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Plasminogen activators are serine proteases that catalyze the conversion of plasminogen to plasmin. The plasminogen activating activity within a tissue is tightly regulated. Cell synthesis of plasminogen activators is modulated by various cytokines. Once secreted, the activities of the plasminogen activator enzymes are modulated by a complex network of molecular interactions. The plasminogen activator proteolytic cascade has been implicated in the regulation of cell growth and differentiation, both of which occur continually in normal human epidermis. The keratinocyte is the predominant cell type that comprises the epidermis. The psoriatic lesion is characterized by epidermal thickening consequent to increased proliferation of the basal and some suprabasal keratinocytes. In situ zymography studies suggest that the focal increases in tissue-type antigen are reflected in a coordinate induction of its enzymatic activity. Studies from several laboratories demonstrate that increased plasminogen activator activity is characteristic of psoriatic epidermis.

The interaction between human endothelial cells and leukocytes during immunological and inflammatory responses is in part mediated through the release of soluble mediators. We report that cultured human umbilical vein endothelial cells secrete IL-6 when stimulated with lipopolysaccharide. The monokines, IL-1 and TNF-alpha, were potent inducers of IL-6, whereas lymphotoxin was only effective at much higher concentrations. IFN gamma also was a strong stimulus of IL-6 production, but TGF-beta did not have an effect at doses modulating other endothelial cell functions. Endothelial cell IL-6 was active as hybridoma-plasmacytoma growth factor and as B-cell and hepatocyte stimulating factor. Endothelial IL-6 activity was neutralized by a specific antibody to IL-6 and it was shown by immunoprecipitation to be identical in size to human fibroblast-derived IL-6. IL-6 did not have a detectable effect on several endothelial cell functions, including proliferation, adherence of leukocytes, and synthesis of PGE2, TPA, and PAI-1. As IL-6 is probably an important regulator of host defense responses, production of this cytokine by endothelial cells may contribute to the pathogenesis of various inflammatory and immunologic diseases.

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.

SummaryLeptin 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 αIIbβ3, as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p

In summary, studies of the expression of fibrinolytic genes in the vessel wall suggest an active, ongoing proteolytic process, the activity of which is dependent on the relative amounts of tPA, uPA, and PAI-1 secreted and locally deposited. Disturbances in the balance of pro- and antifibrinolytic activity in atherosclerotic vessels have considerable potential for influencing both intra- and extravascular fibrinolytic events and may be causally related to the development of vascular disease.

In this chapter, we summarize our studies on plasminogen activator inhibitor 1 (PAI-1), tissue factor, and transforming growth factor beta (TGF-beta) expression in obesity, using genetically obese mice as a model. These studies emphasize the key role played by the adipocyte, a cell whose numbers, size, and metabolic activity are grossly altered in obesity/NIDDM. They also implicate multiple cytokines, hormones, and growth factors in the abnormal expression of these and perhaps other hemostatic genes by adipocytes in obesity/NIDDM. These studies demonstrate that tumor necrosis factor alpha (TNF-alpha) plays a central role in the expression of hemostatic genes in this disorder.

We investigated the interaction between the urokinase receptor (uPAR) and the integrin αvβ3. Vitronectin (VN) induces cell migration by binding to αvβ3, but expression of the uPAR boosts its efficacy. Thus, uPAR may regulate VN-induced cell migration by interacting laterally with αvβ3. 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 αvβ3 and activates αvβ3-dependent signaling pathways such as the Janus kinase/Stat pathway. Moreover, D2A disrupts uPAR-αvβ3 and uPAR-α5β1 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 αvβ3-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.

The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316-324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up-regulation may be a critical component in the reorganization of the vascular bed in response to angiogenic stimuli.

Summary. Background and objectives: Cardiovascular/thrombotic diseases are frequently induced by a variety of stressors. Obese patients are susceptible to thrombotic diseases associated with stress, but the underlying mechanism is still unknown. We have begun to investigate the expression of a primary inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), in association with tissue thrombosis, using restraint-stressed obese mice. Methods and results: We analyzed the expression of PAI-1 after restraint (immobilization) stress in genetically obese mice in comparison with their lean counterparts. Dramatic increases in PAI-1 antigen in plasma and in tissue extracts were observed in the obese mice exposed to restraint stress. The induction of PAI-1 mRNA by stress in the tissues was also pronounced in the stressed obese mice as compared with the lean mice, especially in the hearts and adipose tissues. In situ hybridization analysis revealed that strong signals for PAI-1 mRNA were localized in the adipocytes, cardiovascular endothelial cells, and renal glomerular cells of the stressed obese mice. Histological examination revealed that renal glomerular fibrin deposition was detected only in the obese mice after 2 h of restraint stress. Conclusions: Obesity enhances the stress-mediated PAI-1 induction in the blood and tissues. This phenomenon may be associated with the increased risk of stress-induced renal fibrin deposition in obese subjects.

SummaryAlthough 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.

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.

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.

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.

Objectives— 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.

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.

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 , β3-adrenergic receptor, and peroxisome proliferator-activated receptor γ). These results suggest that elevated MCP-1 may induce adipocyte dedifferentiation and contribute to pathologies associated with hyperinsulinemia and obesity, including type II diabetes.

