Biomedical subjects

Sandip M Kanse

Publications and source records attributed to Sandip M Kanse.

11 recordsLinked to original sources

PubMed2006

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.

Actins↗

PubMed2006

A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding.

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.

Animals↗

PubMed2005

Characterisation and partial purification of Schistosoma mansoni egg-derived pro-angiogenic factor.

Pathogenesis of Schistosoma mansoni infection is due to the accumulation of eggs in the liver and intestine of the host followed by granuloma formation and fibrosis at the site of egg embolism. Nevertheless, total hepatic blood flow and hepatic function appear to be maintained by neovascularisation of the periportal fibrotic tissue. Here we demonstrate that intact live eggs, excretory/secretory products of eggs and the extracts of homogenised eggs stimulate the proliferation and migration of endothelial cells. Formation of endothelial capillary-like outgrowths, as well as, the activation of p42/p44 mitogen-activated protein kinase signal transduction pathway was stimulated by egg extracts, whereas other stages of the schistosome life-cycle did not exhibit such activity. We have characterised and partially purified the pro-angiogenic factor. The active pro-angiogenic component was fast-acting, heat-stable, protease-resistant, weakly heparin-binding and a non-lipid. It could be extracted with acidified ethanol or a hot acetic acid solution and could be partially purified by reverse-phase HPLC. The S. mansoni egg-derived pro-angiogenic factor, described here, may directly contribute to vascular remodelling in the liver and illustrates how helminth parasites may modulate angiogenesis.

Acetic Acid↗

PubMed2004

Promotion of leukocyte adhesion by a novel interaction between vitronectin and the beta2 integrin Mac-1 (alphaMbeta2, CD11b/CD18).

OBJECTIVE: The leukocyte integrin Mac-1 (alphaMbeta2, CD11b/CD18) binds a number of ligands and counter-receptors and thereby is a major determinant in regulation of leukocyte adhesion and extravasation. Vitronectin (VN) is an adhesion-promoting factor that is abundantly present as matrix molecule in vascular diseases such as atherosclerosis. Until now, only an indirect interaction between Mac-1 and VN via the urokinase receptor (urokinase plasminogen activator receptor) was known. We now propose that Mac-1 and VN can directly interact with each other. METHODS AND RESULTS: In an in vitro system with purified components, Mac-1 specifically bound the multimeric matrix form of VN but not the monomeric plasma form. Using various competitors, the interaction domains in Mac-1 and VN were localized. Mac-1-expressing but not untransfected Chinese hamster ovary cells adhered strongly on VN. Introduction of a GFFKR deletion in the alphaM subunit of Mac-1, which increases the constitutive activation of the integrin, led to increased adhesion on VN. Peripheral human blood neutrophils adhered and migrated on multimeric VN in a Mac-1-dependent manner. CONCLUSIONS: These results show that there is a specific integrin-affinity-regulated interaction between Mac-1 and the matrix form but not the plasma form of VN that may significantly participate in leukocyte adhesion and extravasation.

Animals↗

PubMed2004

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.

Arteriosclerosis↗

PubMed2003

Reciprocal regulation of urokinase receptor (CD87)-mediated cell adhesion by plasminogen activator inhibitor-1 and protease nexin-1.

Protease nexin-1 (PN-1) and plasminogen activator inhibitor-1 (PAI-1) are serine protease inhibitors that bind to the extracellular matrix protein vitronectin (VN) with high affinity. PAI-1 is known to inhibit cell adhesion and migration by binding to VN and inhibiting the interaction with integrins or the urokinase receptor (uPAR). Unexpectedly, PN-1 was found to increase the association between VN and uPAR in the presence of enzymatically active uPA. Through this mechanism PN-1 also stimulated uPAR-dependent cell adhesion to immobilized VN. In contrast to PAI-1, PN-1 did not influence VN binding to integrins or integrin-mediated cell adhesion. Upon adhesion of monocytes to VN there was an accumulation of uPAR and PN-1 at the interface between the cell and the matrix, whereas on fibronectin (FN) both components were distributed evenly over the whole cell as visualized by confocal microscopy. Immunohistochemistry of atherosclerotic vessels indicated that PN-1 was found associated with smooth muscle cells, macrophages and platelets. In some regions of the diseased vessels PN-1 was in close proximity to VN and uPAR, but no PN-1 was present in normal vessels. These results indicate a novel function of PN-1 linked to complex formation with uPA that leads to the regulation of VN-dependent adhesion of leukocytes.

Amyloid beta-Protein Precursor↗

PubMed2003

New aspects of integrin-mediated leukocyte adhesion in inflammation: regulation by haemostatic factors and bacterial products.

Leukocyte recruitment to sites of inflammation, infection or vascular injury is a complex event, depending on a tightly coordinated sequence of leukocyte-endothelial- and leukocyte-platelet interactions, which are controlled by the expression and activation of various adhesion receptors and protease systems. The present review will focus on novel aspects of the regulation of integrin-dependent leukocyte adhesion by haemostatic factors and bacterial products. In particular, after a short overview of leukocyte recruitment, the review (i) will focus on the crosstalk between haemostatic factors and adhesion molecules with respect to leukocyte extravasation based on the paradigms of the urokinase receptor and high molecular weight kininogen, (ii) will provide information on novel mechanisms for the regulation of leukocyte recruitment by bacterial proteins, on the basis of the anti-inflammatory role of Staphylococcus aureus extracellular adhesive protein and (iii) will draw attention to the junctional adhesion molecules, a novel family of adhesive receptors that are counter-receptors for leukocyte integrins and mediate vascular cell interactions. The better understanding of the interactions between vascular cells and particularly of integrin-dependent leukocyte adhesion may lead to the development of novel therapeutical concepts in inflammatory vascular disorders.

