Time-dependent ultrastructural changes during venous thrombogenesis and thrombus resolution.

BACKGROUND: Deep vein thrombosis is a common vascular event that can result in debilitating morbidity and even death due to pulmonary embolism. Clinically, patients with faster resolution of a venous thrombus have improved prognosis, but the detailed structural information regarding changes that occur in a resolving thrombus over time is lacking. OBJECTIVES: To define the spatial-morphologic characteristics of venous thrombus formation, propagation, and resolution at the submicron level over time. METHODS: Using a murine model of stasis-induced deep vein thrombosis along with scanning electron microscopy and immunohistology, we determine the specific structural, compositional, and morphologic characteristics of venous thrombi formed after 4 days and identify the changes that take place during resolution by day 7. Comparison is made with the structure and composition of venous thrombi formed in mice genetically deficient in plasminogen activator inhibitor type 1. RESULTS: As venous thrombus resolution progresses, fibrin exists in different structural forms, and there are dynamic cellular changes in the compositions of leukocytes, platelet aggregates, and red blood cells. Intrathrombus microvesicles are present that are not evident by histology, and red blood cells in the form of polyhedrocytes are an indicator of clot contraction. Structural evidence of fibrinolysis is observed early during thrombogenesis and is accelerated by plasminogen activator inhibitor type 1 deficiency. CONCLUSION: The results reveal unique, detailed ultrastructural and compositional insights along with documentation of the dynamic changes that occur during accelerated resolution that are not evident by standard pathologic procedures and can be applied to inform diagnosis and effectiveness of thrombolytic treatments to improve patient outcomes.

Matriptase drives dissemination of ovarian cancer spheroids by a PAR-2/PI3K/Akt/MMP9 signaling axis.

The transmembrane serine protease matriptase is a key regulator of both barrier-disruptive and protective epithelial cell-cell interactions. Elevated matriptase is a consistent feature of epithelial ovarian cancers (OvCa), where multicellular spheroids shed from the primary tumor into the peritoneal cavity are critical drivers of metastasis. Dynamic cell-to-cell adhesive contacts are required for spheroid formation and maintenance. Here, we show that overactive matriptase, reflected in an increased ratio of matriptase to its inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), disrupts cell-cell contacts to produce loose prometastatic spheroids that display increased mesothelial cell adhesion and submesothelial invasion. We show that these activities are dependent on the matriptase activation of a protease-activated receptor-2 (PAR-2) signaling pathway involving PI3K/Akt and MMP9-induced disruption of cell-cell adhesion by the release of the soluble E-cadherin ectodomain. These data reveal a novel pathological connection between matriptase activation of PAR-2 and disruption of cell-cell adhesion, and support the clinical investigation of this signaling axis as a therapeutic strategy for aggressive metastatic OvCa.

Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin.

Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Testisin/Prss21 deficiency causes increased vascular permeability and a hemorrhagic phenotype during luteal angiogenesis.

Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.

Depletion of CD4 and CD8 Positive T Cells Impairs Venous Thrombus Resolution in Mice.

Resolution of deep venous thrombosis involves coordinated inflammatory processes. T cells regulate inflammation in vivo and modulate vascular remodeling in other settings, but their role in venous thrombus resolution remains undefined. To determine the role of T cells in venous thrombus resolution in vivo, stasis induced thrombi were created by vena cava ligation in outbred CD-1 mice. CD4 and CD8 positive T cells, as determined by flow cytometry, were present in thrombi both during thrombus formation and resolution. Depletion of the CD4 and CD8 positive T cells by antibody treatment selectively impaired thrombus resolution compared to animals treated with isotype control antibodies, without an effect on venous thrombus formation. Quantitation of intra-thrombus macrophage numbers, fibrinolytic marker expression, and gelatinolytic activity by zymography revealed that T cell depletion decreased the number of macrophages, reduced the expression of fibrinolytic marker urokinase plasminogen activator (uPA), and decreased the activity of matrix metalloprotinease-9 (MMP-9). These data implicate CD4 and CD8 positive T cells in functionally contributing to venous thrombus resolution, thus representing a potential therapeutic target, but also underscoring potential risks involved in T cell depletion used clinically for solid organ and hematopoietic transplantation procedures.

Fibrinolysis and Inflammation in Venous Thrombus Resolution.

