Yoshihiko Tashiro

Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice

Plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of a major fibrinolytic factor, tissue-type plasminogen activator, can both promote and inhibit angiogenesis. However, the physiologic role and the precise mechanisms underlying the angiogenic effects of PAI-1 remain unclear. In the present study, we report that pharmacologic inhibition of PAI-1 promoted angiogenesis and prevented tissue necrosis in a mouse model of hind-limb ischemia. Improved tissue regeneration was due to an expansion of circulating and tissue-resident granulocyte-1 marker (Gr-1(+)) neutrophils and to increased release of the angiogenic factor VEGF-A, the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated increased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization and genetic knockout studies indicated that both the improved tissue regeneration and the increase in circulating and ischemic tissue-resident Gr-1(+) neutrophils depended on the activation of tissue-type plasminogen activator and matrix metalloproteinase-9 and on VEGF-A and FGF-2. These results suggest that pharmacologic PAI-1 inhibition activates the proangiogenic FGF-2 and VEGF-A pathways, which orchestrates neutrophil-driven angiogenesis and induces cell-driven revascularization and is therefore a potential therapy for ischemic diseases.

MT1-MMP plays a critical role in hematopoiesis by regulating HIF-mediated chemokine/cytokine gene transcription within niche cells

HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP(-/-) mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.

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.

Plasmin inhibitor reduces T-cell lymphoid tumor growth by suppressing matrix metalloproteinase-9-dependent CD11b + /F4/80 + myeloid cell recruitment

Activation of the fibrinolytic system during lymphoma progression is a well-documented clinical phenomenon. But the mechanism by which the fibrinolytic system can modulate lymphoma progression has been elusive. The main fibrinolytic enzyme, plasminogen (Plg)/plasmin (Plm), can activate matrix metalloproteinases (MMPs), such as MMP-9, which has been linked to various malignancies. Here we provide the evidence that blockade of Plg reduces T-cell lymphoma growth by inhibiting MMP-9-dependent recruitment of CD11b+F4/80+ myeloid cells locally within the lymphoma tissue. Genetic Plg deficiency and drug-mediated Plm blockade delayed T-cell lymphoma growth and diminished MMP-9-dependent CD11b+F4/80+ myeloid cell infiltration into lymphoma tissues. A neutralizing antibody against CD11b inhibited T-cell lymphoma growth in vivo, which indicates that CD11b+ myeloid cells have a role in T-cell lymphoma growth. Plg deficiency in T-cell lymphoma-bearing mice resulted in reduced plasma levels of the growth factors vascular endothelial growth-A and Kit ligand, both of which are known to enhance myeloid cell proliferation. Collectively, the data presented in this study demonstrate a previously undescribed role of Plm in lymphoproliferative disorders and provide strong evidence that specific blockade of Plg represents a promising approach for the regulation of T-cell lymphoma growth.

New functions of the fibrinolytic system in bone marrow cell-derived angiogenesis.

Angiogenesis is a process by which new blood vessels form from preexisting vasculature. This process includes differentiation of angioblasts into endothelial cells with the help of secreted angiogenic factors released from cells such as bone marrow (BM)-derived cells. The fibrinolytic factor plasmin, which is a serine protease, has been shown to promote endothelial cell migration either directly, by degrading matrix proteins such as fibrin, or indirectly, by converting matrix-bound angiogenic growth factors into a soluble form. Plasmin can also activate other pericellular proteases such as matrix metalloproteinases (MMPs). Recent studies indicate that plasmin can additionally alter cellular adhesion and migration. We showed that factors of the fibrinolytic pathway can recruit BM-derived hematopoietic cells into ischemic/hypoxic tissues by altering the activation status of MMPs. These BM-derived cells can function as accessory cells that promote angiogenesis by releasing angiogenic signals. This review will discuss recent data regarding the role of the fibrinolytic system in controlling myeloid cell-driven angiogenesis. We propose that plasmin/plasminogen may be a potential target not only for development of effective angiogenic therapeutic strategies for the treatment of cancer, but also for development of strategies to promote ischemic tissue regeneration.