Although older subjects are susceptible to thrombosis under septic conditions, the underlying molecular mechanisms have not been fully elucidated. Since elevated plasminogen activator inhibitor-1 (PAI-1) primarily contributes to endotoxin-induced thrombosis, we first compared the induction of PAI-1 by lipopolysaccharide (LPS) between young and aged mice. The higher induction of PAI-1 antigen and mRNA with increased renal glomerular fibrin deposition was observed in LPS-treated aged mice compared to young mice. In situ hybridization analysis showed that the aging-associated induction of PAI-1 mRNA by LPS was pronounced in hepatocytes and in renal glomerular cells. The increased magnitude of the response of aged mice to lower doses of LPS was observed in terms of renal glomerular fibrin deposition and PAI-1 mRNA induction in the tissues. Furthermore, older PAI-1 deficient mice treated with LPS developed much less fibrin deposition in kidneys. Importantly, a larger induction of receptor molecules for LPS (eg, CD14 and Toll-like receptor 4) was demonstrated in LPS-treated aged mice as compared with young mice. The enhanced LPS signaling in aged mice was also demonstrated by the marked induction of nuclear factor-κB in the tissues after endotoxin treatment. As a consequence, increases in an inflammatory cytokine, tumor necrosis factor-α, were pronounced in plasma and tissues of LPS-treated aged mice. These results emphasize the key role played by PAI-1 in aging-associated deterioration in this thrombosis model, and suggest that the hyperresponse of PAI-1 gene to LPS results from the enhanced LPS signaling and the subsequent inflammatory response in aged mice.

Background— Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are thought to play critical roles in vascular remodeling after injury, with tPA mediating intravascular clot lysis and uPA modulating cell migration within the vessel wall. In human vascular disease, however, thrombus organization and neointimal formation are closely interrelated processes. This study examines the differential roles of tPA and uPA in these processes in mice. Methods and Results— Carotid artery injury and thrombosis were induced in wild-type (WT), uPA-deficient (uPA −/− ), and tPA-deficient (tPA −/− ) mice with the use of ferric chloride. The expression of uPA and tPA was significantly upregulated in the vessel wall of WT mice 1 week after injury, and compared with WT mice, uPA −/− and tPA −/− mice had lower carotid patency rates after injury. At 3 weeks, only 55% of uPA −/− mouse vessels were patent compared with 81% in tPA −/− mice and 100% in WT mice ( P =0.014). Morphometric analysis of injured arterial segments revealed severe luminal stenosis (62±28%) in uPA −/− mice compared with their tPA −/− (16±12%) and WT (6.3±3.6%, P −/− mice developed a cell-rich multilayered neointima and media, the lumen of uPA −/− vessels remained obstructed with acellular unorganized thrombotic material, and their medial areas did not expand. Conclusions— These results indicate that the roles of uPA and tPA in the arterial response to injury are different and more complex than previously assumed and emphasize the critical role of thrombus organization and resolution in neointimal formation and vascular pathology.

The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.

Plasminogen activator inhibitor-1 (PAI-1) is one of the primary inhibitors of the fibrinolytic system and has been implicated in a variety of thrombotic disorders. In this report, stress-induced changes in murine PAI-1 gene expression were investigated to study the role of this inhibitor in the development of stress-induced hypercoagulability. Restraint stress led to a dramatic induction of plasma PAI-1 antigen and of tissue PAI-1 mRNA with maximum induction in adipose tissues. In situ hybridization analysis of the stressed mice revealed that strong signals for PAI-1 mRNA were localized to hepatocytes, renal tubular epithelial cells, adrenomedullar chromaffin cells, neural cells in the paraaortic sympathetic ganglion, vascular smooth muscle cells, and adipocytes, but not to endothelial cells. These observations indicate that the stress induces the PAI-1 gene expression in a tissue-specific and cell type-specific manner. The induction of PAI-1 mRNA by restraint stress was greater than that observed for heat shock protein, a typical stress protein, suggesting that PAI-1 is one of the most highly induced stress proteins. Importantly, the magnitude of induction of PAI-1 mRNA by stress increased markedly with age, and this increase in PAI-1 correlated with tissue thrombosis in the older stressed mice. Moreover, much less tissue thrombosis was induced by restraint stress in young and aged PAI-1-deficient mice compared with age-matched wild-type mice. These results suggest that the large induction of PAI-1 by stress increases the risk for thrombosis in the older populations, and that the adipose tissue may be involved.

SummaryPlasmin is the major enzyme that dissolves fibrin in the vasculature and the predominant source of its zymogen, plasminogen, is liver. However, plasmin has a broad substrate spectrum and, if present in other tissues, may perform additional functions. We tested the hypothesis that plasminogen is expressed broadly extrahepatically. A sensitive and specific isotopic quantitative RT-PCR assay was developed to detect plasminogen mRNA from total RNA isolated from C57BL/6J mice tissues. Plasminogen mRNA was detected in adrenal, kidney, brain, testis, heart, lung, uterus, spleen, thymus and gut. Of these tissues, adrenal had the highest plasminogen mRNA content. In situ hybridization was utilized to localize plasminogen mRNA expressing cell types. Besides hepatocytes, positive cells were identified in both adrenal and kidney medullae and cortexes. Plasminogen mRNA expression was detected in cerebral, hippocampal and cerebellar neurons. Plasminogen mRNA was detected in cells in early stages of spermatogenesis in testis, present in the cortex and medulla of the thymus and in splenic white and red pulps. Our results suggest that the plasminogen gene is expressed broadly in extrahepatic tissues. Thus, tissues separated by local anatomic barriers as well as tissues accessible to circulating plasminogen have the capacity to provide local sources of plasminogen.

