Recombinant Plasminogen Activator Inhibitor-1 Research Articles

Embedded Human Periodontal Ligament Stem Cells Spheroids Enhance Cementogenic Differentiation via Plasminogen Activator Inhibitor 1.

Spheroids reproduce the tissue structure that is found in vivo more accurately than classic two-dimensional (2D) monolayer cultures. We cultured human periodontal ligament stem cells (HPLSCs) as spheroids that were embedded in collagen gel to examine whether their cementogenic differentiation could be enhanced by treatment with recombinant human plasminogen activator inhibitor-1 (rhPAI-1). The upregulated expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP), established cementoblast markers, was observed in the 2D monolayer HPLSCs that were treated with rhPAI-1 for 3 weeks compared with that in the control and osteogenic-induction medium groups. In the embedded HPLSC spheroids, rhPAI-1 treatment induced interplay between the spheroids and collagenous extracellular matrix (ECM), indicating that disaggregated HPLSCs migrated and spread into the surrounding ECM 72 h after three-dimensional (3D) culture. Western blot and immunocytochemistry analyses showed that the CEMP1 expression levels were significantly upregulated in the rhPAI-1-treated embedded HPLSC spheroids compared with all the 2D monolayer HPLSCs groups and the 3D spheroid groups. Therefore, 3D collagen-embedded spheroid culture in combination with rhPAI-1 treatment may be useful for facilitating cementogenic differentiation of HPLSCs.

Role of plasminogen activator inhibitor-1 in oral tongue squamous cell carcinoma: An immunohistochemical and in vitro analysis

ObjectiveTo evaluate the influence of plasminogen activator inhibitor-1 (PAI-1) on the biological behavior and prognosis of oral tongue squamous cell carcinoma (OTSCC). MethodsImmunoexpression of PAI-1 was analyzed in 60 OTSCC specimens and classified as low-expression (≤50% of positive cells) or high-expression (>50%). In vitro effects of recombinant human PAI-1 (rhPAI-1) were assessed through functional assays on the OTSCC-derived cell line SCC-25. Three cell groups were evaluated: G0 (control), G10 (10 nM rhPAI-1), and G20 (20 nM rhPAI-1). ResultsHigh membrane expression of PAI-1 was associated with tumor budding (p = 0.046) and high-risk cases (p = 0.043). Cytoplasmic and membrane expression of PAI-1 was not associated with patient survival. Cell viability (p = 0.020) and progression to the S-phase of the cell cycle (p = 0.024) were higher in G10 and G20 at 24 h. The percentages of apoptotic/necrotic cells were not affected by rhPAI-1. The presence of rhPAI-1 increased cell migration (p = 0.039) and invasion (p = 0.039) after 24 and 72 h, respectively. ConclusionOur findings indicate the involvement of PAI-1 in the biological behavior of OTSCC, although its expression may not predict patient survival. The in vitro results suggest that PAI-1 stimulates cell proliferation, migration and invasion and may contribute to the aggressive phenotype of OTSCC.

Lupeol suppresses plasminogen activator inhibitor-1-mediated macrophage recruitment and attenuates M2 macrophage polarization

Tumor-associated macrophages (TAMs) are closely related with poor prognosis of cancers. The current study investigated whether lupeol regulates TAMs by focusing on the recruitment and polarization of macrophages. We found that lupeol suppressed the recruitment of THP-1 macrophages (THP-1 cells differentiated into macrophages) towards H1299 lung carcinoma cells by inhibiting plasminogen activator inhibitor-1 (PAI-1) production from H1299 cells. The reduced migration of THP-1 macrophages by lupeol was recovered by adding recombinant human PAI-1 as a chemoattractant. Knockdown of PAI-1 or treatment of tiplaxtinin, a PAI-1 inhibitor, in H1299 cells abrogated the chemotaxis of macrophages. Furthermore, lupeol suppressed the interleukin (IL)-4- and IL-13-induced M2 macrophage polarization. The mRNA expression of M2 macrophage markers and the phosphorylation of signal transducer and activator of transcription 6 (STAT6) were commonly decreased by lupeol in RAW264.7 cells. In addition, lupeol-suppressed M2 macrophage polarization led to the reduced migration of Lewis lung carcinoma (LLC) cells. Taken together, our results suggest that lupeol attenuates PAI-1-mediated macrophage recruitment towards cancer cells and inhibits M2 macrophage polarization.

Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome.

Enhanced expression of PAI-1 (plasminogen activator inhibitor-1) has been implicated in atherosclerosis formation in humans with obesity and metabolic syndrome. However, little is known about the effects of pharmacological targeting of PAI-1 on atherogenesis. This study examined the effects of pharmacological PAI-1 inhibition on atherosclerosis formation in a murine model of obesity and metabolic syndrome. Approach and Results: LDL receptor-deficient (ldlr-/-) mice were fed a Western diet high in cholesterol, fat, and sucrose to induce obesity, metabolic dysfunction, and atherosclerosis. Western diet triggered significant upregulation of PAI-1 expression compared with normal diet controls. Addition of a pharmacological PAI-1 inhibitor (either PAI-039 or MDI-2268) to Western diet significantly inhibited obesity and atherosclerosis formation for up to 24 weeks without attenuating food consumption. Pharmacological PAI-1 inhibition significantly decreased macrophage accumulation and cell senescence in atherosclerotic plaques. Recombinant PAI-1 stimulated smooth muscle cell senescence, whereas a PAI-1 mutant defective in LRP1 (LDL receptor-related protein 1) binding did not. The prosenescent effect of PAI-1 was blocked by PAI-039 and R2629, a specific anti-LRP1 antibody. PAI-039 significantly decreased visceral adipose tissue inflammation, hyperglycemia, and hepatic triglyceride content without altering plasma lipid profiles. Pharmacological targeting of PAI-1 inhibits atherosclerosis in mice with obesity and metabolic syndrome, while inhibiting macrophage accumulation and cell senescence in atherosclerotic plaques, as well as obesity-associated metabolic dysfunction. PAI-1 induces senescence of smooth muscle cells in an LRP1-dependent manner. These results help to define the role of PAI-1 in atherosclerosis formation and suggest a new plasma-lipid-independent strategy for inhibiting atherogenesis.

