Recovery of Prostacyclin Production by De-endothelialized Rabbit Aorta CRITICAL ROLE OF NEOINTIMAL SMOOTH MUSCLE CELLS

A B S T R A C T Prostacyclin (PGI2) synthetic capacity was assayed at the surface of aortas at various intervals after removal of endothelium with a balloon catheter. Results were correlated with morphologic changes in the vessel wall seen by light microscopy, scanning and transmission electron microscopy. To assay PGI2 synthetic capacity, we applied an incubation chamber to the luminal surface of the aortas; after arachidonic acid stimulation we assayed the PGI2 synthesized with a bioassay and radioimmunoassay. PGI2 synthesis in deendothelialized aortas was determined immediately after balloon-catheter injury and at intervals of 1 h and 2, 4, 15, 35, and 70 d. PGI2 synthesis was low at 1 h and increased over time with levels at 35 and 70 d reaching that of normal artery. Scanning and transmission electron microscopy of de-endothelialized areas showed persistent absence of endothelium with formation of a neointima composed of smooth muscle cells. De-endothelialized aorta was covered with adherent platelets shortly after injury, however several days later only a few platelets adhered to the denuded surface. Results indicated that (a) endothelium is responsible for nearly all PGI2 production at the luminal surface of the normal aorta, (b) de-endothelialized muscular neointima synthesized increasing quantities of PGI2 with time after injury, and (c) increase of PGI

The Annexin A2/S100A10 System in Health and Disease: Emerging Paradigms

Since its discovery as a src kinase substrate more than three decades ago, appreciation for the physiologic functions of annexin A2 and its associated proteins has increased dramatically. With its binding partner S100A10 (p11), A2 forms a cell surface complex that regulates generation of the primary fibrinolytic protease, plasmin, and is dynamically regulated in settings of hemostasis and thrombosis. In addition, the complex is transcriptionally upregulated in hypoxia and promotes pathologic neoangiogenesis in the tissues such as the retina. Dysregulation of both A2 and p11 has been reported in examples of rodent and human cancer. Intracellularly, A2 plays a critical role in endosomal repair in postarthroplastic osteolysis, and intracellular p11 regulates serotonin receptor activity in psychiatric mood disorders. In human studies, the A2 system contributes to the coagulopathy of acute promyelocytic leukemia, and is a target of high-titer autoantibodies in patients with antiphospholipid syndrome, cerebral thrombosis, and possibly preeclampsia. Polymorphisms in the human ANXA2 gene have been associated with stroke and avascular osteonecrosis of bone, two severe complications of sickle cell disease. Together, these new findings suggest that manipulation of the annexin A2/S100A10 system may offer promising new avenues for treatment of a spectrum of human disorders

Transforming growth factor‐β1 is a heparin‐binding protein: Identification of putative heparin‐binding regions and isolation of heparins with varying affinity for TGF‐β1

Previous studies indicated that a major factor in heparin\u27s ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor‐β1 (TGF‐β1). Heparin appeared to bind directly to TGF‐β1 and to prevent the association of TGF‐β1 with α2‐macroglobulin (α2‐M). The present studies indicate that 20–70% of iodinated TGF‐β1 binds to heparin‐Sepharose and the retained fraction is eluted with ∼0.37 M NaCI. Native, unlabelled platelet TGF‐β1, however, is completely retained by heparin‐Sepharose and eluted with 0.9–1.2 M NaCI. Using synthetic peptides, the regions of TGF‐β1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF‐β1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin‐binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF‐β1, were found to bind to heparin‐Sepharose and were eluted with 0.15 M‐0.30 M NaCI. Only two of these regions were capable of blocking the binding of heparin to 125I‐TGF‐β. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF‐β1. The ability of TGF‐β1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF‐β1 may be a factor in some of the diverse biological actions of heparin. © 1992 Wiley‐Liss, Inc. Copyright © 1992 Wiley‐Liss, Inc

Transforming growth factor‐β1 stimulates macrophage urokinase expression and release of matrix‐bound basic fibroblast growth factor

Macrophage expression of urokinase‐type plasminogen activator (uPA) appears to play a role in their release of matrix‐bound basic fibroblast growth factor (bFGF) and transforming growth factor‐β (TGF‐β). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF‐β1 on macrophage uPA expression. TGF‐β1 stimulated in a dose‐ and time‐dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin‐1α, tumor necrosis factor‐α, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF‐β1 led to a rapid and sustained increase in the steady‐state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF‐β1‐induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitior H7 markedly reduced the ability of TGF‐β1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF‐β1 to upregulate macrophage uPA expression. TGF‐β1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane‐bound plasmin than control cells. Preincubation of TGF‐β1 with either serum or methylamine‐modified α2‐macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF‐β1‐primed macrophages were cultured on matrices containing bound125I‐bFGF, their release of 125I‐bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF‐β to induce macrophage uPA expression and the plasmin‐dependent release of matrix‐bound bFGF may provide an indirect mechanism by which TGF‐β stimulates angiogenesis. © 1993 Wiley‐Liss, Inc. Copyright © 1993 Wiley‐Liss, Inc

