Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide.

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.

Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor.

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.

Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators.

Cell extracts and conditioned media (CM) from cultured bovine aortic endothelial cells (BAEs) were fractionated by PAGE in the presence SDS, and plasminogen activator (PA) activity was localized by fibrin autography. Multiple molecular weight forms of PA were detected in both preparations. Cell-associated PAs had Mr of 48,000, 74,000, and 100,000 while secreted PAs showed Mr of 52,000, 74,000, and 100,000. A broad zone of activity (Mr 80,000-100,000) also was present in both cellular fractions. In addition, PAs of Mr 41,000 and 30,000 appeared upon prolonged incubation or repeated freezing and thawing of the samples, and probably represent degradation products of higher molecular weight forms. This complex lysis pattern was not observed when CM was subjected to isoelectric focusing. Instead, only two classes of activator were resolved, one at pH 8.5, the other at 7.6. Analysis of focused samples by SDS PAGE revealed that the activity at pH 8.5 resulted exclusively from the Mr 52,000 form; all other forms were recovered at pH 7.6. The activity of the Mr 52,000 form was neutralized by anti-urokinase IgG but was not affected by antitissue activator IgG indicating that it is a urokinaselike PA. The activities of the Mr 74,000-100,000 forms were not affected by anti-urokinase. They were blocked by antitissue activator suggesting that all the forms in this group were tissue-type PAs. The multiple forms of PA were differentially sensitive to inactivation by diisopropylfluorophosphate (DFP). Treatment of CM with 10 mM DFP for 2 h at 37 degrees C only partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton PA. The activity of the Mr 100,000 form was not affected by this treatment, or by treatment with 40 mM DFP. Thus, cultured BAEs produce multiple, immunologically distinct forms of PA which differ in size, charge, and sensitivity to DFP. These forms include both urokinaselike and tissue-activator-like PAs. The possibility that one of these forms is a zymogen is discussed.

Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells.

Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAI-1) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R.R., T.J. Podor, E. Dunne, J. Mimuro, and D.J. Loskutoff. J. Cell Biol. 110:155-163), analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAI-1 and then with gold-conjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (less than 1-2% of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor alpha (TNF alpha; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 micrograms/ml, 24 h) resulted in an increased staining of PAI-1 not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAI-1 with purified PAI-1, or treatment of HUVECs with tissue-type plasminogen activator (2.5 micrograms/ml, 2 h, 4 degrees C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonist-activated HUVECs by 80-95%. The topographical location of PAI-1 on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNF alpha- or LPS-activated HUVECs revealed a general increase in PAI-1 staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAI-1-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immuno-electron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAI-1. These increases in surface PAI-1 could be detected by 3 h and continued over a 24-h period. The expression of PAI-1 on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.

The majority of type 1 plasminogen activator inhibitor associated with cultured human endothelial cells is located under the cells and is accessible to solution-phase tissue-type plasminogen activator.

The interactions between exogenously added tissue-type plasminogen activator (t-PA) and the active form of type 1 plasminogen activator inhibitor (PAI-1) produced by and present in cultured human umbilical vein endothelial cells (HUVECs) were investigated. Immunoblotting analysis of the conditioned media obtained from monolayers of HUVECs treated with increasing concentrations of t-PA (less than or equal to 10 micrograms/ml) revealed a dose-dependent formation of both t-PA/PAI-1 complexes, and of a 42,000-Mr cleaved or modified form of the inhibitor. Immunoradiometric assays indicated that t-PA treatment resulted in a fourfold increase in PAI-1 antigen present in the conditioned media. This increase did not result from the release of PAI-1 from intracellular stores, but rather reflected a t-PA-dependent decrease in the PAI-1 content of the Triton X-100 insoluble extracellular matrix (ECM). Although the rate of t-PA-mediated release of PAI-1 was increased by the removal of the monolayer, similar quantities of PAI-1 were removed in the presence or absence of the cells. These results suggest that the cells only represent a semipermeable barrier between ECM-associated PAI-1 and exogenous t-PA. Treatment of HUVECs with t-PA (1 microgram/ml, 2 h) to deplete the ECM of PAI-1 did not affect the subsequent rate of PAI-1 production and deposition into the ECM. Immunogold electron microscopy of HUVECs not only confirmed the location of PAI-1 primarily in the region between the culture substratum and ventral cell surface but failed to demonstrate significant (less than 1%) PAI-1 on the cell surface. Thus, the majority of PAI-1 associated with cultured HUVEC monolayers is present under the cells in the ECM and is accessible to solution-phase t-PA.

