Tissue type plasminogen activator (tPA) is a key enzyme in the fibrinolytic cascade. In this paper we report that tPA contains 2 independent epitopes that exert opposite effects on blood vessel tone. Low concentrations of tPA (1 nM) inhibit the phenylephrine (PE)-induced contraction of isolated aorta rings. In contrast, higher concentrations (20 nM) stimulate the contractile effect of PE. The 2 putative vasoactive epitopes of tPA are regulated by the plasminogen activator inhibitor-1 (PAI-1) and by a PAI-1-derived hexapeptide that binds tPA. TNK-tPA, a tPA variant in which the PAI-1 docking site has been mutated, stimulates PE-induced vasoconstriction at all concentrations used. The stimulatory, but not the inhibitory, effect of tPA on the contraction of isolated aorta rings was abolished by anti-low-density lipoprotein receptor-related protein/␣ 2 -macroglobulin receptor (LRP) antibodies. Administering tPA or TNK-tPA to rats regulates blood pressure and cerebral vascular resistance in a dose-dependent mode. In other in vivo experiments we found that the vasopressor effect of PE is more pronounced in tPA knockout than in wildtype mice. Our findings draw attention to a novel role of tPA and PAI-1 in the regulation of blood vessel tone that may affect the course of ischemic diseases.

Key Points Closed head trauma sequentially releases tPA followed by uPA from injured brain. Increased uPA is responsible for delayed intracerebral hemorrhage, which is prevented by a tPA variant that inhibits uPA activity.

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an ϳ10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.

Angiogenesis, the growth of new blood vessels, is a complex biological process that is orchestrated by several growth factors and components of the extracellular matrix, including fibronectin (FN) and its receptor the integrin alpha5beta1. Angiogenesis is a critical part of inflammation and wound repair, but the mechanism by which vascular proliferation and migration is regulated by inflammatory cells is not completely understood. We have previously shown that human neutrophil peptides (HNPs), also known as alpha-defensins, which are secreted in high concentrations when neutrophils are activated, bind specifically to FN in the extracellular matrix and inhibit plasminogen activation. Therefore, we asked whether HNPs act as a link between inflammation and angiogenesis. Alpha5beta1-mediated endothelial cell adhesion and migration to FN, both under control conditions and under stimulation by vascular endothelial growth factor (VEGF), were inhibited specifically and in a dose-dependent manner by HNPs, whereas endothelial cell adhesion and migration to other components of the extracellular matrix, such as vitronectin, collagen, or fibrinogen/fibrin were not. Consistent with this finding, HNPs bound to and promoted the binding of fibronectin to alpha5beta1 integrin in arginine-glycine-aspartic acid (RGD)-independent manner. HNPs also completely inhibited VEGF-induced proliferation and induced apoptosis of endothelial cells in a dose-dependent manner. Moreover, HNPs inhibited capillary tube formation in three-dimensional fibrin-matrices as well as neovascularization in vivo in the chicken chorioallantoic membrane assay. Taken together, these data indicate that HNPs can regulate angiogenesis by affecting endothelial cell adhesion and migration in an FN-dependent manner as well as endothelial cell proliferation. These findings provide new insight into the role of inflammatory cells in angiogenesis and might provide a platform for developing a novel class of anti-angiogenesis drugs.

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes involving cell adhesion and migration in addition to fibrinolysis. In a previous study we found that two-chain urokinase plasminogen activator (tcuPA) stimulates phenylephrine-induced vasoconstriction of isolated rat aortic rings. In the present paper we report that uPA ؊/؊ mice have a significantly lower mean arterial blood pressure than do wild type mice and that aortic rings from uPA ؊/؊ mice show an attenuated contractile response to phenylephrine. In contrast, the blood pressure of urokinase receptor knockout (uPAR ؊/؊ ) mice and the response of their isolated aortic rings to phenylephrine were normal, indicating that the effect of uPA on vascular contraction is independent of uPAR. Addition of mouse and human uPA almost completely reversed both the impaired vascular contractility and the lower arterial blood pressure in vivo. The in vitro and in vivo effects of infused uPA on aortic contractility and the restoration of normal blood pressure in uPA ؊/؊ mice were prevented by antibody to low-density lipoprotein receptor-related protein/␣ 2 -macroglobulin receptor (LRP). A modified form of uPA that lacks the kringle failed to restore the blood pressure in uPA ؊/؊ mice, notwithstanding having a longer half-life in the circulation. Ligands that regulate the interaction of uPA with LRP, such as PAI-1 or the PAI-1-derived peptide (EEIIMD), abolished the vasoactivity of tcuPA in vitro and in vivo. These studies identify a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility.Urokinase plasminogen activator (uPA) 1 is a multifunctional protein that has been implicated in several physiological and pathological processes, including fibrinolysis. Transgenic mice with a targeted disruption in the uPA gene (uPA Ϫ/Ϫ ) are prone to form thrombi when exposed to endotoxin (1) or hypoxia (2, 3) or when the uPA gene is disrupted in otherwise healthy tPA Ϫ/Ϫ mice (1, 4). We recently reported that clearance of pulmonary microemboli in uPA Ϫ/Ϫ mice is delayed, despite the presence of an intact tPA system (5).uPA has also been implicated in other pathophysiological processes, such as pulmonary inflammation and repair, in which the relationship to fibrinolysis is less clear. For example, uPA Ϫ/Ϫ mice are more susceptible to lethal pulmonary infection (6) and to the development of pulmonary fibrosis (7), endpoints that might reflect contribution of uPA in cell adhesion (8) and migration to these phenotypes. However, the mechanism by which uPA is involved in these processes has not been established.In a previous study we found that uPA enhances phenylephrine and endothelin-induced vasoconstriction of aortic rings isolated from rats (9). The possibility that uPA contributes to the regulation of vascular tone may help to explain some of the phenotypic changes described in uPA Ϫ/Ϫ mice and perhaps provide a broader understanding of the role pl...

Rationale: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. a-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species. Objectives: To study the role of a-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of a-defensins. Methods: Transgenic mice expressing polymorphonuclear leukocyte a-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoproteinrelated receptor (LRP) in permeability was examined. Measurements and Main Results: Acid aspiration induced neutrophil migration and release of a-defensins into lung parenchyma and airways. ALI was more severe in a-defensin-expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, a-defensinexpressing mice showed greater disruption of capillary-epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP. Conclusions: a-Defensins mediate ALI through LRP-mediated loss of capillary-epithelial barrier function, suggesting a potential new approach to intervention.

We have previously identified ␣-defensin in association with medial smooth muscle cells (SMCs) in human coronary arteries. In the present paper we report that ␣-defensin, at concentrations below those found in pathological conditions, inhibits phenylephrine (

The clinical use of tissue-type plasminogen activator (tPA) in the treatment of stroke is profoundly constrained by its serious side effects. We report that the deleterious effects of tPA on cerebral edema and intracranial bleeding are separable from its fibrinolytic activity and can be neutralized. A hexapeptide (EEIIMD) corresponding to amino acids 350-355 of plasminogen activator inhibitor type 1 (PAI-1) abolished the tPA-induced increase in infarct size and intracranial bleeding in both mechanical and embolic models of stroke in rats, and reduced brain edema and neuronal loss after traumatic brain injury in pigs. These experiments suggest mechanisms to reduce the neurotoxic effects of tPA without compromising its fibrinolytic activity, through the use of selective antagonists and new tPA formulations.