Although aging accompanies specific pathological changes, including thrombosis and organ sclerosis, the underlying mechanisms of these processes remain to be elucidated. In the present study, we analyzed the gene expression of plasminogen activator inhibitor-1 (PAI-1), a key molecule in the development of thrombosis, in a murine model of aging, klotho mutant ( kl/kl) mice. Active PAI-1 antigen in plasma and PAI-1 mRNA in several tissues were strikingly elevated in kl/kl mice as compared with wild-type mice. This increased PAI-1 expression was age dependent and linked to the development of ectopic calcification and glomerular fibrin deposition in the kidneys. In situ hybridization analysis of kl/kl mice demonstrated that strong signals for PAI-1 mRNA were localized in renal tubular epithelial cells, cardiomyocytes, adrenal medullar cells, and smooth muscle and endothelial cells in Mönckeberg's arteriosclerotic vessels. Renal glomerular fibrin deposition, as evaluated immunohistochemically, was occasionally observed only in kl/kl mice, and the number of fibrin-positive glomeruli increased as the kl/kl mice aged. These observations suggest that in the process of aging the PAI-1 gene expression is increased, contributing to the development of thrombosis.

Obesity is associated with increased cardiovascular morbidity and mortality and with elevated circulating levels of the satiety factor leptin. This study provides evidence for a direct link between leptin and the risk for thrombotic complications in obese individuals. For example, although arterial injury provokes thrombosis in both lean and obese (ob/ob) mice, the time to complete thrombotic occlusion is significantly delayed in the ob/ob mice, and the thrombi formed are unstable and frequently embolize. The ob/ob mice lack leptin, and intraperitoneal administration of leptin to these mice before injury restores the phenotype of lean mice by shortening the time to occlusion, stabilizing the thrombi, and decreasing the patency rate. The thrombi that form when leptin receptor-deficient obese (db/db) mice are injured also are unstable. However, in this instance, leptin has no effect. Platelets express the leptin receptor, and leptin potentiates the aggregation of platelets from ob/ob but not db/db mice in response to known agonists. These results reveal a novel receptor-dependent effect of leptin on platelet function and hemostasis and provide new insights into the molecular basis of cardiovascular complications in obese individuals. The results suggest that these prothrombotic properties should be considered when developing therapeutic strategies based on leptin.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

Plasminogen activator inhibitor-1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin. It inhibits the adhesion of U937 cells to vitronectin by competing with the urokinase receptor (uPAR; CD87) on these cells for binding to the same domain. Although the inhibitor also blocks integrin-mediated cell adhesion, the molecular basis of this effect is unclear. In this study, the effect of the inhibitor on the adhesion of a variety of cells (e.g., U937, MCF7, HT-1080, and HeLa) to vitronectin was assessed, and the importance of the SMB domain in these interactions was determined. Although PAI-1 blocked the adhesion of all of these cells to vitronectin-coated wells, it did not block adhesion to a variant of vitronectin which lacked the SMB domain. Interestingly, HT-1080 and U937 cells attached avidly to microtiter wells coated with purified recombinant SMB (which does not contain the RGD sequence), and this adhesion was again blocked by the inhibitor. These results affirm that PAI-1 can inhibit both uPAR- and integrin-mediated cell adhesion, and demonstrate that the SMB domain of vitronectin is required for these effects. They also show that multiple cell types can employ uPAR as an adhesion receptor. The use of purified recombinant SMB should help to further define this novel adhesive pathway, and to delineate its relationship with integrin-mediated adhesive events. © 2001 Wiley-Liss, Inc.

SummaryPrevious studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.

Although the association between insulin resistance and cardiovascular risk is well established, the underlying molecular mechanisms are poorly understood. The antifibrinolytic molecule plasminogen activator inhibitor 1 (PAI-1) is a cardiovascular risk factor that is consistently elevated in insulin-resistant states such as obesity and non-insulin-dependent diabetes mellitus (NIDDM). The strong positive correlation between this elevated PAI-1 and the degree of hyperinsulinemia not only implicates insulin itself in this increase, but also suggests that PAI-1 is regulated by a pathway that does not become insulin resistant. The data in this report supports this hypothesis. We show that insulin stimulates PAI-1 gene expression in metabolically insulin-resistant ob/ob mice and in insulin-resistant 3T3-L1 adipocytes. Moreover, we provide evidence that glucose transport and PAI-1 gene expression are mediated by different insulin signaling pathways. These observations suggest that the compensatory hyperinsulinemia that is frequently associated with insulin-resistant states, directly contribute to the elevated PAI-1. These results provide a potential mechanism for the abnormal increases in cardiovascular risk genes in obesity, NIDDM, and polycystic ovary disease.

Thickening of the glomerular basement membrane (GBM) results from excessive accumulation of extracellular matrix (ECM) proteins following glomerular injury. We studied the temporal relationship between the expression of growth factors, ECM accumulation, ECM degrading proteinases, and their inhibitors in a rat model of anti-GBM antibody (Ab) glomerulonephritis (GN) by the RNase protection assay and immunohistochemistry. There were two- or fourfold increases in the expression of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF) A and B chain mRNAs 4 days after anti-GBM Ab administration. These changes were temporally associated with increased accumulation of α1(III) and α2(IV) collagens, fibronectin, and heparan sulfate proteoglycan along the GBM. The increase in matrix accumulation was associated with little or no increases in the proteinases, urokinase plasminogen activator (u-PA) and transin, respectively. There was a 1.6× increase in the u-PA/28s mRNA ratio on day 4 in rats with anti-GBM Ab GN, but this was not associated with an increase in u-PA biologic activity. By comparison, the mRNAs of the proteinase inhibitors, plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase (TIMP) were 5× greater than that of control rats on day 4. PAI-1 mRNA correlate with increased biologic activity. These data demonstrate a temporal association between TGF-β1 and PDGF expression and matrix accumulation within the GBM in anti-GBM Ab GN. In addition, it suggest that this matrix accumulation results from an imbalance between matrix synthesis and degradation.