Contribution of the fibrinolytic pathway to hematopoietic regeneration.

Hematopoietic stem cells (HSCs) can differentiate and proliferate in response to hematopoietic stress (e.g., myelosuppression, infections, and allergic reactions), thereby ensuring a well‐regulated supply of mature and immature hematopoietic cells within the circulation and prompt adjustment of blood cell levels within normal ranges. The recovery of tissues and organs from hematopoietic stress (e.g., myelosuppression or ionizing irradiation) is dependent on two cell types: resident HSCs which repopulate the bone marrow (BM) cavity, and stromal cells. BM regeneration critically depends on the release of soluble factors from cells such as stromal cells, a process regulated by proteases. Two proteolytic systems, the fibrinolytic system and the matrix metalloproteinases (MMPs), have recently been shown to be involved in this process (Heissig B, 2007, Cell Stem Cell 1: 658–670). The plasminogen/plasmin system is mostly recognized for its fibrinolytic activity, but it is also involved in processes such as cell invasion, chemotaxis, growth factor activity modulation, and tissue remodeling. This review focuses on the role of plasmin and its activators as key players in controlling the hematopoietic stress response after myelosuppression (hematopoietic regeneration). Aspects of plasmin regulation, especially regulation of its ability to activate MMPs and the functional consequences of this enzyme activation, such as plasmin‐mediated release of biologically relevant cytokines from the matrix and cell surfaces, will be discussed. J. Cell. Physiol. 221: 521–525, 2009.

Inhibition of plasmin attenuates murine acute graft-versus-host disease mortality by suppressing the matrix metalloproteinase-9-dependent inflammatory cytokine storm and effector cell trafficking

The systemic inflammatory response observed during acute graft-versus-host disease (aGVHD) is driven by proinflammatory cytokines, a ‘cytokine storm’. The function of plasmin in regulating the inflammatory response is not fully understood, and its role in the development of aGVHD remains unresolved. Here we show that plasmin is activated during the early phase of aGVHD in mice, and its activation correlated with aGVHD severity in humans. Pharmacological plasmin inhibition protected against aGVHD-associated lethality in mice. Mechanistically, plasmin inhibition impaired the infiltration of inflammatory cells, the release of membrane-associated proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and Fas-ligand directly, or indirectly via matrix metalloproteinases (MMPs) and alters monocyte chemoattractant protein-1 (MCP-1) signaling. We propose that plasmin and potentially MMP-9 inhibition offers a novel therapeutic strategy to control the deadly cytokine storm in patients with aGVHD, thereby preventing tissue destruction.

Fibrinolytic crosstalk with endothelial cells expands murine mesenchymal stromal cells

Tissue plasminogen activator (tPA), aside from its vascular fibrinolytic action, exerts various effects within the body, ranging from synaptic plasticity to control of cell fate. Here, we observed that by activating plasminogen and matrix metalloproteinase-9, tPA expands murine bone marrow-derived CD45(-)TER119(-)Sca-1(+)PDGFRα(+) mesenchymal stromal cells (PαS-MSCs) in vivo through a crosstalk between PαS-MSCs and endothelial cells. Mechanistically, tPA induces the release of Kit ligand from PαS-MSCs, which activates c-Kit(+) endothelial cells to secrete MSC growth factors: platelet-derived growth factor-BB (PDGF-BB) and fibroblast growth factor 2 (FGF2). In synergy, FGF2 and PDGF-BB upregulate PDGFRα expression in PαS-MSCs, which ultimately leads to PαS-MSC expansion. These data show a novel mechanism by which the fibrinolytic system expands PαS-MSCs through a cytokine crosstalk between niche cells.

