Plasminogen activator inhibitors regulate cell adhesion through a uPAR-dependent mechanism.

Binding of type-1 plasminogen activator inhibitor (PAI-1) to cell surface urokinase (uPA) promotes inactivation and internalization of adhesion receptors (e.g., urokinase receptor (uPAR), integrins) and leads to cell detachment from a variety of extracellular matrices. In this report, we begin to examine the mechanism of this process. We show that neither specific antibodies to uPA, nor active site inhibitors of uPA, can detach the cells. Thus, cell detachment is not simply the result of the binding of macromolecules to uPA and/or of the inactivation of uPA. We further demonstrate that another uPA inhibitor, protease nexin-1 (PN-1), also stimulates cell detachment in a uPA/uPAR-dependent manner. The binding of both inhibitors to uPA leads to the specific inactivation of the matrix-engaged integrins and the subsequent detachment of these integrins from the underlying extracellular matrix (ECM). This inhibitor-mediated inactivation of integrins requires direct interaction between uPAR and those integrins since cells attached to the ECM through integrins incapable of binding uPAR do not respond to the presence of either PAI-1 of PN-1. Although both inhibitors initiate the clearance of uPAR, only PAI-1 triggers the internalization of integrins. However, cell detachment by PAI-1 or PN-1 does not depend on the endocytosis of these integrins since cell detachment was also observed when clearance of these integrins was blocked. Thus, PAI-1 and PN-1 induce cell detachment through two slightly different mechanisms that affect integrin metabolism. These differences may be important for distinct cellular processes that require controlled changes in the subcellular localization of these receptors.

Contribution of leptin receptor N-linked glycans to leptin binding.

The extracellular domain of the human leptin receptor (Ob-R) contains 20 potential N-glycosylation sites whose role in leptin binding remains to be elucidated. We found that a mammalian cell-expressed sOb-R (soluble Ob-R) fragment (residues 22-839 of the extracellular domain) bound leptin with a dissociation constant of 1.8 nM. This binding was inhibited by Con A (concanavalin A) or wheatgerm agglutinin. Treatment of sOb-R with peptide N-glycosidase F reduced leptin binding by approximately 80% concurrently with N-linked glycan removal. The human megakaryoblastic cell line, MEG-01, expresses two forms of the Ob-R, of approx. 170 and 130 kDa molecular mass. Endo H (endoglycosidase H) treatment and cell culture with alpha-glucosidase inhibitors demonstrated that N-linked glycans are of the complex mature type in the 170 kDa form and of the high-mannose type in the 130 kDa form. Both isoforms bound leptin, but not after peptide N-glycosidase F treatment. An insect-cell-expressed sOb-R fragment, consisting of the Ig (immunoglobulin), CRH2 (second cytokine receptor homology) and FNIII (fibronectin type III) domains, bound leptin with affinity similar to that of the entire extracellular domain, but this function was abolished after N-linked glycan removal. The same treatment had no effect on the leptin-binding activity of the isolated CRH2 domain. Our findings show that N-linked glycans within Ig and/or FNIII domains regulate Ob-R function, but are not involved in essential interactions with the ligand.

Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgamma2 and A2.

Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of alpha(IIb)beta(3), as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cgamma2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A(2) axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity.

The reduced, denatured somatomedin B domain of vitronectin refolds into a stable, biologically active molecule.

The high-affinity binding site in human vitronectin (VN) for plasminogen activator inhibitor-1 (PAI-1) has been localized to the NH(2)-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44). A number of published structural and biochemical studies show conflicting results for the disulfide bonding pattern and the overall fold of the SMB domain, possibly because this domain may undergo disulfide shuffling and/or conformational changes during handling. Here we show that bacterially expressed recombinant SMB (rSMB) can be refolded to a single form that shows maximal activity in binding to PAI-1 and to a conformation-dependent monoclonal antibody (mAb 153). The oxidative refolding pathway of rSMB can be followed in the presence of glutathione redox buffers. This approach allowed the isolation and analysis of a number of intermediate folding species and of the final stably folded species at equilibrium. Competitive surface plasmon resonance analysis demonstrated that the stably refolded rSMB regained biological activity since it bound efficiently to PAI-1 and to mAb 153. In contrast, none of the folding intermediates bound to PAI-1 or to mAb 153. We also show by NMR analysis that the stably refolded rSMB is identical to the material used for the solution structure determination [Kamikubo et al. (2004) Biochemistry 43, 6519] and that it binds specifically to mAb 153 via an interface that includes the three aromatic side chains previously implicated in binding to PAI-1.