Obesity is associated with a cluster of abnormalities, including hypertension, insulin resistance, hyperinsulinemia, and elevated levels of both plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta (TGF-beta). Although these changes may increase the risk for accelerated atherosclerosis and fatal myocardial infarction, the underlying molecular mechanisms remain to be defined. Although tumor necrosis factor alpha (TNF-alpha) has been implicated in the insulin resistance associated with obesity, its role in other disorders of obesity is largely unknown. In this report, we show that in obese (ob/ob) mice, neutralization of TNF-alpha or deletion of both TNF receptors (TNFRs) results in significantly reduced levels of plasma PAI-1 antigen, plasma insulin, and adipose tissue PAI-1 and TGF-beta mRNAs. Studies in which exogenous TNF-alpha was infused into lean mice lacking individual TNFRs indicate that TNF-alpha signaling of PAI-1 in adipose tissue can be mediated by either the p55 or the p75 TNFR. However, TNF-alpha signaling of TGF-beta mRNA expression in adipose tissue is mediated exclusively via the p55 TNFR. Our results suggest that TNF-alpha is a common link between the insulin resistance and elevated PAI-1 and TGF-beta in obesity. The chronic elevation of TNF-alpha in obesity thus may directly promote the development of the complex cardiovascular risk profile associated with this condition.

IntroductionObesity is a major public health problem in Western societies with substantial economic consequences. In the United States alone, over 30% of the population is defined as clinically obese, and thereby, are subject to increased risk for obesity-associated disorders, including cardiovascular disease (CVD), hypertension, insulin resistance, and non-insulin dependent diabetes mellitus (NIDDM).1,2 In spite of the magnitude of this problem, the molecular changes in obesity that promote these conditions are far from resolved. The fact that human obesity is a polygenic disorder with complex environmental and behavioral characteristics has made obesity research one of the more difficult areas of investigation in the medical sciences.1,3 However, recent studies of genetically obese mice and rats have produced breakthroughs in several areas of obesity-related research, primarily because obesity in these animals appears to be monogenic (i.e., to result from mutations in single genes). In this regard, five different genes, all mapped to different chromosomal locations, have been shown to cause distinct syndromes of spontaneous obesity with severe insulin resistance in mice.3 These include the obese (ob), diabetes (db), tubby (tub), lethal yellow (Ay) and fat (fat) mutations. Genes encoding intercellular adhesion molecule 1 (ICAM-1) and the leukocyte integrin αmβ2 (MAC-1) also have been implicated in the regulation of adipose tissue mass.4 These animal studies, together with studies of cultured adipocytes, have provided fundamental new information about the factors and cells that may be responsible for the altered hemostatic balance leading to increased cardiovascular risk in obesity/NIDDM.In this chapter, we summarize our studies on plasminogen activator inhibitor 1 (PAI-1), tissue factor, and transforming growth factor (TGF)-β expression in obesity, using genetically obese mice as a model. These studies emphasize the key role played by the adipocyte, a cell whose numbers, size, and metabolic activity are grossly altered in obesity/NIDDM. They also implicate multiple cytokines, hormones, and growth factors in the abnormal expression of these and perhaps other hemostatic genes by adipocytes in obesity/NIDDM (Fig. 1). These studies emphasize the key role played by tumor necrosis factor (TNF)-α in the expression of hemostatic genes in this disorder.

Activated protein C (APC) acts as an anticoagulant by inhibiting coagulation factors Va and VIIIa. Although the liver appears to be the primary site of protein C (PC) synthesis, the demonstration that other components of this system are produced extrahepatically raises the possibility that PC itself is synthesized in other tissues. We therefore used quantitative reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry to screen various murine tissues for PC expression. Relatively high levels of PC mRNA were detected in the kidney (35% of liver) and testis (22% of liver). PC mRNA and antigen were demonstrated in tubular epithelial cells in the renal cortex, in spermatogenic cells in the testis, and in epithelial cells in the epididymis. Low but significant levels of PC mRNA were detected in the epididymis (1.7% of the level in liver), brain (1.1% of liver), and lung (0.8% of liver). PC antigen was demonstrated in bronchial epithelial cells in the lung, in pyramidal neurons in the cerebrum, and in Purkinje cells in the cerebellum. The extrahepatic expression of PC mRNA (ie, in the kidney) was significantly decreased in mice with renal disease (eg, in MRL lpr/lpr mice with autoimmune lupus nephritis, in db/db mice with diabetic nephropathy, and in endotoxin-treated mice with acute renal injury). The decreased renal expression of PC may contribute to the increased procoagulant potential of the kidney during septic and inflammatory processes and to the progression of kidney disease associated with these conditions.