Plasminogen activator inhibitor-1 regulates macrophage-dependent postoperative adhesion by enhancing EGF-HER1 signaling in mice

Adhesive small bowel obstruction remains a common problem for surgeons. After surgery, platelet aggregation contributes to coagulation cascade and fibrin clot formation. With clotting, fibrin degradation is simultaneously enhanced, driven by tissue plasminogen activator–mediated cleavage of plasminogen to form plasmin. The aim of this study was to investigate the cellular events and proteolytic responses that surround plasminogen activator inhibitor (PAI‐1; Serpine1) inhibition of postoperative adhesion. Peritoneal adhesion was induced by gauze deposition in the abdominal cavity in C57BL/6 mice and those that were deficient in fibrinolytic factors, such as Plat−/− and Serpine1−/−. In addition, C57BL/6 mice were treated with the novel PAI‐1 inhibitor, TM5275. Some animals were treated with clodronate to deplete macrophages. Epidermal growth factor (EGF) experiments were performed to understand the role of macrophages and how EGF contributes to adhesion. In the early phase of adhesive small bowel obstruction, increased PAI‐1 activity was observed in the peritoneal cavity. Genetic and pharmacologic PAI‐1 inhibition prevented progression of adhesion and increased circulating plasmin. Whereas Serpine1−/− mice showed intra‐abdominal bleeding, mice that were treated with TM5275 did not. Mechanistically, PAI‐1, in combination with tissue plasminogen activator, served as a chemoattractant for macrophages that, in turn, secreted EGF and up‐regulated the receptor, HER1, on peritoneal mesothelial cells, which led to PAI‐1 secretion, further fueling the vicious cycle of impaired fibrinolysis at the adhesive site. Controlled inhibition of PAI‐1 not only enhanced activation of the fibrinolytic system, but also prevented recruitment of EGF‐secreting macrophages. Pharmacologic PAI‐1 inhibition ameliorated adhesion formation in a macrophage‐dependent manner.—Honjo, K., Munakata, S., Tashiro, Y., Salama, Y., Shimazu, H., Eiamboonsert, S., Dhahri, D., Ichimura, A., Dan, T., Miyata, T., Takeda, K., Sakamoto, K., Hattori, K., Heissig, B. Plasminogen activator inhibitor‐1 regulates macrophage‐dependent postoperative adhesion by enhancing EGF‐HER1 signaling in mice. FASEB J. 31, 2625–2637 (2017). www.fasebj.org

Plasminogen deficiency attenuates postnatal erythropoiesis in male C57BL/6 mice through decreased activity of the LH-testosterone axis.

Novel roles for the serine protease plasmin have been implicated recently in physiological and pathological processes. However, whether plasmin is involved in erythropoiesis is not known. In the present study, we studied the consequences of plasminogen deficiency on erythropoiesis in plasminogen-deficient (Plg knockout [KO]) mice. Erythroid differentiation was attenuated in male Plg KO mice and resulted in erythroblastic accumulation within the spleen and bone marrow, with increased apoptosis in the former, erythrocytosis, and splenomegaly, whereas similar erythropoietic defect was less prominent in female Plg KO mice. In addition, erythrocyte lifespan was shorter in both male and female Plg KO mice. Erythropoietin levels were compensatory increased in both male and female Plg KO mice, and resulted in a higher frequency of burst-forming units-erythroid within the spleen and bone marrow. Surprisingly, we found that male Plg KO mice, but not their female counterparts, exhibited normochromic normocytic anemia. The observed sex-linked erythropoietic defect was attributed to decreased serum testosterone levels in Plg KO mice as a consequence of impaired secretion of the pituitary luteinizing hormone (LH) under steady-state condition. Surgical castration causing testosterone deficiency and stimulating LH release attenuated erythroid differentiation and induced anemia in wild-type animals, but did not further decrease the hematocrit levels in Plg KO mice. In addition, complementation of LH using human choriogonadotropin, which increases testosterone production, improved the erythropoietic defect and anemia in Plg KO mice. The present results identify a novel role for plasmin in the hormonal regulation of postnatal erythropoiesis by the LH-testosterone axis.