Sandip M. Kanse

Urokinase receptor: a molecular organizer in cellular communication.

In a variety of cell types, the glycolipid-anchored urokinase receptor (uPAR) is colocalized pericellularly with components of the plasminogen activation system and endocytosis receptors. uPAR is also coexpressed with caveolin and members of the integrin adhesion receptor superfamily. The formation of functional units with these various proteins allows the uPAR to mediate the focused proteolysis required for cell migration and invasion and to contribute both directly and indirectly to cell adhesive processes in a non-proteolytic fashion. This dual activity, together with the initiation of signal transduction pathways by uPAR, is believed to influence cellular behaviour in angiogenesis, inflammation, wound repair and tumor progression/metastasis and open up the way for uPAR-based therapeutic approaches.

Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes

Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted β2-integrin and urokinase receptor–mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2–3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented β2-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.

Isolation and characterization of the circulating form of human endostatin.

Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10–20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C‐terminal fragment of collagen α1(XVIII) was guided by MALDI‐MS and the exact molecular mass determined by ESI‐MS was found to be 18 494 Da. N‐terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1–3 and 2–4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no anti‐proliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.

Induction of Vascular SMC Proliferation by Urokinase Indicates a Novel Mechanism of Action in Vasoproliferative Disorders

The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor-independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.

The G534E polymorphism of the gene encoding the factor VII–activating protease is associated with cardiovascular risk due to increased neointima formation

The G534E polymorphism (Marburg I [MI]) of factor VII–activating protease (FSAP) is associated with carotid stenosis and cardiovascular disease. We have previously demonstrated that FSAP is present in atherosclerotic plaques and it is a potent inhibitor of vascular smooth muscle proliferation and migration in vitro. The effect of wild-type (WT)- and MI-FSAP on neointima formation in the mouse femoral artery after wire-induced injury was investigated. Local application of WT-FSAP led to a 70% reduction in the neointima formation, and this effect was dependent on the protease activity of FSAP. MI-FSAP did not inhibit neointima formation in vivo. This is due to a reduced proteolytic activity of MI-FSAP, compared to WT-FSAP, toward platelet-derived growth factor BB, a key mediator of neointima development. The inability of MI-FSAP to inhibit vascular smooth muscle accumulation explains the observed linkage between the MI-polymorphism and increased cardiovascular risk. Hence, FSAP has a protective function in the vasculature, and analysis of MI polymorphism is likely to be clinically relevant in restenosis.

Enzymatically Modified, Nonoxidized LDL Induces Selective Adhesion and Transmigration of Monocytes and T-Lymphocytes Through Human Endothelial Cell Monolayers

Circulating monocytes and T lymphocytes extravasate through the endothelium at sites of developing atheromatous lesions, where they tend to accumulate and mediate the progression of the disease. We have previously demonstrated the presence of an enzymatically degraded, nonoxidized form of LDL (E-LDL) in early human fatty streaks, which possesses major biological properties of an atherogenic lipoprotein. The effects of E-LDL on human endothelial cells have now been studied with respect to adhesion and transmigration of monocytes and T lymphocytes. E-LDL induced a rapid and dose-dependent selective adhesion of monocytes and T lymphocytes to endothelial cell monolayers within 30 minutes of incubation. Maximal increases in the number of adherent monocytes (8-fold) and of adherent T lymphocytes (4-fold) were observed after treatment with 50 microg/mL E-LDL. E-LDL was more active than oxidized LDL (ox-LDL), whereas native LDL produced only minor adhesive effects. Both E-LDL and ox-LDL enhanced transmigration of monocytes and of T lymphocytes through endothelial monolayers. Again, E-LDL was more potent than ox-LDL, inducing transmigration to a similar extent as N-formyl-Met-Leu-Phe. In endothelial cells, E-LDL stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1), platelet-endothelial cells adhesion molecule-1 (PECAM-1), P-selectin, and E-selectin with distinct kinetics. Analyses with blocking antibodies indicated that ICAM-1 and P-selectin together mediated approximately 70% of cell adhesion, whereas blocking of PECAM-1 had no effect on adhesion but reduced transmigration to less than 50% of controls. E-LDL also upregulated expression of ICAM-1 in human aortic smooth muscle cells, and this correlated with increased adhesion of T lymphocytes. E-LDL is thus able to promote the selective adhesion of monocytes and T lymphocytes to the endothelium, stimulate transmigration of these cells, and foster their retention in the vessel wall by increasing their adherence to smooth muscle cells. These findings underline the potential significance of E-LDL in the pathogenesis of atherosclerosis.

Factor VII-activating protease (FSAP): Vascular functions and role in atherosclerosis

FSAP is a plasma serine protease for which a potential role in the regulation of coagulation and fibrinolysis is postulated, based on its property to activate factor VII (FVII) as well as pro-urokinase (uPA). In clinical studies, the G534E single nucleotide polymorphism (Marburg I) of FSAP has been linked to late complications of atherothrombosis and is associated with a low proteolytic activity, particularly, towards pro-uPA. This has stimulated much interest in a search for additional functions of FSAP in the cardiovascular system. FSAP is a potent inhibitor of vascular smooth muscle cell proliferation and migration in vitro and local application of FSAP (but not Marburg I variant) in animal models reduces neointima formation. This is due to a reduced proteolytic activity of the variant isoform towards platelet derived growth factor-BB, a key mediator of neointima development. Moreover, appreciable quantities of FSAP are localized to unstable atherosclerotic plaques and may contribute to plaque instability. These data indicate that the cellular regulatory effects of FSAP may be more important than its influence on haemostasis. In this review the contribution of FSAP to vascular fibroproliferative inflammatory diseases in the context of pericellular proteolysis of the extracellular matrix, growth factor activity and haemostasis will be highlighted.