Plasminogen activator inhibitor 1 is the primary physiological inhibitor of plasminogen activation in vivo, and elevations in plasma PAI-1 appear to compromise normal fibrin clearance mechanisms and promote thrombosis. PAI-1 is dramatically upregulated in obesity, a complex condition associated with increased risk for myocardial infarction, accelerated atherosclerosis, hypertension, glucose intolerance, insulin resistance, hyperinsulinemia, and NIDDM. In spite of the apparent link between elevated PAI-1 levels and thrombotic disease, little is known about the origin of this plasma inhibitor in obesity/NIDDM or about the signals that control its biosynthesis. Potential insights into the underlying molecular events have come from recent studies of genetically obese mice and of cultured adipocytes. These studies are reviewed here.* They emphasize the key role played by the adipocyte, a cell whose numbers, size, and metabolic activity are grossly altered in obesity/NIDDM. They also suggest that multiple cytokines, hormones, and growth factors may be involved, and they raise the possibility that the abnormal expression of other hemostatic genes by adipocytes in obesity/NIDDM may also contribute to the cardiovascular complications of this disorder. In this regard, our preliminary studies indicate that TF gene expression is elevated in the adipose tissues of the obese mouse. PAI-1 appears to be the primary physiological inhibitor of plasminogen activation in blood, since it is the only PAI found complexed with single-chain tissue-type PA in carefully collected human plasma, and the second-order rate constant for its interaction with tissue-type PA and urokinase-type PA (≈3.5×107 [mol/L] −1 · s−1) is at least two orders of magnitude higher than that of other PAIs.1 2 3 4 The normal concentration of PAI-1 protein in human plasma ranges from …

We previously demonstrated CD14 gene expression in myeloid and epithelial cells of the mouse and showed that expression of the CD14 gene in both is modulated by lipopolysaccharide (LPS). Here we test the hypothesis that the induction of CD14 in these cells is an indirect effect of LPS, one mediated by tumor necrosis factor alpha (TNF-alpha). TNF-alpha induced a transient increase in levels of CD14 in plasma with a peak at 6 to 8 h, and this increase in levels of CD14 antigen in plasma was accompanied by increased levels of CD14 mRNA in lung, liver, and kidney. Moreover, in situ hybridization studies revealed that CD14 mRNA was induced in both myeloid cells and epithelial cells, the same cells that respond to LPS. Pretreatment of mice with anti-TNF antiserum reduced the LPS-mediated increase in levels of CD14 in plasma and significantly reduced the level of induction of CD14 mRNA in selected epithelial cells in the kidney and liver. The antiserum did not appear to block LPS-mediated induction in myeloid cells in the tissues examined. In C3H/HeJ mice, the epithelial response to LPS was markedly attenuated whereas the response to TNF-alpha was normal. Thus, regulation of CD14 gene expression by LPS differs in epithelial and myeloid cells, with the epithelial responses in kidney and liver being mediated, in part, by TNF-alpha.

Vitronectin (Vn) regulates proteolytic enzyme systems, as well as cell migration and tissue remodelling. These processes have been implicated in the pathogenesis of atherosclerosis. In this study, the distribution of Vn antigen in apparently normal and atherosclerotic human blood vessels was evaluated. Normal and diseased vessels showed Vn immunostaining in the lamina elastica interna and externa, and in strand-like structures in the adventitia. In most of these instances, the Vn antigen appeared to be located in the proximity of elastin. In pulmonary arteries, Vn staining was additionally detected in the media. The intima was devoid of Vn antigen in all vessels studied. In general, there was increased deposition of Vn antigen in the atherosclerotic arteries. In particular, strong Vn staining was apparent in amorphous material adjacent to cholesterol clefts and in acellular fibrous tissue, in plaques present in the carotic artery and aorta. Collagen layers and fresh fibrin depositions were devoid of Vn antigen. In spite of the abundance of Vn immunostaining throughout the normal and diseased vessel wall, the Vn transcript was not detectably by in situ hybridization. These results indicate that Vn is a constituent of the normal vessel wall and raise the possibility that increased local deposition of Vn may be related to the development of atherosclerotic vascular disease.

The disturbances in the balance of pro- and antifibrinolytic activity, as observed in AAA and obesity, respectively, have considerable potential for influencing both intra- and extravascular fibrinolytic events and may be causally related to the development of vascular disease. For example, the wall of the aortic atherosclerotic aneurysm seems to host an uneven distribution and imbalanced expression of the various components of the fibrinolytic system. The sites of increased proteolytic activity may contribute to localized neovascularization and promote the rapid breakdown of ECM components, which result in mural weakening and eventual rupture of untreated aortic aneurysms. On the other hand, the disturbance of the normal hemostatic balance observed in obesity appears to result from the elevated expression of PAI-1 by the adipose tissue. Our data strongly suggest that the adipocyte is one of the primary cells in the adipose tissue capable of expressing PAI-1 both in obesity, and in response to cytokines and hormones like TNF-alpha and insulin. Since both TNF-alpha and insulin are known to increase in obesity, the elevated levels of PAI-1 observed in the plasma of obese individuals may result from TNF-alpha and/or insulin induction of PAI-1 in the adipose tissue itself.

Tumor necrosis factor-α (TNF-α) is chronically elevated in the adipose tissue from obese humans and mice. This increase in TNF-α contributes to the insulin resistance, elevated plasminogen activator inhibitor-1 (PAI-1) levels, and cardiovascular complications associated with obesity and noninsulin-dependent diabetes (NIDDM). PAI-1 gene expression in adipose tissue is also stimulated by transforming growth factor-β (TGF-β). Experiments were performed to determine whether TGF-β is regulated by TNF-α and elevated in obesity. The concentration of TGF-β and PAI-1 mRNA in murine adipose tissue and cultured 3T3-L1 adipocytes was determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), and the cellular localization of these molecules was evaluated using in situ hybridization and cell fractionation. Total TGF-β protein was determined by employing an ELISA assay. TGF-β mRNA and protein were increased in the adipose tissue from two different strains of genetically obese mice (i.e., ob/ob and db/db), compared with their lean counterparts. This increase in TGF-β may result from TNF-α since TNF-α increased TGF-β mRNA expression in the adipose tissue of lean mice and stimulated TGF-β production by cultured adipocytes. Administration of TGF-β increased PAI-1 antigen in the plasma and PAI-1 mRNA in the adipocytes of lean mice, and enhanced the rate of PAI-1 synthesis by adipocytes in vitro. TNF-α contributes to the elevated TGF-β expression demonstrated in the adipose tissue of obese mice. A potential role for TGF-β in the increased PAI-1 and vascular pathologies associated with obesity/NIDDM is suggested.

