Chiemi Nishida

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.

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.

Hes1 promotes blast crisis in chronic myelogenous leukemia through MMP-9 upregulation in leukemic cells

High levels of HES1 expression are frequently found in BCR-ABL(+) chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC-like disease; however, the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-κB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of HES1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, CMPs expressing BCR-ABL and Hes1 secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC-like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells.

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.