Is plasminogen activator inhibitor-1 the molecular switch that governs urokinase receptor-mediated cell adhesion and release?

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.

Effect of thrombin on the fibrinolytic activity of cultured bovine endothelial cells.

The vascular endothelium is a rich source of plasminogen activator (PA) and thus of blood vessel-associated fibrinolytic activity. Cultured bovine aortic endothelial cells were employed to determine if components of the coagulation system interact with the endothelium to modify expression of this activity. The addition of thrombin to these cultures led to a rapid decline in intracellular PA activity, with as little as 3 ng/ml, or 0.1 nM thrombin causing a 50% decrease within 30 min. Thrombin inactivated with diisopropylflurophosphate or hirudin did not elicit the response. Although control cultures secreted high levels of PA, no PA activity could be detected in the media surrounding the thrombin-treated cells. This loss of activity did not appear to result from direct inactivation of PA by thrombin. These observations indicate that the fibrinolytic potential of cultured endothelial cells is rapidly suppressed by trace amounts of thrombin. The generation of thrombin at sites of vascular injury may have a similar effect on the endothelium.

Regulation of murine type 1 plasminogen activator inhibitor gene expression in vivo. Tissue specificity and induction by lipopolysaccharide, tumor necrosis factor-alpha, and transforming growth factor-beta.

The regulation of type 1 plasminogen activator inhibitor (PAI-1) gene expression was studied in vivo employing a murine model system. Nuclease protection analysis revealed relatively high concentrations of PAI-1 mRNA in the aorta, adipose tissue, heart, and lungs of untreated CB6 (BalbC X C57B16) mice. Treatment of CB6 mice with LPS, TNF-alpha, or transforming growth factor-beta (TGF-beta) increased the steady-state levels of PAI-1 mRNA within 3 h in all tissues examined. However, the greatest responses to TGF-beta were observed in adipose tissue and the kidney, while LPS and TNF-alpha strongly stimulated PAI-1 gene expression in the liver, kidney, lung, and adrenals. In C3H/HeJ mice, which exhibit defective TNF-alpha release in response to LPS, the response of the PAI-1 gene to LPS was severely attenuated. However, injection of these mice with TNF-alpha increased PAI-1 mRNA in a tissue-specific pattern strikingly similar to that observed in LPS-treated CB6 mice. These results demonstrate that the PAI-1 gene is regulated in a complex and tissue-specific manner in vivo, and suggest a role for TNF-alpha in the response of the PAI-1 gene to sepsis.

Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator.

The primary hypothesis of this report is that the formation and subsequent removal of fibrin in specific tissues during pathologic processes reflects temporal changes in the local expression of key procoagulant and fibrinolytic genes. To begin to test this hypothesis, we have used quantitative PCR assays and in situ hybridization analysis to examine the effects of endotoxin on the expression of specific genes in murine tissues, and to relate these changes to fibrin deposition/dissolution using immunohistochemical approaches. Endotoxin caused large increases in plasminogen activator inhibitor-1 mRNA and modest increases in tissue factor mRNA in most tissues examined. However, fibrin was only detected in the kidneys and adrenals of endotoxin-treated mice, and it was transient. Unexpectedly, changes in urokinase-type plasminogen activator mRNA but not tissue-type plasminogen activator mRNA correlated with fibrin deposition/dissolution in these tissues. Pretreatment of mice with the fibrinolytic inhibitor epsilon-aminocaproic acid before endotoxin increased both the number of fibrin-positive tissues and the duration of fibrin deposition in the kidneys and adrenals. These results suggest that the absence of fibrin in some tissues reflects ongoing local fibrinolysis, and that increases in plasminogen activator inhibitory and tissue fac- tor gene expression and decreases in urokinase-type plasminogen activator expression are necessary for tissue-specific fibrin deposition. Changes in tissue-type plasminogen activator gene expression do not appear to be essential for fibrin deposition/dissolution in this murine model of sepsis.

Distribution and regulation of plasminogen activator inhibitor-1 in murine adipose tissue in vivo. Induction by tumor necrosis factor-alpha and lipopolysaccharide.

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.

