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.

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.

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.

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.

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.