymography analyses revealed that several angiogenic and antiangiogenic proteins, including leptin, TNFalpha, acidic fibroblast growth factor (FGFa), interferon-gamma, and matrix metalloprotease (MMP)-2 and MMP-9, were present in the ADMs. Gene expression of most of these angiogenic factors was induced in the adipose tissue of diet-induced obese mice. Furthermore, leptin, TNFalpha, and MMP-2 were up-regulated at the protein level in the adipocyte fractions prepared from epididymal adipose tissues of high-fat-diet-induced obese mice. ADMs induced cell migration and tube formation of human umbilical vein endothelial cells, which were partially suppressed by neutralizing antibodies to leptin, TNFalpha, or FGFa but not to interferon-gamma. Supporting these data, a mixture of leptin, TNFalpha, and FGFa induced tube formation. ADMs also promoted cell invasion of human umbilical vein endothelial cells through Matrigel, which was suppressed by the addition of the MMP inhibitor 1,10'-phenanthroline and a neutralizing antibody to MMP-2 but not to MMP-9. These results suggest that ADMs are associated with multiple angiogenic factors and play a role in angiogenesis in adipose tissue.

A role for niches in hematopoietic cell development.

Stem cells reside in a physical niche, a particular microenvironment. The organization of cellular niches has been shown to play a key role in regulating normal stem cell differentiation, maintenance and regeneration. Hematopoietic stem cells (HSC) emerge at distinct allocation territories during ontogenesis, notably the aorto-gonadal region, the fetal liver. Adult HSC expand and differentiate exclusively in the bone marrow (BM). They can be mobilized into the blood stream. This implies that stem cells are not autonomous units of development; rather, tissue specific niches control their destiny. Interaction of HSCs with their stem cell niches is critical for adult hematopoiesis in the BM. A niche is composed of stromal cells, which either through direct cell-to-cell contact or via release of soluble factors maintain the typical features of stem cells, mainly stem cell quiescence, maintenance or expansion. HSCs are keeping the balance of the quiescence and the self-renewal in the stem cell niche, and are maintaining long-term hematopoiesis.Therefore, an understanding of cellular and chemical architecture of the stem cell niche is vital in understanding stem cell behavior. This review summarizes the recent developments in our understanding of the stem cell niche with particular focus on the HSC niche.