OBJECTIVEIn white adipose tissue, adipocytes and adipocyte precursor cells are enmeshed in a dense network of type I collagen fibrils. The fate of this pericellular collagenous web in diet-induced obesity, however, is unknown. This study seeks to identify the genetic underpinnings of proteolytic collagen turnover and their association with obesity progression in mice and humans.RESEARCH DESIGN AND METHODSThe hydrolysis and degradation of type I collagen at early stages of high-fat diet feeding was assessed in wild-type or MMP14 (MT1-MMP)-haploinsufficient mice using immunofluorescent staining and scanning electron microscopy. The impact of MMP14-dependent collagenolysis on adipose tissue function was interrogated by transcriptome profiling with cDNA microarrays. Genetic associations between MMP14 gene common variants and obesity or diabetes traits were examined in a Japanese cohort (n = 3,653).RESULTSIn adult mice, type I collagen fibers were cleaved rapidly in situ during a high-fat diet challenge. By contrast, in MMP14 haploinsufficient mice, animals placed on a high-fat diet were unable to remodel fat pad collagen architecture and display blunted weight gain. Moreover, transcriptional programs linking type I collagen turnover with adipogenesis or lipogenesis were disrupted by the associated decrease in collagen turnover. Consistent with a key role played by MMP14 in regulating high-fat diet–induced metabolic programs, human MMP14 gene polymorphisms located in proximity to the enzyme's catalytic domain were closely associated with human obesity and diabetes traits.CONCLUSIONSTogether, these findings demonstrate that the MMP14 gene, encoding the dominant pericellular collagenase operative in vivo, directs obesogenic collagen turnover and is linked to human obesity traits.

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 tissuetype 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. IntroductionApproximately 500 to 1000 people per million per year are diagnosed with critical ischemia of the limb, which in most cases results in serious morbidity and mortality. Therapeutic restoration of blood flow by, for example, the induction of the formation of new capillaries (angiogenesis) is the ultimate goal for critical limb ischemia patients. Growth of new blood vessels in the adult occurs through angiogenesis or arteriogenesis (vessel maturation via recruitment of smooth muscle cells) and vasculogenesis (mobilization of BM-derived cells). 1,2 In contrast to promising results from animal studies, administration of proangiogenic factors such as fibroblast growth factor 2 (FGF-2, also known as basic FGF) or VEGF-A failed to induce significant improvement in ischemia in several phase 1 clinical trials. 3 The plasminogen activation system and matrix metalloproteinases (MMPs), which can cleave growth factors, growth factor receptors, and adhesion molecules and mediate the extracellular matrix degradation that is necessary for cell migration, are widely recognized as being involved in the process of angiogenesis. 2,4 Although plasminogen activator inhibitor-1 (PAI-1) is one of the primary regulators of the fibrinolytic system, it also has dramatic effects on cell adhesion, detachment, and migration 5 and can inhibit cellular migration by affecting cell adhesion. 6,7 PAI-1-deficient (PAI-1 Ϫ/Ϫ ) mice showed improved vascular wound healing in models of perivascular electric or transluminal mechanical injury 8 due to improved migration of PAI-1 Ϫ/Ϫ smooth muscle cells. The 52-kDa serine protease inh...

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.

Key Points• tPA expands mesenchymal stromal cells (MSCs) in the bone marrow by a cytokine (KitL and PDGF-BB) crosstalk with endothelial cells.• Pharmacologic inhibition of receptor tyrosine kinases (c-Kit and PDGFRa) impairs tPA-mediated MSC proliferation.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

Adipogenesis is directed by both transcriptional network and posttranslational modification of chromatin structure. Although adipogenesis in vivo proceeds in collagen-rich extracellular matrix (ECM) environments, the impact of ECM proteins and their modifying enzymes on the epigenetic regulation of adipogenesis has been largely unknown. We aimed to define the role of fibrillar type I collagen and its modifying enzymes in regulating adipogenic chromatin signatures and gene regulation in the in vivo-like settings. Adipogenic cocktail induces a robust increase in the level of protranscriptional acetylated histone H3 at lysine 9 (H3K9ac) within 24 h. When cultured atop fibrillar type I collagen gel, however, H3K9ac levels in differentiating 3T3-L1 cells are substantially reduced. The suppression of adipogenic histone mark in differentiating 3T3-L1 cells is type I collagen density dependent and released by heat denaturing of the subjacent collagen substratum, pointing to the critical role played by the triple-helical structure of type I collagen. By probing adipogenic collagenolysis with a series of proteinase inhibitors, matrix metalloproteinase (MMP) family members are found to be responsible for adipogenic collagenolysis. At the same time, MMP inhibitor specifically blocked the adipogenic induction of H3K9ac. By targeting individual MMP using small interfering RNA oligos, MMP14 was identified as the major adipogenic MMP critical for H3K9 acetylation. Consistently, MMP14-null adipose tissues display diminished protranscriptional histone mark H3K9ac while maintaining repressive histone mark tri-methylated histone H3 at lysine 9 (H3K9me3). Taken together, MMP14-dependent collagenolysis plays the major role in regulating adipogenic histone marks by releasing the epigenetic constraints imposed by fibrillar type I collagen.

Treatment of rat pituitary GH(4)C(1) cell membranes with calpain, a calcium-activated cysteine protease, increased adenylate cyclase activity, and this activity was inhibited by a calpain inhibitor, leupeptin. Calpain treatment potentiated the activity of guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but did not attenuate MnCl(2) action on adenylate cyclase, suggesting that calpain acted at the G-protein level, rather than directly on adenylate cyclase. This calpain stimulation of adenylate cyclase was inhibited by an antibody raised against the C-terminal portion of G(s)alpha, but not by anti-G(i)2alpha or anti-Gbeta antibodies. Furthermore, it was shown that G(s)alpha is more susceptible to calpain-mediated proteolysis than G(i)2alpha or Gbeta. Therefore the stimulatory effect of calpain on adenylate cyclase is due to the cleavage of G(s)alpha in GH(4)C(1) cell membranes. Proteolysis of G(s)alpha by micro-calpain involved sequential cleavages at two sites, resulting in the generation of a 39 kDa fragment first, and then a 20 kDa fragment, from the C-terminus. Treatment of GH(4)C(1) cell membranes with cholera toxin increased the rate of cleavage. Cholera toxin treatment of intact GH(4)C(1) cells induced the translocation of calpain from the cytosol to the membranes, a hallmark of calpain activation. In addition, treatment of intact GH(4)C(1) cells with a calpain-specific inhibitor, benzyloxycarbonyl-Leu-leucinal, blocked the increased cAMP production and the down-regulation of G(s)alpha, which were produced by cholera toxin or pituitary adenylate cyclase-activating polypeptide. These results suggest that calpain sustains adenylate cyclase in an active form through the cleavage of G(s)alpha to an active G(s)alpha fragment. This is a novel calpain-dependent activation mechanism of G(s)alpha and, thus, of adenylate cyclase in rat pituitary cells.

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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.