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.

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.

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.

Recanalization and flow regulate venous thrombus resolution and matrix metalloproteinase expression in vivo.

OBJECTIVE: We examined the role of thrombus recanalization and ongoing blood flow in the process of thrombus resolution by comparing two murine in vivo models of deep venous thrombosis. METHODS: In CD1 mice, we performed surgical inferior vena cava ligation (stasis thrombosis), stenosis (thrombosis with recanalization), or sham procedure. We analyzed thrombus weight over time as a measure of thrombus resolution and quantified the messenger RNA and protein levels of membrane-type matrix metalloproteinases (MT-MMPs) as well as effectors of the plasmin complex at days 4, 8, and 12 after surgery. RESULTS: Despite similar initial thrombus size, the presence of ongoing blood flow (stenosis model) was associated with a 45.91% subsequent improvement in thrombus resolution at day 8 and 12.57% at day 12 compared with stasis thrombosis (ligation model). Immunoblot and real-time polymerase chain reaction analysis demonstrated a difference in MMP-2 and MMP-9 activity at day 8 between the two models (P = .03 and P = .006, respectively) as well as a difference in MT2-MMP gene expression at day 8 (P = .044) and day 12 (P = .03) and MT1-MMP protein expression at day 4 (P = .021). Histologic analyses revealed distinct areas of recanalization in the thrombi of the stenosis model compared with the ligation model as well as the recruitment of inflammatory cells, especially macrophages, and a focal pattern of localized expression of MT1-MMP and MT3-MMP proteins surrounding the areas of recanalization in the stenosis model. CONCLUSIONS: Recanalization and ongoing blood flow accelerate deep venous thrombus resolution in vivo and are associated with distinct patterns of MT1-MMP and MT3-MMP expression and macrophage localization in areas of intrathrombus recanalization.

A phylogenomic profile of hemerythrins, the nonheme diiron binding respiratory proteins.

BACKGROUND: Hemerythrins, are the non-heme, diiron binding respiratory proteins of brachiopods, priapulids and sipunculans; they are also found in annelids and bacteria, where their functions have not been fully elucidated. RESULTS: A search for putative Hrs in the genomes of 43 archaea, 444 bacteria and 135 eukaryotes, revealed their presence in 3 archaea, 118 bacteria, several fungi, one apicomplexan, a heterolobosan, a cnidarian and several annelids. About a fourth of the Hr sequences were identified as N- or C-terminal domains of chimeric, chemotactic gene regulators. The function of the remaining single domain bacterial Hrs remains to be determined. In addition to oxygen transport, the possible functions in annelids have been proposed to include cadmium-binding, antibacterial action and immunoprotection. A Bayesian phylogenetic tree revealed a split into two clades, one encompassing archaea, bacteria and fungi, and the other comprising the remaining eukaryotes. The annelid and sipunculan Hrs share the same intron-exon structure, different from that of the cnidarian Hr. CONCLUSION: The phylogenomic profile of Hrs demonstrated a limited occurrence in bacteria and archaea and a marked absence in the vast majority of multicellular organisms. Among the metazoa, Hrs have survived in a cnidarian and in a few protostome groups; hence, it appears that in metazoans the Hr gene was lost in deuterostome ancestor(s) after the radiata/bilateria split. Signal peptide sequences in several Hirudinea Hrs suggest for the first time, the possibility of extracellular localization. Since the alpha-helical bundle is likely to have been among the earliest protein folds, Hrs represent an ancient family of iron-binding proteins, whose primary function in bacteria may have been that of an oxygen sensor, enabling aerophilic or aerophobic responses. Although Hrs evolved to function as O2 transporters in brachiopods, priapulids and sipunculans, their function in annelids remains to be elucidated. Overall Hrs exhibit a considerable lack of evolutionary success in metazoans.

Globin gene family evolution and functional diversification in annelids.

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.

Gene structure and molecular phylogeny of the linker chains from the giant annelid hexagonal bilayer hemoglobins.

Giant extracellular hexagonal bilayer hemoglobin (HBL-Hb), found only in annelids, is an approximately 3500-kDa heteropolymeric structure involved in oxygen transport. The HBL-Hbs are comprised of globin and linker chains, the latter being required for the assembly of the quaternary structure. The linker chains, varying in size from 225 to 283 amino acids, have a conserved cysteine-rich domain within their N-terminal moiety that is homologous to the cysteine-rich modules constituting the ligand binding domain of the low-density lipoprotein receptor (LDLR) protein family found in many metazoans. We have investigated the gene structure of linkers from Arenicola marina, Alvinella pompejana, Nereis diversicolor, Lumbricus terrestris, and Riftia pachyptila. We found, contrary to the results obtained earlier with linker genes from N. diversicolor and L. terrestris, that in all of the foregoing cases, the linker LDL-A module is flanked by two phase 1 introns, as in the human LDLR gene, with two more introns in the 3' side whose positions varied with the species. In addition, we obtained 13 linker cDNAs that have been determined experimentally or found in the EST database LumbriBASE. A molecular phylogenetic analysis of the linker primary sequences demonstrated that they cluster into two distinct families of linker proteins. We propose that the common gene ancestor to annelid linker genes exhibited a four-intron and five-exon structure and gave rise to the two families subsequent to a duplication event.

The multigenic family of the extracellular hemoglobin from the annelid polychaete Arenicola marina.

The extracellular hemoglobin of the lugworm Arenicola marina which inhabits on the intertidal area, a sulfide-rich environment, comprises eight globin chains previously determined by mass spectrometry. We have cloned and sequenced five of the globin components. The deduced amino-acid sequences exhibit an extracellular signal peptide and two cysteine residues involved in an internal disulfide bond. The molecular weights calculated from the globin primary structures obtained from complete cDNA sequences are in good agreement with the mass spectrometry values obtained with the native hemoglobin. Phylogenetic analysis has allowed assigning the five A. marina sequences to the different globin sub-families. Two of the globins were found to be A2 globin chains lacking the cysteine residues proposed to be involved in the binding of hydrogen sulfide by such hemoglobin. We discuss the unusual absence of these cysteines in the light of their invariant occurrence in the A2 subfamily of hemoglobins from annelids inhabiting sulfide-rich environments.

Novel dissociation mechanism of a polychaetous annelid extracellular haemoglobin.

The extracellular haemoglobin of the marine polychaete, Arenicola marina, is a hexagonal bilayer haemoglobin of approximately 3600 kDa, formed by the covalent and noncovalent association of many copies of both globin subunits (monomer and trimer) and nonglobin or 'linker' subunits. In order to analyse the interactions between globin and linker subunits, dissociation and reassociation experiments were carried out under whereby Arenicola hexagonal bilayer haemoglobin was exposed to urea and alkaline pH and the effect was followed by gel filtration, SDS/PAGE, UV-visible spectrophotometry, electrospray-ionization MS, multiangle laser light scattering and transmission electron microscopy. The analysis of Arenicola haemoglobin dissociation indicates a novel and complex mechanism of dissociation compared with other annelid extracellular haemoglobins studied to date. Even though the chemically induced dissociation triggers partial degradation of some subunits, spontaneous reassociation was observed, to some extent. Parallel dissociation of Lumbricus haemoglobin under similar conditions shows striking differences that allow us to propose a hypothesis on the nature of the intersubunit contacts that are essential to form and to hold such a complex quaternary structure.