Plasminogen activator inhibitor-1 is elevated in obesity and may be a risk factor for obesity/NIDDM related cardiovascular disease. In spite of this, little is known about the tissue and cellular origin of elevated PAI-1 in obesity or of the mediators and molecular mechanisms that regulate it. We have begun to systematically address these issues using genetically obese (ob/ob, db/db) mice. Plasma PAI-1 levels were 5-fold higher in obese mice compared to their lean counterparts. Subsequent RT-PCR and in situ hybridization studies suggest that the increased plasma PAI-1 originates primarily from the adipocyte in response to chronically elevated levels of tumor necrosis factor-alpha (TNF-alpha), insulin, and transforming growth factor-beta (TGF-beta). Thus, the signals and mechanisms that lead to elevated plasma PAI-1 observed in obesity are complex, and appear to involve interactions between multiple mediators and the adipose tissue itself.

The conformation and degree of multimerization of vitronectin (Vn) appears to be of critical importance for its functions, but little is known about the underlying mechanisms that control Vn multimerization. We report that Vn secreted by cultured hepatoma cells is present as a mixture of monomeric and multimeric forms. A single protein of Mr 45,000 co-purified with hepatoma cell-derived Vn, which was immunologically identified as type 1 plasminogen activator inhibitor (PAI-1). The possibility that PAI-1 may modulate Vn multimerization was investigated. The addition of active PAI-1 to unfractionated plasma containing Vn monomers resulted in the formation of covalently and noncovalently associated Vn multimers and expression of conformationally sensitive epitopes. In contrast, inactive forms of PAI-1 did not efficiently induce Vn multimerization and conformational change. Gel filtration analysis revealed that Vn remained multimeric after dissociation from PAI-1. Vn multimers were also assembled using purified monomeric Vn and PAI-1, suggesting that a plasma cofactor was not required to induce Vn multimerization. This study provides insights into physiological mechanism responsible for the generation of homomultimeric Vn, a multimeric form of Vn that is not in complex with other proteins and which expresses a functional repertoire distinct from that of plasma Vn.

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.

Although elevated plasminogen activator inhibitor-1 (PAI-1) is associated with obesity and may be a risk factor for cardiovascular disease, the mechanism(s) that lead to this elevation, and the tissue/cellular origins of this increase, remain to be defined. In this report, we have addressed these questions using genetically obese mice (ob/ob) and their lean counterparts (+/?). PAI-1 activity and antigen levels were determined using a tissue-type plasminogen activator (t-PA) binding assay and Western blotting. The concentration of PAI-1 mRNA in tissues was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and the cellular localization of PAI-1 was evaluated using in situ hybridization, immunohistochemistry, and cell fractionation. PAI-1 activity was approximately 4-fold higher in plasma from ob/ob mice than in that obtained from their lean counterparts, and this difference increased further with age (i.e., 6-fold at 3 months). PAI-1 mRNA levels were elevated 4- to 5-fold in the adipose tissues of obese mice, and these differences in mRNA also increased with age. The elevated PAI-1 mRNA in the adipose tissues of obese mice was localized to mature adipocytes as well as to vascular smooth muscle cells and occasional endothelial cells. Obesity is often associated with hyperinsulinemia, and acute injection of insulin into lean mice increased PAI-1 mRNA 6- to 8-fold in the epididymal fat in cells that morphologically resembled adipocytes. Insulin did not increase PAI-1 in large vessel endothelial or smooth muscle cells. The adipocyte response to insulin was confirmed in cell culture studies where PAI-1 synthesis by mature 3T3-L1 adipocytes was increased 5- to 6-fold by insulin. Our results suggest that elevated PAI-1 associated with obesity may result in part from insulin-induced induction of PAI-1 specifically by adipocytes within the fat itself.

Although elevated plasma plasminogen activator inhibitor 1 (PAI-1) is associated with obesity, very little is known about its tissue or cellular origin, or about the events that lead to increased PAI-1 levels under obese conditions. Since TNF-alpha is increased in rodents both during obesity and in response to endotoxin treatment, we examined the effects of these agents on PAI-1 gene expression in the adipose tissue of CB6 mice. In untreated mice, PAI-1 mRNA was detected in both mature adipocytes and in stromal vascular cells. Both TNF-alpha and endotoxin significantly increased PAI-1 mRNA in the adipose tissue, peaking at 3-8 h. In situ hybridization analysis of adipose tissue from untreated mice revealed a weak signal for PAI-1 mRNA only in the smooth muscle cells within the vascular wall. In contrast, after endotoxin or TNF-alpha treatment, PAI-1 mRNA also was detected in adipocytes and in adventitial cells of vessels. Endotoxin also induced PAI-1 in endothelial cells, while TNF-alpha additionally induced it in smooth muscle cells. Mature 3T3-L1 adipocytes in culture also expressed PAI-1 mRNA, and its rate of synthesis was also upregulated by TNF-alpha. These studies suggest that the adipose tissue itself may be an important contributor to the elevated PAI-1 levels observed in the plasma under obese conditions.

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.