Leptin-dependent platelet aggregation and arterial thrombosis suggests a mechanism for atherothrombotic disease in obesity.

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.

Detection and partial characterization of an inhibitor of plasminogen activator in human platelets.

In this study, we demonstrate the presence of a previously undescribed fibrinolytic inhibitor in human serum. It has an apparent molecular weight of 50,000 and is not detected in serum derived from platelet-poor plasma, suggesting that it originates from platelets. This conclusion is supported by a number of observations. For example, extracts of washed, gel-filtered human platelets contain an inhibitor of similar activity and size, and physiological concentrations of thrombin induce its release from the platelets. Moreover, the kinetics and dose dependency of this release are similar to those observed for the release of platelet factor 4, and the release of both molecules is blocked by pretreating the platelets with prostaglandin E1 and theophylline. Mixing experiments, which were devised to investigate the specificity of the inhibitor, showed that the fibrinolytic activity initiated by both urokinase and tissue-type plasminogen activator was blocked by platelet releasate in a dose-dependent manner. In both cases, the amount of inhibition increased when the releasates were preincubated with the purified activators, indicating a direct interaction between the activators and an inhibitor(s). The inhibitory activity was removed by preincubating the releasates with antiserum prepared against an antiactivator purified from cultured bovine aortic endothelial cells. These results indicate that platelets contain an inhibitor which is released by thrombin, inhibits both urokinase and tissue-type plasminogen activator, and is immunologically similar to an inhibitor produced by endothelial cells. This molecule may represent a new class of inhibitors, the antiactivators, which function together with alpha 2-antiplasmin to regulate the fibrinolytic system of the blood. Its release from platelets by thrombin may protect the growing thrombus against premature dissolution initiated by plasminogen activators released by the endothelium.

Regulation of the fibrinolytic system of cultured human vascular endothelium by interleukin 1.

We examined the effects of human interleukin 1 (IL-1) on the production of fibrinolytic components by cultured human vascular endothelium. Conditioned media collected from IL-1-treated (5 U/ml, 24 h) monolayers exhibited decreased tissue-type plasminogen activator (tPA) activity and increased plasminogen activator inhibitor (PAI) activity, as assessed by fibrin and reverse fibrin-autography. Quantitative immunological assays revealed a 35% decrease in tPA antigen and a 360% increase in active PAI antigen, after incubation for 24 h with 0.6 U/ml IL-1. Maximal effects (approximately 50% decrease in tPA antigen; 400-800% increase in active PAI antigen) were observed with 2.5-5 U/ml IL-1. Changes in tPA and PAI reached a maximum at approximately 24 h and persisted for greater than 48 h. IL-1 induction of endothelial procoagulant activity was more rapid and transient, peaking by 6 h and subsiding by 24 h. Natural monocyte-derived IL-1 and two species of recombinant IL-1 had comparable effects. Heat and polymyxin-B treatments differentiated IL-1 actions from those of endotoxin, which promoted similar endothelial alterations. IL-1 effects on endothelial procoagulant and fibrinolytic activities may contribute to the generation and maintenance of fibrin in pathophysiological settings in vivo.

Human melanoma cells derived from lymphatic metastases use integrin alpha v beta 3 to adhere to lymph node vitronectin.

Human melanoma is a highly metastatic cancer and the regional lymph nodes are generally the first site of metastasis. Adhesion to cryostat sections of human lymph nodes was therefore studied using two human melanoma models established from lymph node metastases, namely, MeWo cell lines of diverse metastatic potentials and a highly metastatic cell line of recent origin designated MIM/8. We found a good correlation between the metastatic potentials of the melanoma cells as measured in nude mice and their ability to adhere to cryostat sections of human lymph nodes. When adhesion to immobilized extracellular matrix proteins was measured, a significant increase in adhesion, which correlated with increased metastasis, was seen mainly on vitronectin and to a lesser extent on fibronectin. The adhesion to vitronectin and to the frozen sections were specifically blocked by an RGD-containing peptide, mAb 661 to vitronectin and mAb LM609 to integrin alpha v beta 3. FACS analysis revealed a significant and specific increase in cell surface expression of alpha v beta 3 on the metastatic cells as compared to the parent line. Together these results suggest that the adhesion of melanoma cells to lymph node vitronectin via the alpha v beta 3 receptor plays a role in the process of lymphatic dissemination.

