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.

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.

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.

Tumor suppressor protein p53 negatively regulates ischemia-induced angiogenesis and arteriogenesis.

OBJECTIVE: The tumor suppressor protein p53 regulates angiogenesis and is a key regulatory mediator of cellular apoptosis, proliferation, and growth. p53 expression is induced in response to ischemia; however, its role in regulating ischemia-induced angiogenesis and arteriogenesis remains undefined. The objective of this study was to define the role of p53 in regulating ischemia-induced angiogenesis and arteriogenesis and to identify mechanisms by which this regulation occurs in vivo. METHODS: Surgically induced hindlimb ischemia or mesenteric artery ligation was performed in wild-type (p53+/+) and p53 knockout (p53-/-) mice. Limb perfusion and revascularization were assessed by laser Doppler perfusion imaging, capillary density, and collateral artery development. Mesenteric collateral artery flow and development were determined by arterial flow measurement and by histologic analysis, respectively. An in vitro aortic ring assay was performed on p53+/+ and p53-/- aortic tissue to evaluate endothelial function. The p53 inhibitor and activator pifithrin-α and quinacrine, respectively, were used to modulate p53 activity in vivo after ischemia. RESULTS: Absence of p53 in mice resulted in increased limb perfusion (P < .05), capillary density (P < .05), and collateral artery development (P < .05) after induction of hindlimb ischemia. In the nonischemic mesenteric artery ligation model of arteriogenesis, p53 expression was induced in collateral arteries and increased arterial blood flow in mice lacking p53 (P < .05). Lack of p53 decreased apoptosis in ischemic hindlimb tissue (P < .05) and increased proangiogenic factors hypoxia-inducible factor 1α and vascular endothelial growth factor (VEGF). Endothelial cell outgrowth in vitro increased in the absence of p53 (P < .05). Pharmacologic augmentation of p53 expression after ischemia impaired perfusion and collateral artery formation and decreased VEGF levels (P < .05). Conversely, inhibition of p53 with pifithrin-α augmented limb perfusion (P < .05) and collateral artery formation (P < .05) and increased protein levels of hypoxia-inducible factor 1α and VEGF. Pharmacologic augmentation and inhibition of p53 had no significant effect in mice lacking p53. CONCLUSIONS: p53 negatively regulates ischemia-induced angiogenesis and arteriogenesis. Inhibition of p53 increases ischemia-induced arteriogenesis and limb perfusion and thus represents a potential therapeutic strategy for arterial occlusive disease.

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of ß-aminopropionitrile-induced Aortic Aneurysm and Dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in ß-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

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.

Matrix Metalloproteinase 9 (MMP-9) Regulates Vein Wall Biomechanics in Murine Thrombus Resolution.

OBJECTIVE: Deep venous thrombosis is a common vascular problem with long-term complications including post-thrombotic syndrome. Post-thrombotic syndrome consists of leg pain, swelling and ulceration that is related to incomplete or maladaptive resolution of the venous thrombus as well as loss of compliance of the vein wall. We examine the role of metalloproteinase-9 (MMP-9), a gene important in extracellular remodeling in other vascular diseases, in mediating thrombus resolution and biomechanical changes of the vein wall. METHODS AND RESULTS: The effects of targeted deletion of MMP-9 were studied in an in vivo murine model of thrombus resolution using the FVB strain of mice. MMP-9 expression and activity significantly increased on day 3 after DVT. The lack of MMP-9 impaired thrombus resolution by 27% and this phenotype was rescued by the transplantation of wildtype bone marrow cells. Using novel biomechanical techniques, we demonstrated that the lack of MMP-9 significantly decreased thrombus-induced loss of vein wall compliance. Biomechanical analysis of the contribution of individual structural components showed that MMP-9 affected the elasticity of the extracellular matrix and collagen-elastin fibers. Biochemical and histological analyses correlated with these biomechanical effects as thrombi of mice lacking MMP-9 had significantly fewer macrophages and collagen as compared to those of wildtype mice. CONCLUSIONS: MMP-9 mediates thrombus-induced loss of vein wall compliance by increasing stiffness of the extracellular matrix and collagen-elastin fibers during thrombus resolution. MMP-9 also mediates macrophage and collagen content of the resolving thrombus and bone-marrow derived MMP-9 plays a role in resolution of thrombus mass. These disparate effects of MMP-9 on various aspects of thrombus illustrate the complexity of individual protease function on biomechanical and morphometric aspects of thrombus resolution.

Characterization of the chandipura virus leader RNA-phosphoprotein interaction using single tryptophan mutants and its detection in viral infected cells.