Factor VII-activating protease (FSAP) inhibits growth factor-mediated cell proliferation and migration of vascular smooth muscle cells

The factor VII activating protease (FSAP) is a serine‐protease present in human plasma that serves to activate single‐chain plasminogen activators, as well as coagulation factor VII. FSAP was localized within atherosclerotic lesions, and a genetic polymorphism in FSAP is associated with carotid stenosis. Hence, this study was conducted to gain broader insights into the cellular effects of FSAP on vascular smooth muscle cells (VSMC). DNA synthesis and cell proliferation assays revealed an inhibitory action of FSAP on platelet‐derived growth factor BB (PDGF‐BB)‐mediated proliferation of VSMC. FSAP also inhibited PDGF‐BB‐induced migration of VSMC. These cellular effects of FSAP could be neutralized by an anti‐FSAP mAb as well as by protease inhibitors such as aprotinin or a chloromethylketone inhibitor. Moreover, unfractionated heparin promoted the antiproliferative effect of FSAP on VSMC and was essential for the inhibition of VSMC migration. FSAP inhibited PDGF‐BB binding to human VSMC and concomitantly blocked PDGF‐BB‐dependent phosphorylation of mitogen activated protein kinase p42/p44 and tyrosine phosphorylation of other proteins. These results unravel a new function of FSAP as an inhibitor of the proatherogenic phenotype of vascular smooth muscle.

Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen

Proteolytic cleavage of single‐chain, high molecular weight kininogen (HK) by kallikrein releases the short‐lived vasodilator bradykinin and leaves behind a two‐chain, high molecular weight kininogen (HKa) reported to bind to the β2‐integrin Mac‐1 (CR3, CD11b/CD18, αMβ2) on neutrophils and exert antiad‐hesive properties by binding to the urokinase receptor (uPAR) and vitronectin. We define the molecular mechanisms for the antiadhesive effects of HK related to disruption of β2‐integrin‐mediated cellular interactions in vitro and in vivo. In a purified system, HK and HKa inhibited the binding of soluble fibrinogen and ICAM‐1 to immobilized Mac‐1, but not the binding of ICAM‐1 to immobilized LFA‐1 (CD11a/CD18, αLβ2). This inhibitory effect could be attributed to HK domain 5 and to a lesser degree to HK domain 3, consistent with the requirement of both domains for binding to Mac‐1. Accordingly, HK, HKa, and domain 5 inhibited the adhesion of Mac‐1 but not LFA‐1‐transfected K562 human erythroleukemic cells to ICAM‐1. Moreover, adhesion of human monocytic cells to fibrinogen and to human endothelial cells was blocked by HK, HKa, and domain 5. By using peptides derived from HK domain 5, the sequences including amino acids H475‐G497 (and to a lesser extent, G440‐H455) were identified as responsible for the antiadhesive effect, which was independent of uPAR. Finally, administration of domain 5 into mice, followed by induction of thioglycol‐late‐provoked peritonitis, decreased the recruitment of neutrophils by ~70% in this model of acute inflammation. Taken together, HKa (and particularly domain 5) specifically interacts with Mac‐1 but not with LFA‐1, thereby blocking Mac‐1‐dependent leukocyte adhesion to fibrinogen and endothelial cells in vitro and in vivo and serving as a novel endogenous regulator of leuko‐cyte recruitment into the inflamed tissue.—Chavakis, T., Kanse, S. M., Pixley, R. A., May, A. E., Isordia‐Salas, I., Colman, R. W., Preissner, K. T. Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J. 15, 2365–2376 (2001)

Receptor-independent role of the urokinase-type plasminogen activator during arteriogenesis.

To define the role of the plasminogen activators (PAs) urokinase PA (uPA) and tissue PA (tPA) as well as the uPA receptor (uPAR) in arteriogenesis, we investigated their impact in a rabbit and mouse model of adaptive collateral artery growth. Collateral artery growth was induced by occlusion of the femoral artery in rabbit and wild‐type (WT) mice and in mice with targeted inactivation of uPA (uPA−/−), tPA (tPA−/−), or uPAR (uPAR−/−). Northern blot results revealed a significant up‐regulation of uPA but not uPAR or tPA in the early phase of arteriogenesis in rabbit and WT mice. This up‐regulation on RNA level was followed by an increased protein level and enzymatic activity. Impaired perfusion recovery upon femoral artery ligation was observed by laser Doppler analysis in vivo in uPA‐deficient mice but not in uPAR or tPA deficiency compared with WT mice. Immunohistochemical studies revealed an association of leukocyte infiltration with arteriogenesis in WT mice that was strongly reduced in uPA−/− but not in uPAR‐ or tPA‐deficient mice. We conclude that arteriogenesis is promoted by an uPA‐mediated infiltration of leukocytes that is not dependent on uPAR.