Activation of rabbit Hageman factor by homogenates of cultured rabbit endothelial cells.

Rabbit Hageman factor was proteolytically cleaved and activated by a homogenate prepared from cultured rabbit endothelial cells. Cleavage of radiolabeled Hageman factor was monitored by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Endothelial cell-mediated cleavage of Hageman factor was demonstrated both in a purified system and in plasma, was time and concentration dependent, and was associated with formation of the characteristic 28,000 M(r) form of active Hageman factor. The rate of cleavage of Hageman factor was not affected by Triton X-100 (Rohm and Haas, Co., Philadelphia, Pa.), hexadimethrine bromide (Polybrene, Aldrich Chemical Co., Inc., Milwaukee, Wis.), hirudin, soybean trypsin inhibitor, or antisera to plasminogen or prekallikrein. However, cleavage was enhanced by kaolin, and was inhibited by diisopropyl-fluorophosphate. The enzyme responsible for cleavage of Hageman factor was localized to the 100,000-g-sedimentable, subcellular fraction of the endothelial cell homogenate and was relatively specific, because neither radiolabeled rabbit Factor XI nor rabbit prekallikrein were themselves proteolytically cleaved by the endothelial cell homogenate. However, when these molecules were incubated with the homogenate in the presence of Hageman factor, both Factor XI and prekallikrein were cleaved, demonstrating that Hageman factor had been activated by the endothelial cell homogenate. Furthermore, the kallikrein generated by endothelial cell homogenate-activated Hageman factor was capable of liberating kinin from high molecular weight kininogen as measured by bioassay. Cultured rabbit endothelial cells, therefore, possess the capacity to activate Hageman factor by proteolysis. This may be one mechanism for Hageman factor activation in vivo.

Astrocytes are the primary source of tissue factor in the murine central nervous system. A role for astrocytes in cerebral hemostasis.

Hemostasis in the brain is of paramount importance because bleeding into the neural parenchyma can result in paralysis, coma, and death. Consistent with this sensitivity to hemorrhage, the brain contains large amounts of tissue factor (TF), the major cellular initiator of the coagulation protease cascades. However, to date, the cellular source for TF in the central nervous system has not been identified. In this study, analysis of murine brain sections by in situ hybridization demonstrated high levels of TF mRNA in cells that expressed glial fibrillary acidic protein, a specific marker for astrocytes. Furthermore, primary mouse astrocyte cultures and astrocyte cell lines from mouse, rat, and human constitutively expressed TF mRNA and functional protein. These data indicated that astrocytes are the primary source of TF in the central nervous system. We propose that astrocytes forming the glia limitans around the neural vasculature and deep to the meninges are intimately involved in controlling hemorrhage in the brain. Finally, we observed an increase in TF mRNA expression in the brains of scrapie-infected mice. This modulation of TF expression in the absence of hemorrhage suggested that TF may function in processes other than hemostasis by altering protease generation in normal and diseased brain.

Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse.

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.

Protein C inhibitor is expressed in tubular cells of human kidney.

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.

Expression of fibrinolytic genes in atherosclerotic abdominal aortic aneurysm wall. A possible mechanism for aneurysm expansion.

Expansion of atherosclerotic abdominal aortic aneurysm (AAA) has been attributed to remodeling of the extracellular matrix by active proteolysis. We used in situ hybridization to analyze the expression of fibrinolytic genes in aneurysm wall from eight AAA patients. All specimens exhibited specific areas of inflammatory infiltrates with macrophage-like cells expressing urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) mRNA. Type 1 PA inhibitor (PAI-1) mRNA was expressed at the base of the necrotic atheroma of all specimens and also within some of the inflammatory infiltrates where it frequently colocalized in regions containing u-PA and t-PA mRNA expressing cells. However, in these areas, the cellular distribution of the transcripts for t-PA and u-PA extended far beyond the areas of PAI-1 expression. These observations suggest a local ongoing proteolytic process, one which is only partially counteracted by the more restricted expression of PAI-1 mRNA. An abundance of capillaries was also obvious in all inflammatory infiltrates and may reflect local angiogenesis in response to active pericellular fibrinolysis. The increased fibrinolytic capacity in AAA wall may promote angiogenesis and contribute to local proteolytic degradation of the aortic wall leading to physical weakening and active expansion of the aneurysm.

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition.

Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.