Molecular mechanisms of tumor necrosis factor-alpha-mediated plasminogen activator inhibitor-1 expression in adipocytes.

Increased expression of plasminogen activator inhibitor -1 (PAI-1) in adipose tissues is thought to contribute to both the cardiovascular and metabolic complications associated with obesity. Tumor necrosis factor alpha (TNF-alpha) is chronically elevated in adipose tissues of obese rodents and humans and has been directly implicated to induce PAI-1 in adipocytes. In this study, we used 3T3-L1 adipocytes to examine the mechanism by which TNF-alpha up-regulates PAI-1 in the adipocyte. Acute (3 h) and chronic (24 h) exposure of 3T3-L1 adipocytes to TNF-alpha induces PAI-1 mRNA by increasing the rate of transcription of the PAI-1 gene, and de novo protein synthesis is not required for this process. Although the p44/42 and PKC signaling pathways appear to be significant in the induction of PAI-1 mRNA in response to acute treatment with TNF-alpha, the more dramatic induction of PAI-1 mRNA observed in response to chronic exposure of adipocytes to TNF-alpha was mediated by these and additional signaling molecules, including p38, PI3-kinase, tyrosine kinases, and the transcription factor NF-kappaB. Moreover, the dramatic increase in PAI-1 observed after chronic exposure of adipocytes to TNF-alpha was accompanied by increased metabolic insulin resistance. Finally, we demonstrate that the PKC pathway is also central for PAI-1 induction in response to insulin and transforming growth factor-beta (TGF-beta), two additional molecules which are elevated in obesity and shown to directly induce PAI-1 in the adipocyte. The understanding of the mechanism of regulating PAI-1 expression in the adipocytes at the molecular level provides new insight to help identify novel targets in fighting the pathological complications of obesity.

Domain 2 of the urokinase receptor contains an integrin-interacting epitope with intrinsic signaling activity: generation of a new integrin inhibitor.

We investigated the interaction between the urokinase receptor (uPAR) and the integrin alphavbeta3. Vitronectin (VN) induces cell migration by binding to alphavbeta3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with alphavbeta3. In contrast, cells expressing a uPAR mutant lacking domain 2 do not migrate in response to VN. This effect is overcome by D2A, a synthetic peptide derived from the sequence of domain 2. In addition, D2A has chemotactic activity that requires alphavbeta3 and activates alphavbeta3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-alphavbeta3 and uPAR-alpha5beta1 co-immunoprecipitation, indicating that it can bind both of these integrins. We also identify the chemotactically active epitope harbored by peptide D2A. Mutating two glutamic acids into two alanines generates peptide D2A-Ala, which lacks chemotactic activity but inhibits VN-, FN-, and collagen-dependent cell migration. In fact, the GEEG peptide has potent chemotactic activity, and the GAAG sequence has inhibitory capacities. In summary, we have identified an integrin-interacting sequence located in domain 2 of uPAR, which is also a new chemotactic epitope that can activate alphavbeta3-dependent signaling pathways and stimulate cell migration. This sequence thus plays a pivotal role in the regulation of uPAR-integrin interactions. Moreover, we describe a novel, very potent inhibitor of integrin-dependent cell migration.

Historical analysis of PAI-1 from its discovery to its potential role in cell motility and disease.

Although plasminogen activator inhibitor 1 (PAI-1) is one of the primary regulators of the fibrinolytic system, it also has dramatic effects on cell adhesion, detachment and migration. PAI-1 also differs from other serine protease inhibitors (serpins) in that it is a trace protein in plasma, it has a short half-life in vivo, its synthesis is highly regulated, and it binds to the adhesive glycoprotein vitronectin (VN) with high affinity and specificity. These unique and diverse properties of PAI-1 probably account for the many observations in the literature that correlate abnormalities in PAI-1 gene expression with a variety of pathological conditions. In this review, we discuss the discovery, origin, properties and regulation of PAI-1, and then speculate about its potential role in vascular disease, fibrosis, obesity and the metabolic syndrome, and cancer.

Unexpected role of plasminogen activator inhibitor 1 in cell adhesion and detachment.