Bleomycin-induced lung injury is an established murine model of human pulmonary fibrosis. Although procoagulant molecules (e.g., tissue factor [TF]) and fibrinolytic components (e.g., urokinase [u-PA] and type 1 plasminogen activator inhibitor [PAI-1]) have been detected in alveolar fluid from injured lungs, the origin of these molecules remains unknown. We therefore examined the expression of procoagulant and fibrinolytic components in relation to the distribution of parenchymal fibrin in bleomycin-injured lungs. Extravascular fibrin localized to the alveolar and extracellular matrix in injured lung tissue. Injured lung tissue extracts contained elevated levels of PAI-1 activity and decreased levels of u-PA activity. Whole lung PAI-1 and TF mRNAs were dramatically induced by lung injury. In situ hybridization of injured lungs revealed that PAI-1, u-PA, and TF mRNAs were induced within the fibrin-rich fibroproliferative lesions, primarily in fibroblast-like and macrophagelike cells, respectively, while TF mRNA was also induced in perilesional alveolar cells. Taken together, these observations suggest that the induction of PAI-1 and TF gene expression plays and important role in the formation and persistence of extracellular fibrin in bleomycin injured murine lungs.

Vitronectin (Vn) is not only a major adhesive glycoprotein in plasma but also regulates cell-mediated proteolytic enzyme cascades, including the complement, coagulation, and fibrinolytic systems. This broad functional activity suggests that Vn may also play a critical role in development. To begin to investigate this possibility, we studied Vn gene expression during murine embryogenesis. In situ hybridization analysis of embryonic tissues revealed Vn mRNA primarily in the liver and the central nervous system (CNS). In the liver, Vn mRNA was detected by day 10, the level increasing at later developmental stages. In the CNS, Vn mRNA was also detected as early as day 10 and was confined to the floor plate. However, as development proceeded, high levels of Vn transcripts became prominent in the meninges of the cortex and spinal cord, and in close proximity to brain capillaries. The perikarya of most neurons lacked Vn mRNA. Unexpectedly, high levels of Vn mRNA were associated with capillaries of the CNS, but not with blood vessels of peripheral organs. These results indicate that Vn is expressed in a spatially and temporally distinct pattern during murine embryogenesis, and suggest that the Vn transcript may be a CNS-specific vascular marker.

Expression of type 1 plasminogen activator inhibitor in renal tissue in murine lupus nephritis. Many renal diseases are associated with fibrin deposition in the glomeruli, a situation that reflects an abnormality in the balance between the coagulation and fibrinolytic systems. We recently demonstrated that normal mouse kidney contains very low levels of type 1 plasminogen activator inhibitor (PAI-1), a potent anti-fibrinolytic protein, but that during endotoxemia, large amounts of PAI-1 protein and mRNA are expressed in glomerular and peritubular endothelial cells. These results raise the possibility that overexpression of PAI-1 in the glomerulus may contribute to the ongoing pathology seen in renal disease. To directly investigate this possibility, we studied PAI-1 expression in MRL/ lpr mice, using in situ hybridization and immunohistochemistry. Female MRL/ lpr mice develop early onset lupus glomerulonephritis (GN), a disease in which fibrin deposition is detected in the glomerulus and in which anti-coagulation therapy improves the prognosis. We detected very low levels of PAI-1 mRNA and antigen in the smooth muscle cells of renal vessels and in the renal papilla of 16 control mice. In contrast, PAI-1 was expressed in relatively high levels throughout the kidneys of 33 out of 34 diseased mice, both within the glomerulus and also in tubules and vessels. Moreover, the level of PAI-1 in the tissues seemed to correlate with the severity of the disease. PAI-1 expression was localized to endothelial cells, parietal epithelial cells, tubular epithelial cells and infiltrating mononuclear cells in the tubulointerstitium. None of these cells express detectable levels of PAI-1 in the normal kidney. The inappropriate expression of PAI-1 in the kidneys of mice with lupus GN suggests that this important inhibitor of fibrinolysis may play a role in the pathogenesis of this disease process.

Protein C inhibitor (PCI) is a serpin that inhibits a number of proteases. PCI is found in urine and binds to kidney epithelial cells. To determine if kidney is a source of PCI, cDNA was produced from human kidney total RNA. Sequencing and restriction mapping showed identity between kidney and liver PCI cDNA sequences. Similar cDNAs were obtained from rhesus monkey kidney and liver RNAs. Conditioned medium from the rhesus monkey kidney cell line CCL7.1 was analyzed on immunoblots, showing a 57,000-D protein band that comigrated with human plasma PCI. Immunohistochemical staining and in situ hybridization of human kidney tissue sections showed that kidney PCI antigen and RNA were confined to tubular cells. The findings are consistent with the idea that PCI is synthesized and localized in kidney tissue where it may provide protease inhibitory activity and suggest that complexes of PCI with urokinase found in human urine may be produced locally in the kidney.

A competitive polymerase chain reaction (PCR) assay was developed to quantitate vitronectin (Vn) mRNA in murine tissues using a synthetic RNA as an external standard. Although the liver contained the highest concentration of Vn mRNA, significant levels were also detected in the brain (25-fold less) and in adipose tissue, heart, and skeletal muscle (100-fold less than liver). Lower concentrations also were detected in the lung, uterus, testis, and thymus, and little or no Vn mRNA could be detected in kidney, spleen, and blood. These results indicate that significant amounts of Vn mRNA are produced in extrahepatic organs. The regulation of Vn gene expression in vivo was studied in a murine model system in which acute systemic inflammation was induced by endotoxin administration. Plasma Vn levels increased 2- to 3-fold within 16 h after endotoxin administration and remained elevated for up to 72 h. This increase appeared to result from increased synthesis in the liver since the steady-state level of hepatic Vn mRNA increased 4-fold after endotoxin administration. Moreover, Vn mRNA levels in heart, lung, and brain were not significantly increased by endotoxin. These results suggest that Vn gene expression in vivo is regulated in a tissue-specific manner and identify Vn as a novel acute phase reactant.