Clinical observations and accumulating laboratory evidence support a complex interplay between coagulation, inflammation, innate immunity and fibrinolysis in venous thromboembolism (VTE). VTE, which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), and the subsequent complications of post-thrombotic syndrome (PTS), are significant causes of morbidity and mortality in patients. Clinical risk factors for VTE include cancer, major trauma, surgery, sepsis, inflammatory bowel disease, paralysis, prolonged periods of immobility, and aging. Abnormalities in venous blood flow or stasis initiates the activation of endothelial cells, and in concert with platelets, neutrophils and monocytes, propagates VTE in an intact vein. In addition, inflammatory cells play crucial roles in thrombus recanalization and restoration of blood flow via fibrinolysis and vascular remodeling. Faster resolution of the thrombus is key for improved disease prognosis. While in the clinical setting, anticoagulation therapy is successful in preventing propagation of venous thrombi, current therapies are not designed to inhibit inflammation, which can lead to the development of PTS. Animal models of DVT have provided many insights into the molecular and cellular mechanisms involved in the formation, propagation, and resolution of venous thrombi as well as the roles of key components of the fibrinolytic system in these processes. Here, we review the recent advances in our understanding of fibrinolysis and inflammation in the resolution of VTE.

Correction to: PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

The affiliation of Erik W. Martin is corrected in this paper.

PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis. KEY MESSAGES: Testisin inhibits metastatic ovarian tumor burden and ascites production. Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion. PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells.

Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2-Mediated Signaling: Co-Conspirators in Cancer Progression.

Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.

Serpins in Venous Thrombosis and Venous Thrombus Resolution.

Several serpins function as potent inhibitors of thrombolytic serine proteases. Venous thrombosis is a common and debilitating condition whose incidence is on the rise. Studies using genetically modified mice and inhibitors have shown that the plasminogen activator inhibitors (PAI), PAI-1 and PAI-2, are primary regulators of plasminogen activation and contribute to regulating the resolution of experimental venous thrombi, via inflammatory mechanisms, vascular remodeling, and inhibition of fibrinolysis. Therapies to accelerate venous thrombus resolution would be beneficial, since delayed or incomplete clot resolution frequently leads to postthrombotic syndrome, a long-term complication associated with debilitating limb swelling, pain, and recurrent skin ulceration. Here we describe a useful and reproducible mouse model for the study of venous thrombus resolution involving ligation of the inferior vena cava and elucidation of the molecular and cellular determinants of venous thrombus formation and resolution.

Improving Cancer Detection and Treatment with Liquid Biopsies and ptDNA.

Liquid biopsy, or the capacity to noninvasively isolate and analyze plasma tumor DNA (ptDNA) using blood samples, represents an important tool for modern oncology that enables increasingly safe, personalized, and robust cancer diagnosis and treatment. Here, we review advances in the development and implementation of liquid biopsy approaches, and we focus on the capacity of liquid biopsy to noninvasively detect oncological disease and enhance early detection strategies. In addition to noting the distinctions between mutation-targeted and mutation-agnostic approaches, we discuss the potential for genomic analysis and longitudinal testing to identify somatic lesions early and to guide intervention at more manageable disease stages.

Inflammatory cytokines down-regulate the barrier-protective prostasin-matriptase proteolytic cascade early in experimental colitis.

Compromised gastrointestinal barrier function is strongly associated with the progressive and destructive pathologies of the two main forms of irritable bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD). Matriptase is a membrane-anchored serine protease encoded by suppression of tumorigenicity-14 (ST14) gene, which is critical for epithelial barrier development and homeostasis. Matriptase barrier-protective activity is linked with the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin, which is a co-factor for matriptase zymogen activation. Here we show that mRNA and protein expression of both matriptase and prostasin are rapidly down-regulated in the initiating inflammatory phases of dextran sulfate sodium (DSS)-induced experimental colitis in mice, and, significantly, the loss of these proteases precedes the appearance of clinical symptoms, suggesting their loss may contribute to disease susceptibility. We used heterozygous St14 hypomorphic mice expressing a promoter-linked ß-gal reporter to show that inflammatory colitis suppresses the activity of the St14 gene promoter. Studies in colonic T84 cell monolayers revealed that barrier disruption by the colitis-associated Th2-type cytokines, IL-4 and IL-13, down-regulates matriptase as well as prostasin through phosphorylation of the transcriptional regulator STAT6 and that inhibition of STAT6 with suberoylanilide hydroxamic acid (SAHA) restores protease expression and reverses cytokine-induced barrier dysfunction. Both matriptase and prostasin are significantly down-regulated in colonic tissues from human subjects with active ulcerative colitis or Crohn's disease, implicating the loss of this barrier-protective protease pathway in the pathogenesis of irritable bowel disease.