Plasminogen activator inhibitor 1 (PAI-1) is the primary physiological inhibitor of plasminogen activation in vivo, and thus it is one of the main regulators of the fibrinolytic system. In this regard, individuals with elevated PAI-1 seem to have an increased risk for thrombotic disease, whereas those lacking the inhibitor develop a lifelong bleeding diathesis. Unexpectedly, recent observations demonstrate that cancer patients with high PAI-1 levels have a poor prognosis for survival. This correlation with metastatic disease may be related to the observation that high PAI-1 levels decrease the adhesive strength of cells for their substratum, and that this de-adhesive activity of PAI-1 is not related to its role as a protease inhibitor. Initial insights into potential mechanisms by which PAI-1 regulates the attachment, detachment, and migration of cells are addressed in this review.

Inhibition of endogenous leptin protects mice from arterial and venous thrombosis.

OBJECTIVE: Human obesity is associated with an increased risk for arterial and venous thrombosis and with elevated levels of leptin in the blood. Leptin administration promotes arterial thrombosis in mice, and leptin-deficient ob/ob mice have an attenuated thrombotic response to injury. Thus, endogenous leptin may regulate arterial and venous thrombosis in vivo. Experiments were performed to test this hypothesis. METHODS AND RESULTS: A leptin-neutralizing antibody was administered intravenously into wild-type mice 15 minutes before carotid artery injury with ferric chloride. The antibody-treated mice demonstrated prolonged times to thrombotic occlusion and formed unstable, embolizing thrombi. Thus, inhibiting leptin converted the thrombotic phenotype of wild-type mice into one that closely resembled that of ob/ob mice. The effect of leptin inhibition on venous thrombosis and pulmonary embolism was also investigated. Injection of a mixture of collagen and epinephrine into the jugular vein induced fatal pulmonary embolism in >90% of the control wild-type mice but in <40% of their antibody-treated counterparts. Histological analysis revealed that the antibody significantly reduced the number of occlusive thrombi in the pulmonary vessels. CONCLUSIONS: Inhibition of circulating leptin protects against arterial and venous thrombosis in mice and possibly in hyperleptinemic obese individuals.

Disulfide bonding arrangements in active forms of the somatomedin B domain of human vitronectin.

The N-terminal cysteine-rich somatomedin B (SMB) domain (residues 1-44) of the human glycoprotein vitronectin contains the high-affinity binding sites for plasminogen activator inhibitor-1 (PAI-1) and the urokinase receptor (uPAR). We previously showed that the eight cysteine residues of recombinant SMB (rSMB) are organized into four disulfide bonds in a linear uncrossed pattern (Cys(5)-Cys(9), Cys(19)-Cys(21), Cys(25)-Cys(31), and Cys(32)-Cys(39)). In the present study, we use an alternative method to show that this disulfide bond arrangement remains a major preferred one in solution, and we determine the solution structure of the domain using NMR analysis. The solution structure shows that the four disulfide bonds are tightly packed in the center of the domain, replacing the traditional hydrophobic core expected for a globular protein. The few noncysteine hydrophobic side chains form a cluster on the outside of the domain, providing a distinctive binding surface for the physiological partners PAI-1 and uPAR. The hydrophobic surface consists mainly of side chains from the loop formed by the Cys(25)-Cys(31) disulfide bond, and is surrounded by conserved acidic and basic side chains, which are likely to contribute to the specificity of the intermolecular interactions of this domain. Interestingly, the overall fold of the molecule is compatible with several arrangements of the disulfide bonds. A number of different disulfide bond arrangements were able to satisfy the NMR restraints, and an extensive series of conformational energy calculations performed in explicit solvent confirmed that several disulfide bond arrangements have comparable stabilization energies. An experimental demonstration of the presence of alternative disulfide conformations in active rSMB is provided by the behavior of a mutant in which Asn(14) is replaced by Met. This mutant has the same PAI-1 binding activity as rVN1-51, but its fragmentation pattern following cyanogen bromide treatment is incompatible with the linear uncrossed disulfide arrangement. These results suggest that active forms of the SMB domain may have a number of allowed disulfide bond arrangements as long as the Cys(25)-Cys(31) disulfide bond is preserved.

Angiogenesis in an in vivo model of adipose tissue development.