Elevated plasma levels of type 1 plasminogen activator inhibitor (PAI- 1) have been implicated in mediating the fibrin deposition and occlusive lesions that occur within the placental vasculature in preeclampsia (PE) and intrauterine growth retardation (IUGR). In this report we identify the cells within the normal-appearing villous tissue that are responsible for the local production of PAI-1 in women with PE and IUGR. Levels for another fibrinolytic inhibitor (ie, type 2 plasminogen activator inhibitor [PAI-2]) were determined for comparative purposes. Elevated levels of PAI-1 were detected in placenta extracts from PE/IUGR patients (121 +/- 38 ng/mg, n = 8) when compared with the levels in placenta extracts from normal women (43 +/- 17 ng/mg, n = 10) or women with IUGR but not PE (51 +/- 22 ng/mg, n = 11). Immunohistochemical analysis of paraffin sections showed an increased immunoreactivity for PAI-1 in the placental villous syncytiotrophoblasts from PE/IUGR women compared with the immunostaining of placental samples from the normal or IUGR group. In contrast, antigen levels and immunostaining for PAI-2 were reduced in the placentas harvested from not only the PE/IUGR women (209 +/- 144 ng/mg) but also the IUGR group (169 +/- 106 ng/mg) in comparison with the PAI-2 levels in normal placentas (535 +/- 98 ng/mg). To document that the increased immunoreactivity for PAI-1 in PE/IUGR syncytiotrophoblasts was mediated by an increased production of PAI-1 within these cells, in situ hybridization analysis was performed. A strong positive signal for PAI-1 mRNA in villous syncytiotrophoblasts from PE patients (n = 5) was obtained after 2 weeks of exposure to the NTB2 emulsion in comparison with the weak signal for PAI-1 mRNA that required a 10-week exposure of the normal placenta sections (n = 10). Northern blotting for PAI-1 mRNA showed that both transcripts (ie, 3.2 and 2.3 kb) were elevated in samples of two PE patients in comparison with the PAI-1 mRNA transcripts present in a normal placenta and an IUGR placental sample. These results show increased PAI-1 and mRNA levels in placentas from PE patients and raise the possibility that localized elevated levels of PAI-1 may play a role in the initiation of placental damage, as well as in the thrombotic complications associated with this disease.

BACKGROUND Chronic thromboembolic pulmonary hypertension is the result of nonresolving pulmonary emboli that lead to chronic obstruction of the central pulmonary arteries. METHODS AND RESULTS To determine if the failure to lyse pulmonary thromboemboli is caused by the local expression of the primary inhibitor of tissue-type plasminogen activator (type 1 plasminogen activator inhibitor, PAI-1), levels of PAI-1 antigen and mRNA were analyzed by immunohistochemistry and in situ hybridization in specimens harvested from a series of patients during pulmonary thromboendarterectomies. Red, fibrin-rich thrombi within the thromboendarterectomy specimens were lined with a single layer of endothelial cells exhibiting high levels of PAI-1 antigen. Quantitation of the in situ hybridization signal revealed that a significant increase in PAI-1 mRNA was present in the endothelial cells lining the fresh thrombi in comparison to the signal present in the endothelial cells from noninvolved areas of patients' pulmonary arteries (n = 16, P < .001). In contrast, tissue-type plasminogen activator antigen levels were low in all samples. Yellowish-white thrombi were composed of smooth muscle cells and endothelial cells in numerous vessels that stained prominently for PAI-1 antigen. Both types of cells within the highly organized tissues also exhibited elevated PAI-1 mRNA levels in comparison to patient pulmonary artery specimens that were free of thrombus (n = 16, P < .02). CONCLUSIONS The prevalence of PAI-1 expression within pulmonary thromboemboli suggests that this inhibitory may play a role in the stabilization of vascular thrombi.

The plasminogen activator (PA) proteolytic cascade has been implicated in the regulation of cell activities, including proliferation and differentiation, both of which occur continuously in normal human epidermis and are aberrant in psoriatic epidermis. To elucidate further the mechanisms by which PA is regulated in epidermis, we evaluated the levels of PA inhibitors type 1 (PAI-1) and type 2 (PAI-2) in normal and psoriatic epidermis. PAI-2, but not PAI-1, was detectable by mRNA, antigen, and activity assays, indicating that PAI-2 is the predominant epidermal PA inhibitor. In situ hybridization revealed that PAI-2 mRNA occurred throughout normal epidermis, although the signal was most intense in the granular layers. Similarly, PAI-2 antigen was most prominent in the granular layers; its distribution in these differential layers was along the cell periphery. Diffuse, fainter staining for PAI-2 was also detected in the basal cells and in some spinous layers of normal epidermis. Extracts of normal epidermis contained PA inhibitory activity identified as PAI-2 by immunoprecipitation with specific antibody. In psoriatic epidermis, PAI-2 mRNA and antigen were most prominent in the more superficial layers beneath the cornified cells. As with normal epidermis, PAI-2 assumed a pericellular distribution in the psoriatic cells. These data demonstrate that PAI-2 is constitutively expressed in vivo by keratinocytes in human epidermis and indicate that this protein is the predominant inhibitor of PA activity in normal and psoriatic human epidermis.