THP‐1 macrophage membrane‐bound plasmin activity is up‐regulated by transforming growth factor‐β1 via increased expression of urokinase and the urokinase receptor

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor‐b̃1 (TGF‐b̃1) on uPA, uPA receptor, and plasminogen receptor expression by human THP‐1 macrophage was examined. TGF‐b̃1 induction of uPA expression by THP‐1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF‐b̃1 led to a dose‐ and time‐dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF‐b̃1. The differential response exhibited by suspension and adherent THP‐1 cells may reflect differences in their expression of TGF‐b̃1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF‐b̃1‐induced alterations in uPA receptor expression by adherent THP‐1 cells were examined by quantitating membrane‐bound uPA activity. Membrane‐bound uPA activity increased three‐fold when cells were incubated with TGF‐b̃1. The increase in membrane‐uPA activity expressed by TGF‐b̃1‐treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF‐b̃1 primed cells with exogenous uPA did not lead to an increase in membrane‐bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF‐b̃1‐treated cells. Following incubation with plasminogen, membrane‐bound plasmin activity increased three‐fold in TGF‐b̃1‐treated cells. However, no change in immunoreactive membrane‐bound plasmin(ogen) was observed. In addition, binding of 125I‐Lys‐plasminogen to THP‐1 cells was not affected by TGF‐b̃1 treatment. We conclude that TGF‐b̃1 stimulates membrane‐bound plasmin activity, without affecting plasminogen receptor expression, through the up‐regulation of uPA and the uPA receptor expression. © 1995 Wiley‐Liss, Inc. Copyright © 1995 Wiley‐Liss, Inc

Exposure of cryptic domains in the α1-chain of laminin-1 by elastase stimulates macrophages urokinase and matrix metalloproteinase-9 expression

Degradation of the extracellular matrix leads to the release of fragments, which elicit biological responses distinct from intact molecules. We have reported that α1: Ser2091-Arg2108 a peptide derived from the al-chain of laminin-1, triggers protein kinase C-dependent activation of MAPKerk1/2, leading to the up-regulation of macrophage urokinase type plasminogen activator and matrix metalloproteinase (MMP)-9 expression. Since intact laminin-1 failed to trigger these events, we hypothesized that α1: Ser2091-Arg2108 is cryptic or assumes a conformation not recognized by macrophages. Here we demonstrate that elastase cleavage of laminin-1 generates fragments, which stimulate proteinase expression by RAW264.7 macrophages and peritoneal macrophages. In contrast, fragments generated by MMP-2, MMP-7, or plasmin had no effect on macrophage proteinase expression. Elastase-generated laminin-1 fragments were fractionated by heparin-Sepharose chromatography. Heparin-binding fragments stimulated macrophages\u27 proteinase expression severalfold greater than nonbinding fragments. The heparin binding fragments reacted with antibodies directed against regions of the α1-chain including α1: Ser2091-Arg2108 and the globular domain. A peptide from the first loop of the globular domain (α1: Ser2179-Ser2198) triggered the phosphorylation of MAPKerk1/2 and stimulated the expression of macrophage urokinase type plasminogen activator and MMP-9. Moreover, a heparin-binding fraction isolated from an aortic aneurysm contained fragments of α1-chain and stimulated macrophages\u27 proteinase expression. Based on these data, we conclude that cryptic domains in the COOH-terminal portion of the α1-chain of laminin are exposed by proteolysis and stimulate macrophages\u27 proteinase expression

Fucoidan is a non-anticoagulant inhibitor of intimal hyperplasia

We previously reported that heparin inhibits the proliferation of fibroblasts and vascular smooth muscle cells (SMC), in part, by binding to and increasing the antiproliferative activity of transforming growth factor-β1 (TGF-β1). We now report that certain other polyanions which are structurally distinct from heparin, such as fucoidan and polyinosinic acid, are more avid ligands for TGF-β1 and more potent antiproliferative agents than heparin. Fucoidan possessed more potent antiproliferative activity than heparin against rat and bovine aortic SMC in vitro, though possessing much lower anticoagulant activity than heparin. Furthermore, fucoidan suppressed in vivo intimal hyperplasia when continuously infused into rats subjected to balloon-catheter injury. Unlike heparin, which also suppressed intimal hyperplasia, fucoidan did not cause systemic anticoagulation. Thus, fucoidan may be useful as a non-anticoagulant inhibitor of post-angioplasty intimal hyperplasia. © 1992