Obesity is associated with an increased risk for cardiovascular disease and cancer. Angiogenesis is a critical component of these pathological processes, and expanding adipose tissue represents one of the few sites of active angiogenesis in the adult. Despite the potential importance of angiogenesis in obesity, little is known about underlying mechanisms. This problem is magnified by the absence of useful quantitative model systems. In this report, we examine the angiogenic process using the 3T3-F442A model of adipose tissue development. In this model, 3T3-F442A preadipocytes are implanted subcutaneously into athymic Balb/c nude mice. We show that these cells develop into highly vascularized fat pads over the next 14-21 days, and that these fat pads are morphologically similar to normal subcutaneous adipose tissue. Histological studies demonstrate that a new microvasculature is evident as early as 5 days after cell implantation, and real-time quantitative RT-PCR analyses show that the expression of endothelial cell markers and adipogenesis markers increase in parallel during fat pad development. Finally, these preliminary studies suggest that the neovasculature originates by sprouting from larger, host-derived blood vessels that run parallel to peripheral nerves and that endothelial progenitor cells play little, if any, role in this process.

The low density lipoprotein receptor-related protein is a motogenic receptor for plasminogen activator inhibitor-1.

Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

Increased expression of plasminogen activator inhibitor-1 in cardiomyocytes contributes to cardiac fibrosis after myocardial infarction.

Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in tissue fibrosis by inactivating matrix metalloproteinases, which might effect on the progression of left ventricular dysfunction. However, little has been known about the expression of PAI-1 during cardiac remodeling. We used a mouse model of myocardial infarction (MI) by coronary ligation, in which the progression of left ventricular remodeling was confirmed by echocardiography. Histological examination showed that interstitial and perivascular fibrosis progressed in the post-MI (PMI) heart at 4 weeks after the procedure. We observed the dramatic induction of cardiac PAI-1 mRNA and PAI-1 antigen in plasma in the PMI mice, as compared with the sham-operated (sham) mice. In situ hybridization analysis demonstrated that strong signals for PAI-1 mRNA were localized to cardiomyocytes in the border of infarct area and around fibrous lesions, and to perivascular mononuclear cells, which seemed to be mast cells, only in hearts of the PMI mice. Importantly, less development of cardiac fibrosis after MI was observed in mice deficient in PAI-1 as compared to wild-type mice. The mRNA expression of cytokines, transforming growth factor-beta, and tumor necrosis factor-alpha, was also increased in hearts of the PMI mice, but not in the sham mice. These observations suggest that cardiomyocytes and mast cells contribute to the increased PAI-1 expression, resulting in the development of interstitial and perivascular fibrosis in the PMI heart, and that the regional induction of cytokines may be involved in this process.

Leptin promotes vascular remodeling and neointimal growth in mice.

OBJECTIVE: Human obesity is associated with elevated leptin levels and a high risk of death from cardiovascular disease. In the present study, we investigated the effects of leptin on vascular wound healing and arterial lesion growth in mice. METHODS AND RESULTS: Wild-type mice placed on an atherogenic, high-fat diet had elevated (9-fold) leptin levels compared with their counterparts maintained on normal chow, and the former demonstrated significantly enhanced neointimal thickening after carotid artery injury with ferric chloride. The lesions forming in response to injury strongly expressed leptin receptor mRNA and protein. Unexpectedly, the atherogenic diet had no effect on injured vessels from leptin-deficient ob/ob mice despite aggravating obesity, diabetes, and hyperlipidemia in these animals. Daily administration of leptin to ob/ob mice during the 3-week period after injury reversed this phenotype, dramatically increasing neointimal thickness and the severity of luminal stenosis. Exogenous leptin also enhanced lesion growth and increased cellular proliferation in injured arteries from wild-type mice but had no effect on vessels from leptin receptor-deficient db/db mice. CONCLUSIONS: Our results raise the possibility that there might be a direct, leptin receptor-mediated link between the hyperleptinemia in human obesity and the increased risk for cardiovascular complications associated with this condition.

Expression profiling identifies genes that continue to respond to insulin in adipocytes made insulin-resistant by treatment with tumor necrosis factor-alpha.