Myeloid p53 regulates macrophage polarization and venous thrombus resolution by inflammatory vascular remodeling in mice.

Deep venous thrombosis (DVT) remains a common and serious cardiovascular problem with both fatal and long-term consequences. The consequences of DVT include the development of postthrombotic syndrome in 25% to 60% of DVT patients. Despite the clinical importance of venous thrombus resolution, the cellular and molecular mediators involved are poorly understood, and currently there is no molecular therapy to accelerate this process. Several lines of evidence suggest that a complex and interrelated array of molecular signaling processes are involved in the inflammatory vascular remodeling associated with the resolution of DVT. Here, we have identified a role for the tumor suppressor gene p53 in regulating venous thrombus resolution. Using the stasis model of venous thrombosis and resolution in mice, we found that genetic deficiency of p53 or pharmacologic inhibition by pifithrin impairs thrombus resolution and is associated with increased fibrosis and altered expression of matrix metalloproteinase-2. The effect of p53 loss was mediated by cells of the myeloid lineage, resulting in enhanced polarization of the cytokine milieu toward an M1-like phenotype. Furthermore, augmentation of p53 activity using the pharmacological agonist of p53, quinacrine, accelerates venous thrombus resolution in a p53-dependent manner, even after establishment of thrombosis. Together, these studies define mechanisms by which p53 regulates thrombus resolution by increasing inflammatory vascular remodeling of venous thrombi in vivo, and the potential therapeutic application of a p53 agonist as a treatment to accelerate this process in patients with DVT.

Membrane-anchored proteases in endothelial cell biology.

PURPOSE OF REVIEW: The endothelial cell plasma membrane is a metabolically active, dynamic, and fluid microenvironment where pericellular proteolysis plays a critical role. Membrane-anchored proteases may be expressed by endothelial cells as well as mural cells and leukocytes with distribution both inside and outside of the vascular system. Here, we will review the recent advances in our understanding of the direct and indirect roles of membrane-anchored proteases in vascular biology and the possible conservation of their extravascular functions in endothelial cell biology. RECENT FINDINGS: Membrane-anchored proteases belonging to the serine or metalloprotease families contain amino-terminal or carboxy-terminal domains, which serve to tether their extracellular protease domains directly at the plasma membrane. This architecture enables protease function and substrate repertoire to be regulated through dynamic localization in distinct areas of the cell membrane. These proteases are proving to be key components of the cell machinery for regulating vascular permeability, generation of vasoactive peptides, receptor tyrosine kinase transactivation, extracellular matrix proteolysis, and angiogenesis. SUMMARY: A complex picture of the interdependence between membrane-anchored protease localization and function is emerging that may provide a mechanism for precise coordination of extracellular signals and intracellular responses through communication with the cytoskeleton and with cellular signaling molecules.

Essential Components of Cancer Education.

Modern cancer therapy/care involves the integration of basic, clinical, and population-based research professionals using state-of-the-art science to achieve the best possible patient outcomes. A well-integrated team of basic, clinical, and population science professionals and educators working with a fully engaged group of creative junior investigators and trainees provides a structure to achieve these common goals. To this end, the structure provided by cancer-focused educational programs can create the integrated culture of academic medicine needed to reduce the burden of cancer on society. This summary outlines fundamental principles and potential best practice strategies for the development of integrated educational programs directed at achieving a work force of professionals that broadly appreciate the principals of academic medicine spanning the breadth of knowledge necessary to advance the goal of improving the current practice of cancer care medicine.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden.

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.

Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface.

SerpinB2 mediated regulation of macrophage function during enteric infection.

Host defense is an orchestrated response involving changes in the expression of receptors and release of mediators from both immune and structural cells. There is a growing recognition of the important role of proteolytic pathways for the protective immune response to enteric pathogens. Enteric nematode infection induces a type 2 immune response with polarization of macrophages toward the alternatively activated phenotype (M2). The Th2 cytokines, IL-4, and IL-13, induce a STAT6-dependent upregulation of the expression of the protease inhibitor, serpinB2, which protects macrophages from apoptosis. M2 are critical to worm clearance and a novel role for serpinB2 is its regulation of the chemokine, CCL2, which is necessary for monocyte and/or macrophage influx into small intestine during infection. There is a growing list of factors including immune (LPS, Th2 cytokines) as well as hormonal (gastrin, 5-HT) that are linked to increased expression of serpinB2. Thus, serpinB2 represents an immune regulated factor that has multiple roles in the intestinal mucosa.