The phosphoprotein (P protein) of the chandipura virus (CHPV), a negative strand RNA virus, is involved in both transcription and replication of the viral life cycle. Interaction between the P protein and the viral leader (le) RNA under in vitro conditions has been previously reported for CHPV and other negative strand RNA viruses such as the rinderpest virus (RPV). However, till date, the region of the P protein involved in le RNA binding remains undefined. Moreover, the in vivo occurrence of this interaction has not been studied before. Here, we have characterised the P protein-le RNA interaction, using single tryptophan mutants of the P protein. The CHPV P protein contains two tryptophan residues located at amino acid position 105 and 135 respectively. Our previous study showed that Trp 135 is located in a buried region within a less polar environment whereas Trp 105 is more solvent-exposed. In this study we have used steady state and time resolved fluorescence spectroscopy at 298 K to show that the buried tryptophan (Trp 135) is involved in the interaction with the le RNA and the more solvent exposed Trp 105 is only slightly perturbed during this interaction. We also show that Trp 135 is responsible for the dimerization of the CHPV P protein. In addition, we have been able to demonstrate for the first time that the P protein-le RNA interaction is detectable in CHPV-infected Vero-76 cells and this interaction is augmented during the replication phase of the viral cycle.

Elucidation of functional domains of Chandipura virus Nucleocapsid protein involved in oligomerization and RNA binding: implication in viral genome encapsidation.

Chandipura virus, a member of the vesiculovirus genera, has been recently recognized as an emerging human pathogen. Previously, we have shown that Chandipura virus Nucleocapsid protein N is capable of binding to both specific viral leader RNA as well as non-viral RNA sequences, albeit in distinct monomeric and oligomeric states, respectively. Here, we distinguish the regions of N involved in oligomerization and RNA binding using a panel of deletion mutants. We demonstrate that deletion in the N-terminal arm completely abrogates self-association of N protein. Monomer N specifically recognizes viral leader RNA using its C-terminal 102 residues, while oligomerization generates an additional RNA binding surface involving the N-terminal 320 amino acids of N overlapping with a protease resistant core that is capable of forming nucleocapsid like structure and also binding heterogeneous RNA sequences. Finally, we propose a model to explain the mechanism of genome encapsidation of this important human pathogen.

Characterization of the structure of the phosphoprotein of Chandipura virus, a negative stranded RNA virus probing intratryptophan energy transfer using single and double tryptophan mutants.

The phosphoprotein (P protein) of Chandipura virus (CHPV), a negative stranded RNA virus, is involved in both transcription and replication phases of the viral life cycle. The two Tryptophan (Trp) residues of CHPV, located at 105 and 135 respectively and two single Trp mutants W135F and W105F and a double Trp mutant W135F/W105F have been characterized by steady state and time-resolved fluorescence and phosphorescence at 298 K and 77 K. Results indicate that Trp135 is more buried with less polar and more hydrophobic environment whereas the Trp105 is solvent exposed. Quantum yields (capital EF, Cyrillic) suggest that the singlet-singlet (S <--> S) non-radiative energy transfer (ET) from the Trp135 to the Trp105 occurs with 66% efficiency. The simulation of the fluorescence spectra of the WT and the time resolved studies support the results. Lifetime and capital EF, Cyrillic of the single Trp mutants suggest an intrinsic static quenching of the Trp105. The results at 77 K indicate that the ET takes place from the lowest triplet state (T(1)) of the Trp105 to the T(1) of the Trp135 apart from the backward S <--> S ET from the Trp105 to the Trp135. The triplet-triplet (T <--> T) ET implies a distance of <10 A between the Trp105 and the Trp135. Using the crystal structure of Vesicular Stomatitis Virus (VSV) phosphoprotein exhibiting about 34% similarity with the CHPV P protein, a homology modelling of CHPV supports the observed distance between the Trp residues, the S <--> S ET efficiency and the environments of the Trp residues in CHPV.

Reviewing Chandipura: a vesiculovirus in human epidemics.

Chandipura virus, a member of the rhabdoviridae family and vesiculovirus genera, has recently emerged as human pathogen that is associated with a number of outbreaks in different parts of India. Although, the virus closely resembles with the prototype vesiculovirus, Vesicular Stomatitis Virus, it could be readily distinguished by its ability to infect humans. Studies on Chandipura virus while shed light into distinct stages of viral infection; it may also allow us to identify potential drug targets for antiviral therapy. In this review, we have summarized our current understanding of Chandipura virus life cycle at the molecular detail with particular interest in viral RNA metabolisms, namely transcription, replication and packaging of viral RNA into nucleocapsid structure. Contemporary research on otherwise extensively studied family member Vesicular Stomatitis Virus has also been addressed to present a more comprehensive picture of vesiculovirus life cycle. Finally, we reveal examples of protein economy in Chandipura virus life-cycle whereby each viral protein has evolved complexity to perform multiple tasks.

Identification of a region of fast skeletal troponin T required for stabilization of the coiled-coil formation with troponin I.

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca(2+)-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the alpha helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform.