Differential Expression of Urokinase-Type Plasminogen Activator (uPA), Its Receptor (uPA-R), and Inhibitor Type-2 (PAI-2) during Differentiation of Keratinocytes in an Organotypic Coculture System

Cultured keratinocytes resemble migrating keratinocytes under conditions of reepithelialization during wound healing. Such keratinocytes express urokinase-type plasminogen activator (uPA) and its specific receptor (uPA receptor). Receptor-bound uPA activates plasminogen, thus providing plasmin for pericellular proteolysis. uPA is regulated by the plasminogen activator inhibitors PAI-1 and PAI-2. As indicated by immunohistology, neither uPA nor uPA receptor is expressed in normal epidermis. Thus, the down-regulation of uPA and uPA-receptor expression in keratinocytes appears to be an important event in epidermal healing and restoration of a normal epidermal tissue architecture. We have addressed this matter by using a culture and differentiation system for keratinocytes in vitro. Keratinocytes were grown in organotypic cocultures for 4, 7, and 14 days. Frozen sections were analyzed with indirect immunofluorescence staining and overlay zymography, the latter detecting activity of plasminogen activators. While tPA and PAI-I stainings were consistently negative over the entire observation period, uPA and uPA receptor were expressed by basal keratinocytes at Days 4 and 7, but not at Day 14. Accordingly, overlay zymography revealed uPA activity at Days 4 and 7. PAI-2 was found throughout the entire observation period, but with varying distribution: at Days 4 and 7 all suprabasal keratinocytes stained positive for PAI-2. At Day 14, PAI-2-specific stainings were confined to the uppermost cells of the stratum spinosum. Our data demonstrate that uPA and uPA receptor, which are up-regulated in cultured keratinocytes, are down-regulated upon restoration of an epidermis-like structure. The distribution of PAI-2 varied over the observation period and at Day 14 resembled the distribution of PAI-2 in normal epidermis. Taken together, keratinocytes in organotypic coculture behave like keratinocytes in healing wounds in vivo with respect to the expression of the plasminogen activator system.     

Does Plasminogen Activator Inhibitor-1 Drive Lymphangiogenesis?

The purpose of this study is to explore the function of plasminogen activator inhibitor-1 (PAI-1) during pathological lymphangiogenesis. PAI-1, the main physiological inhibitor of plasminogen activators is involved in pathological angiogenesis at least by controlling extracellular proteolysis and by regulating endothelial cell survival and migration. Protease system''s role in lymphangiogenesis is unknown yet. Thus, based on its important pro-angiogenic effect, we hypothesized that PAI-1 may regulate lymphangiogenesis associated at least with metastatic dissemination of cancer cells. To address this issue, we studied the impact of PAI-1 deficiency in various murine models of tumoral lymphangiogenesis. Wild-type PAI-1 proficient mice were used as controls. We provide for the first time evidence that PAI-1 is dispensable for tumoral lymphangiogenesis associated with breast cancers either induced by mammary carcinoma cell injection or spontaneously appearing in transgenic mice expressing the polyomavirus middle T antigen (PymT) under the control of a mouse mammary tumor virus long-terminal repeat promoter (MMTV-LTR). We also investigated inflammation-related lymphatic vessel recruitment by using two inflammatory models. PAI-1 deficiency did neither affect the development of lymphangioma nor burn-induced corneal lymphangiogenesis. These novel data suggest that vascular remodelling associated with lymphangiogenesis and angiogenesis involve different molecular determinants. PAI-1 does not appear as a potential therapeutic target to counteract pathological lymphangiogenesis.     

Plasminogen Activator Inhibitor-1 Suppresses Profibrotic Responses in Fibroblasts from Fibrotic Lungs

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive interstitial scarification. A hallmark morphological lesion is the accumulation of myofibroblasts or fibrotic lung fibroblasts (FL-fibroblasts) in areas called fibroblastic foci. We previously demonstrated that the expression of both urokinase-type plasminogen activator (uPA) and the uPA receptor are elevated in FL-fibroblasts from the lungs of patients with IPF. FL-fibroblasts isolated from human IPF lungs and from mice with bleomycin-induced pulmonary fibrosis showed an increased rate of proliferation compared with normal lung fibroblasts (NL-fibroblasts) derived from histologically “normal” lung. Basal expression of plasminogen activator inhibitor-1 (PAI-1) in human and murine FL-fibroblasts was reduced, whereas collagen-I and α-smooth muscle actin were markedly elevated. Conversely, alveolar type II epithelial cells surrounding the fibrotic foci in situ, as well as those isolated from IPF lungs, showed increased activation of caspase-3 and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct (Ad-PAI-1) suppressed expression of uPA and collagen-I and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased collagen-I and α-smooth muscle actin. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased collagen-I and uPA. These changes were associated with increased Akt/phosphatase and tensin homolog proliferation/survival signals in FL-fibroblasts, which were reversed by transduction with Ad-PAI-1. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of fibrosing lung disease and, in particular, IPF.     

Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1

Thrombosis is a leading cause of death worldwide. Recombinant tissue-type plasminogen activator (tPA) is the Food and Drug Administration-approved thrombolytic drug. tPA is rapidly inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort. Precise details, with atomic resolution, of the molecular interactions between tPA and PAI-1 remain unknown despite previous extensive studies. Here, we report the crystal structure of the tPA·PAI-1 Michaelis complex, which shows significant differences from the structure of its urokinase-type plasminogen activator analogue, the uPA·PAI-1 Michaelis complex. The PAI-1 reactive center loop adopts a unique kinked conformation. The structure provides detailed interactions between tPA 37- and 60-loops with PAI-1. On the tPA side, the S2 and S1β pockets open up to accommodate PAI-1. This study provides structural basis to understand the specificity of PAI-1 and to design newer generation of thrombolytic agents with reduced PAI-1 inactivation.     

Abrogation of Plasminogen Activator Inhibitor-1-Vitronectin Interaction Ameliorates Acute Kidney Injury in Murine Endotoxemia

Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1^−/−) or expressing a PAI-1-mutant (PAI-1^R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1^−/− and PAI-1^R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1^−/− mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.     

Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

Background

We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE).

Methods

Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α₂-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl₂, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405).

Results

Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT.

Conclusion

The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT.     

Plasminogen Activator Inhibitor-1 Represses Integrin- and Vitronectin-Mediated Cell Migration Independently of Its Function as an Inhibitor of Plasminogen Activation

Cell migration involves the integrins, their extracellular matrix ligands, and pericellular proteolytic enzyme systems. We have studied the role of plasminogen activator inhibitor-1 (PAI-1) in cell migration, using human amnion WISH cells and human epidermoid carcinoma HEp-2 cells in an assay measuring migration from microcarrier beads and a modified Boyden-chamber assay. Active, but not latent or reactive center-cleaved, PAI-1 inhibited migration. A PAI-1 mutant without ability to inhibit plasminogen activation was as active as wild-type PAI-1 as a migration inhibitor, showing that inhibition of plasminogen activation was not involved. PAI-1 specifically interfered with integrin- and vitronectin-mediated migration: Migration onto vitronectin-coated but not onto fibronectin-coated surfaces was inhibited by PAI-1, a cyclic RGD peptide inhibited migration, and both cell lines expressed vitronectin-binding α_v-integrins. In addition, active PAI-1, but not latent or reactive center-cleaved PAI-1, inhibited vitronectin binding to integrins in anin vitrobinding assay, without affecting binding of fibronectin. Monoclonal antibodies against the urokinase receptor, another vitronectin binding protein, did not affect cell migration in the beads assay, while some inhibitory effect was observed in the Boyden-chamber assay. We conclude that PAI-1, independently of its role as a proteinase inhibitor, inhibits cell migration by competing for vitronectin binding to integrins, while the interference of PAI-1 with binding of vitronectin to the urokinase receptor may play a secondary role. These data define a novel function for the serpin PAI-1, enabling it to regulate cell migration over vitronectin-rich extracellular matrix in the body.     

Mice deficient in plasminogen activator inhibitor-1 have improved skeletal muscle regeneration

Skeletal muscle possesses a remarkable capacity for regeneration. Although the regulation of this process at the molecular level remains largely undefined, the plasminogen system appears to play a critical role. Specifically, mice deficient in either urokinase-type plasminogen activator (uPA^–/– mice) or plasminogen demonstrate markedly impaired muscle regeneration after injury. In the present study, we tested the hypothesis that loss of the primary inhibitor of uPA, plasminogen activator inhibitor-1 (PAI-1), would improve muscle regeneration. Repair of the extensor digitorum longus muscle was assessed after cardiotoxin injury in wild-type, uPA^–/–, and PAI-1-deficient (PAI-1^–/–) mice. As expected, there was no uPA activity in the injured muscles of uPA^–/– mice, and muscles from these transgenic animals demonstrated impaired regeneration. On the other hand, uPA activity was increased in injured muscle from PAI-1^–/– mice to a greater extent than in wild-type controls. Furthermore, PAI-1^–/– mice demonstrated increased expression of MyoD and developmental myosin after injury as well as accelerated recovery of muscle morphology, protein levels, and muscle force compared with wild-type animals. The injured muscles of PAI-1-null mice also demonstrated increased macrophage accumulation, contrasting with impaired macrophage accumulation in uPA-deficient mice. The extent of macrophage accumulation correlated with both the clearance of protein after injury and the efficiency of regeneration. Taken together, these results indicate that PAI-1 deficiency promotes muscle regeneration, and this protease inhibitor represents a therapeutic target for enhancing muscle regeneration. muscle injury; muscle repair; urokinase-type plasminogen activator; muscle inflammation; macrophage     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

Plasminogen activator inhibitor (type-1) in rat adrenal medulla

Summary Plasminogen activator inhibitor type-1 (PAI-1) was identified in extracts of rat adrenal medulla, and its immunohistochemical localization was studied together with that of tissue-type plasminogen activator (t-PA). By staining of adjacent sections and by doublestaining of the same section we demonstrate that the same cells of the adrenal medulla contain both PAI-1 and t-PA immunoreactivity in the cytoplasm. In addition a few ganglion cells of the adrenal medulla were found to contain PAI-1 but not t-PA. Neither of the components were found in the adrenal cortex. Analysis of extracts from isolated adrenal medulla using reverse zymography showed the presence of a plasminogen activator inhibitor with M _r46000. The inhibitory activity disappeared when the extract was passed through a column with sepharose-coupled anti-PAI-1 IgG, while the run-through from a similar column coupled with preimmune IgG still contained the inhibitor. The present findings suggest that PAI-1 could play a role in the regulation of t-PA activity in the rat adrenal gland medullary cells.     

Effects of a High-Fat Diet on Spontaneous Metastasis of Lewis Lung Carcinoma in Plasminogen Activator Inhibitor-1 Deficient and Wild-Type Mice

This study investigated the effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma (LLC) in plasminogen activator inhibitor-1 deficient (PAI-1^−/−) and wild-type mice. The high-fat diet increased the number of pulmonary metastases by 60% (p<0.01), tumor cross-sectional area by 82% (p<0.05) and tumor volume by 130% (p<0.05) compared to the AIN93G diet. Deficiency in PAI-1 reduced the number of metastases by 35% (p<0.01) compared to wild-type mice. In mice fed the high-fat diet, PAI-1 deficiency reduced tumor cross-sectional area by 52% (p<0.05) and tumor volume by 61% (p<0.05) compared to their wild-type counterparts; however, PAI-1 deficiency affected neither area nor volume in mice fed the AIN93G diet. Adipose and plasma concentrations of PAI-1 were significantly higher in high-fat fed wild-type mice than in their AIN93G-fed counterparts. Adipose and plasma PAI-1 were not detectable in PAI-1^−/− mice regardless of the diet. Mice deficient in PAI-1 showed significantly greater plasma concentrations of monocyte chemotactic protein-1, tumor necrosis factor-α, leptin, vascular endothelial growth factor, tissue inhibitor of metalloproteinase-1 and insulin compared to wild-type mice, indicating a compensatory overproduction of inflammatory cytokines, angiogenic factors and insulin in the absence of PAI-1. We conclude that PAI-1 produced by the host, including that by adipose tissue, promotes high-fat enhanced metastasis of LLC.     

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1

Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC₅₀ values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1.     

Plasminogen activator inhibitor-type 1 in Lewis lung carcinoma

Summary Plasminogen activator inhibitor-type 1 (PAI-1) was identified in extracts of Lewis lung carcinoma, and its immunohistochemical localization was studied together with that of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators. All primary tumors (n=11) contained heterogeneously distributed immunoreactivity against each of the three components. Most often, areas that contained u-PA immunoreactivity also contained PAI-1 immunoreactivity. However, several areas showed a strong u-PA immunoreactivity, but no or low PAI-1 immunoreactivity. The latter staining pattern was only found in periferal areas, and usually in areas with histological signs of tissue destruction. Lung metastases always contained u-PA immunoreactivity, while PAI-1 immunoreactivity was found in most, but not all, metastases. t-PA immunoreactivity was found in a few scattered tumor cells, in primary carcinomas as well as metastases. Controls that included absorption with highly purified antigen preparations and immunoblotting, indicated that all the immunoreactivity represented genuine PAI-1, u-PA and t-PA, respectively. The results are consistent with an assumption that the plasminogen activation system, and particularly u-PA and PAI-1, plays a role in regulation of breakdown of extracellular matrix proteins during invasive growth in this carcinoma.     

Relationship of Plasminogen Activator to Fibrin

THERE are two principal theories of the mechanism of thrombus dissolution by the fibrinolytic system. Alkjaersig et al.¹ suggested that as fibrin polymerizes, plasminogen is adsorbed preferentially to the fibrin and is available in large quantities within a thrombus which is comparatively free of antiplasmin. When an activator enters the circulation it diffuses into the clot converting the plasminogen to plasmin in situ and so promotes lysis. Ambrus and Markus², however, proposed that when plasmin forms in the circulation naturally or during infusion of an activator it is normally bound to the excess antiplasmin present in blood. They suggested that this plasmin/antiplasmin complex is reversible and dissociates in the presence of fibrin, its preferred substrate, so allowing the plasmin to bring about fibrin dissolution by “external lysis”. Neither of these theories, however, is supported by an observed phenomena.     

Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis.

Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.     

表达人PAI-1的重组腺病毒的制备及检测

人纤溶酶原激活剂抑制物1（PAI-1）基因与复制缺陷型腺病毒载体AdCMVHSgD重组,与pJM17质粒共转染293细胞,采用不铺琼脂的方法产生重组病毒.PCR证实PAI-1基因重组进入腺病毒.进一步感染B16（F10）细胞,细胞表面洗提物和上清分别经纤维显示胶和反向纤维蛋白显示胶检测PAI-1抑制纤溶酶原激活剂（PA）的活性.     

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial.     

靶向纤溶酶原激活物抑制剂1的肿瘤治疗研究进展

肿瘤是严重危害人类健康的疾病.研究表明,实体瘤周围环境中的胞外基质蛋白、浸润性免疫细胞和间充质细胞分泌的蛋白质组等均与肿瘤的发生、发展以及肿瘤治疗的耐受性等密切相关.肿瘤微环境中一个重要调控因子,纤溶酶原激活物抑制剂1 (plasminogen activator inhibitor-1,PAI-1),不仅与组织型纤溶酶原激活物(tissue-type plasminogen activators,tPA)构成调节纤溶活性的一对关键物质,而且参与肿瘤的侵袭、浸润和转移等多个环节并扮演重要角色.本文针对近年来PAI-1的结构和功能方面研究新进展及其与肿瘤微环境的相关性进行综述,并提出PAI-1可作为抗肿瘤治疗的重要靶点.同时,本文也分析了PAI-1抑制剂对肿瘤干预的研究现状,指出PAI-1抑制剂对肿瘤治疗的潜在应用价值.