We have employed microarray technology using RNA from normal 3T3-L1 adipocytes and from 3T3-L1 adipocytes made insulin-resistant by treatment with tumor necrosis factor-alpha to identify a new class of insulin-responsive genes. These genes continued to respond normally to insulin even though the adipocytes themselves were metabolically insulin-resistant, i.e. they displayed a significantly decreased rate of insulin-stimulated glucose uptake. Approximately 12,000 genes/expressed sequence tags (ESTs) were screened. Of these, 40 genes/ESTs were identified that became insulin-resistant as expected (e.g. Socs-3, junB, and matrix metalloproteinase-11). However, 61 genes/ESTs continued to respond normally to insulin. Although some of these genes were previously shown to be regulated by insulin (e.g. Glut-1 and beta3-adrenergic receptor), other novel insulin-sensitive genes were also identified (e.g. Egr-1, epiregulin, Fra-1, and ABCA1). Real-time reverse transcription-PCR analysis confirmed the expression patterns of several of the differentially expressed genes. One gene that remained insulin-sensitive in the insulin-resistant adipocytes is the transcription factor Egr-1. Using an antisense strategy, we show that tissue factor and macrophage colony-stimulating factor, two cardiovascular risk factors, are downstream EGR-1 target genes in the adipocyte. Taken together, these data support the hypothesis that some signaling pathways remain insulin-sensitive in metabolically insulin-resistant adipocytes. These pathways may promote abnormal gene expression in hyperinsulinemic states like obesity and type II diabetes and thus may contribute to pathologies associated with these conditions.

Enhanced thrombosis in atherosclerosis-prone mice is associated with increased arterial expression of plasminogen activator inhibitor-1.

OBJECTIVE: This study was undertaken to investigate the origin and pathophysiological importance of plasminogen activator inhibitor (PAI-1) in atherosclerosis. METHODS AND RESULTS: We used the ferric chloride model to induce carotid artery injury in apolipoprotein E knockout (apoE-/-) and wild-type (WT) mice. ApoE-/- mice fed high-fat diet for 4 months developed severe hypercholesterolemia and had significantly elevated plasma PAI-1 levels (2.3+/-0.3 versus 0.6+/-0.1 ng/mL in WT mice; P<0.05). These mice exhibited a prothrombotic phenotype with shortened times to thrombotic arterial occlusion (8.6 versus 11.5 minutes; P<0.001) and reduced recanalization rates (12% versus 51%; P<0.0001) compared with WT mice. In situ hybridization, reverse transcriptase-polymerase chain reaction, and immunohistochemistry showed a significantly upregulated PAI-1 expression in P-selectin-positive (activated) endothelial cells lining normal-appearing arterial segments and within the advanced atherosclerotic lesions of apoE-/- mice. No significant upregulation of PAI-1 expression was found in the other organs studied, and only trace amounts of PAI-1 mRNA were detected in murine platelets. Importantly, deletion of the PAI-1 gene reversed the prothrombotic tendency and reduced neointimal growth after injury in apoE-/- mice despite the persistence of excessive hypercholesterolemia. CONCLUSIONS: These results suggest that increased vascular expression of PAI-1 may contribute to the elevated circulating levels of the inhibitor and be responsible, at least in part, for the prothrombotic phenotype in apoE-/- mice.

Critical role of integrin alpha 5 beta 1 in urokinase (uPA)/urokinase receptor (uPAR, CD87) signaling.

Urokinase-type plasminogen activator (uPA) induces cell adhesion and chemotactic movement. uPA signaling requires its binding to uPA receptor (uPAR/CD87), but how glycosylphosphatidylinositol-anchored uPAR mediates signaling is unclear. uPAR is a ligand for several integrins (e.g. alpha 5 beta 1) and supports cell-cell interaction by binding to integrins on apposing cells (in trans). We studied whether binding of uPAR to alpha 5 beta 1 in cis is involved in adhesion and migration of Chinese hamster ovary cells in response to immobilized uPA. This process was temperature-sensitive and required mitogen-activated protein kinase activation. Anti-uPAR antibody or depletion of uPAR blocked, whereas overexpression of uPAR enhanced, cell adhesion to uPA. Adhesion to uPA was also blocked by deletion of the growth factor domain (GFD) of uPA and by anti-GFD antibody, whereas neither the isolated uPA kringle nor serine protease domain supported adhesion directly. Interestingly, anti-alpha 5 antibody, RGD peptide, and function-blocking mutations in alpha 5 beta 1 blocked adhesion to uPA. uPA-induced cell migration also required GFD, uPAR, and alpha 5 beta 1, but alpha 5 beta 1 alone did not support uPA-induced adhesion and migration. Thus, binding of uPA causes uPAR to act as a ligand for alpha 5 beta 1 to induce cell adhesion, intracellular signaling, and cell migration. We demonstrated that uPA induced RGD-dependent binding of uPAR to alpha 5 beta 1 in solution. These results suggest that uPA-induced adhesion and migration of Chinese hamster ovary cells occurs as a consequence of (a) uPA binding to uPAR through GFD, (b) the subsequent binding of a uPA.uPAR complex to alpha 5 beta 1 via uPAR, and (c) signal transduction through alpha 5 beta 1.