Animals↗

PubMed2002

Urokinase receptor surface expression regulates monocyte adhesion in acute myocardial infarction.

The urokinase receptor (urokinase plasminogen activator receptor; uPAR) regulates monocyte adhesion by direct binding to vitronectin and by forming complexes with integrins. Therefore, possible up-regulation of uPAR in acute myocardial infarction (AMI) may affect monocyte adhesion. In 20 patients with AMI, uPAR surface expression (measured by flow cytometry) was increased compared with that in patients with chronic stable angina (mean +/- SD fluorescence, 179 +/- 96 vs 80 +/- 53; P =.002). Expression of uPAR correlated with activation of beta(2)-integrins lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen 1 (Mac-1), measured by using monoclonal antibodies (mAbs) 24 and CBRM1/5. Isolated mononuclear cells (MNCs) from patients with AMI showed enhanced adhesiveness to human umbilical vein endothelial cells (HUVECs), to fibrinogen (Mac-1 ligand), and to vitronectin (uPAR ligand). Excessive adhesion of MNCs to HUVECs was inhibited by mAbs anti-CD18 (84%), anti-CD11a (51%), and anti-CD11b (57%), indicating a major contribution of LFA-1 and Mac-1. The mAb anti-uPAR R3 blocked adhesion of cells from patients with AMI to vitronectin (95%) but also beta(2)-integrin-mediated adhesion to fibrinogen (79%) and HUVECs (66%). Incubation of monocytic MonoMac6 cells with plasma from patients with AMI enhanced uPAR messenger RNA expression and cell adhesion to HUVECs. Thus, released soluble factors may contribute to enhanced monocyte adhesion in AMI. Mouse pre-B lymphocytes (BAF3 cells) transfected with various amounts of uPAR complementary DNA showed a strong correlation of uPAR expression with beta(2)-integrin-dependent adhesion to intercellular adhesion molecule 1, thus providing evidence for the functional relevance of uPAR up-regulation in an isolated in vitro system. In conclusion, we found that uPAR expression is elevated on monocytes in AMI and contributes to enhanced cell adhesion. Thus, uPAR may be a novel target for prevention of unwanted monocyte recruitment as part of inflammatory cardiovascular processes.

Adult↗

PubMed2002

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 beta(2)-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 beta(2)-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.

Animals↗

PubMed2002

Molecular interactions and functional interference between vitronectin and transforming growth factor-beta.

Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their storage or presentation towards cellular receptors. The multifunctional adhesive glycoprotein vitronectin (VN), which is found in the circulation and widely distributed throughout different tissues, has been implicated in the regulation of vascular cell functions, and these activities could be related to interactions with various growth factors. In vitro, soluble VN interfered with transforming growth factor-beta (TGF-beta) binding to isolated extracellular matrix and was found to associate with TGF-beta1 and TGF-beta2 as well as with other growth factors such as vascular endothelial growth factor, epidermal growth factor, or basic fibroblast growth factor in a saturable manner. In particular, binding of TGF-beta was maximal for the heparin-binding multimeric isoform of VN, whereas VN in a ternary complex with thrombin and antithrombin or plasma VN exhibited weaker binding. Plasminogen activator inhibitor-1 (PAI-1) or heparin, but not desulfated glycosaminoglycans, interfered with binding of VN to TGF-beta, and soluble PAI-1 was able to dissociate VN-bound TGF-beta. Upon limited plasmin proteolysis of VN, only the fragments comprising the intact aminoterminal portion of VN bound to TGF-beta as did a synthetic peptide (amino acids 43 to 62), indicating that TGF-beta and PAI-1 share common binding site(s) on VN. Although VN did not influence TGF-beta bioactivity for mink lung epithelial cells, TGF-beta dose dependently inhibited both urokinase-receptor as well as alpha(v)-integrin-dependent adhesion to VN. This activity of TGF-beta was reminiscent of the antiadhesive function of PAI-1. In atherosclerotic tissue sections, staining patterns of VN and TGF-beta indicated their colocalization. These findings describe VN as a new binding protein for TGF-beta, whereby specific functions of both factors become modulated by this interaction.

Animals↗

PubMedDate not supplied

Variability in the expression of urokinase receptor (CD87) mutants on cells: relevance to cell adhesion.

The urokinase-type plasminogen activator receptor (uPAR, CD87) is a glycosylphosphatidylinositol (GPI)-anchored protein, containing three homologous Ly-6 domains, that mediates integrin-independent cell adhesion by directly binding to extracellular matrix protein vitronectin (VN). To elucidate the structural requirements for the uPAR-dependent cell adhesion on VN, several glycolipid-anchored variants of uPAR were expressed in BAF3 cells, (mouse pre B-lymphocytes) followed by functional analysis. The individual domains of uPAR were expressed at very low levels, the two domain mutants were expressed to a higher level and the wild type uPAR was expressed highly. Point mutations in domain 2 of uPAR have been shown to diminish cellular binding of the ligand urokinase and we observed a lack of VN binding to this mutant. Flow cytometry with a number of monoclonal antibodies indicated that the domain-specific antigenic determinants in these mutants were well preserved. Only the cells expressing the intact uPAR with all three domains adhered strongly to a VN substrate, whereas none of the other transfected cells showed significant cell adhesion. Hence, any alterations in the domain structure of uPAR reduce its expression and only the intact receptor can sustain the direct cell adhesion on VN-rich matrices found at sites of inflammation and injury.

Amino Acid Substitution↗