Monocyte chemoattractant protein 1 in obesity and insulin resistance.

This study identifies monocyte chemoattractant protein 1 (MCP-1) as an insulin-responsive gene. It also shows that insulin induces substantial expression and secretion of MCP-1 both in vitro in insulin-resistant (IR) 3T3-L1 adipocytes and in vivo in IR obese mice (ob/ob). Thus, MCP-1 resembles other previously described genes (e.g., PAI-1 and SREBP-1c) that remain sensitive to insulin in IR states. The hyperinsulinemia that frequently accompanies obesity and insulin resistance may therefore contribute to the altered expression of these and other genes in insulin target tissues. In vivo studies also demonstrate that MCP-1 is overexpressed in obese mice compared with their lean controls, and that white adipose tissue is a major source of MCP-1. The elevated MCP-1 may alter adipocyte function because addition of MCP-1 to differentiated adipocytes in vitro decreases insulin-stimulated glucose uptake and the expression of several adipogenic genes (LpL, adipsin, GLUT-4, aP2, beta3-adrenergic receptor, and peroxisome proliferator-activated receptor gamma). These results suggest that elevated MCP-1 may induce adipocyte dedifferentiation and contribute to pathologies associated with hyperinsulinemia and obesity, including type II diabetes.

Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins.

The binding of urokinase plaminogen activator (uPA) to its cell surface receptor (uPAR; CD87) promotes cell adhesion by increasing the affinity of the receptor for both vitronectin (VN) and integrins. We provide evidence that plasminogen activator inhibitor (PAI)-1 can detach cells by disrupting uPAR-VN and integrin-VN interactions and that it does so by binding to the uPA present in uPA-uPAR-integrin complexes on the cell surface. The detached cells cannot reattach to VN unless their surface integrins are first activated by treatment with MnCl2. Immunoprecipitation and subcellular fractionation experiments reveal that PAI-1 treatment triggers deactivation and disengagement of uPA-uPAR-integrin complexes and their endocytic clearance by the low density lipoprotein receptor-related protein. Transfection experiments demonstrate that efficient cell detachment by PAI-1 requires an excess of matrix-engaged uPA-uPAR-integrin complexes over free engaged integrins and that changes in this ratio alter the efficacy of PAI-1. Together, these results suggest a VN-independent, uPA-uPAR-dependent mechanism by which PAI-1 induces cell detachment. This pathway may represent a general mechanism, since PAI-1 also can detach cells from fibronectin and type-1 collagen. This novel "deadhesive" activity of PAI-1 toward a variety of cells growing on different extracellular matrices may begin to explain why high PAI-1 levels often are associated with a poor prognosis in human metastatic disease.

Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm.

We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E-deficient (apoE-/-) mice treated with angiotensin II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (1.44 mg/kg per day) was implanted subcutaneously into 7- to 11-month-old male mice for 1 month. Ang II induced AAA in 9 (90%) of 10 hyperlipidemic mice deficient in apoE (apoE-/-/uPA+/+ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE-/-/uPA-/- mice) (P<0.05). Although the expansion of the suprarenal aorta was significantly less in apoE-/-/uPA-/- mice than in apoE-/-/uPA+/+ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in 7 (39%) of 18 strain-matched wild-type C57 black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE-/-) mice, in which 8 (100%) of 8 mice developed AAA. Only 1 (4%) of 27 uPA-/- mice developed AAA after Ang II treatment. We conclude the following: (1) uPA plays an essential role in Ang II-induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3) atherosclerosis and/or hyperlipidemia promotes but is not essential for Ang II-induced AAA formation in this model.