PAI-1 overexpression promotes invasion and migration of esophageal squamous carcinoma cells.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. Plasminogen activator inhibitor-1 (PAI-1), encoded by SERPINE1, is highly expressed in various types of tumor tissues, which contributes to cancer progression. The present study explored the role and underlying mechanisms of PAI-1 in ESCC. We found that the PAI-1 protein was extracellularly secreted more from ESCC cells with high PAI-1 expression using Western blotting and enzyme linked immunosorbent assay (ELISA). Knockdown of SERPINE1 expression significantly inhibited the invasion and migration of ESCC KYSE150 and KYSE450 cell lines, which could be restored when adding exogenous human recombinant PAI-1 into the culture medium of the cells stably expressing SERPINE1 shRNA. In vivo experiments showed that SERPINE1 knockdown significantly inhibited xenograft growth and lung metastasis of ESCC cells. Molecular analysis demonstrated that PAI-1 activated AKT and ERK signaling pathways. Co-immunoprecipitation (Co-IP) assays identified that PAI-1 may interact with the membrane receptor LDL receptor related protein 1 (LRP1). These results indicated that overexpression of PAI-1, through interacting with LRP1, might enhance invasion and migration of ESCC cells as well as promote ESCC progression.

The Markedly Increased PAI1 in Obesity Induces a Compensatory Increase of Hepatocyte Tpa Expression By Activating a LRP1-CREB1 Pathway

Tissue-type plasminogen activator (tPA) initiates fibrinolysis, the primary mechanism that dissolves a thrombus. We have shown that in lean mice the hepatocytes maintain a basal level of circulating tPA that influences fibrinolysis in the occurrence of a vessel injury. As a serine protease, tPA is inhibited by the serpin plasminogen activator inhibitor 1 (PAI1). Both PAI1 and tPA which can be produced by hepatocytes, which is important for regulating energy metabolism and is sensitive to metabolic stress. In obesity, despite an increase in plasma tPA, blood tPA activity and fibrinolysis are reduced primarily due to a larger increase in PAI1. However, the source and regulatory mechanism of this phenomena are unknown. Palmitate treatment of human primary hepatocytes causes an increase in tPA mRNA/protein, reflecting the situation in the obese liver. This increase, however, is overcompensated by a larger increase in PAI1 mRNA, resulting in decreased net tPA activity in the cell culture medium. Silencing PAI1 mRNA with si-SERPINE1 in these cells increased tPA activity in the culture medium. Surprisingly, silencing PAI1 also prevented the increase of tPA mRNA induced by palmitate. Building on this observation, we further treated the cells with active recombinant PAI1 (rPAI1), which induced tPA expression. Next, we fed PAI1fl/flmice with a diet-induced obesity (DIO) diet for three months and silenced their hepatic PAI1 using AAV8-TBG-Cre, which inactivates ~95% floxed gene expression in hepatocytes but not in other cell types of the liver or other tissues. We found that while plasma tPA activity was increased in hepatocyte-PAI1 knockout mice, plasma tPA protein concentration was reduced. In other words, reducing hepatocyte PAI1 in obesity stopped the compensatory increase in liver tPA mRNA and protein. Thus, PAI1 induces tPA, which is likely a compensatory response. To explore the mechanism whereby rPAI1 induces tPA, we considered CREB1, as it is a transcription activator of tPA expression in endothelial cells and is expressed in hepatocytes. We found that CREB1 was activated (phosphorylated) by rPAI1 in human primary hepatocytes and that silencing CREB1 prevented the elevated tPA expression induced by rPAI1. Next, we silenced hepatic CREB1 in obese mice by treating DIO diet-fed CREB1fl/flmice with AAV8-TBG-Cre, which blocked the compensatory increase in tPA and thus worsened the impaired fibrinolysis in obesity. LDL receptor-related protein 1 (LRP1), the major cellular receptor of PAI1, can stimulate CREB1 transcription activity in neurons and adipocytes. As LRP1 is expressed on the surface of hepatocytes, we hypothesized that PAI1 activates CREB1 through LRP1-mediated signaling to increase tPA expression in obesity. Indeed, silencing LRP1 with si-LRP1 stopped the increase of tPA expression induced by rPAI1 in human primary hepatocytes. Interestingly, treating the cells with a mutant rPAI1 lacking the LRP1-interacting heparin-binding domain was unable to induce tPA expression. In summary, hepatocyte tPA mRNA/protein is increased in obesity, but this increase is ultimately overcompensated by a larger increase in PAI1, resulting in decreased plasma tPA activity and fibrinolysis. The markedly increased PAI1, through activating LRP1-CREB1 signaling pathway, drives an increase in hepatic tPA expression as a "compensatory" pathway in obesity. Preventing this compensatory pathway in obesity resulted in the worsening of fibrinolysis. Therapeutic boosting of this compensatory pathway may provide novel strategies to restore effective fibrinolysis in obesity. Disclosures No relevant conflicts of interest to declare.

Plasminogen Activator Inhibitor 1 (PAI1) Promotes Actin Cytoskeleton Reorganization and Glycolytic Metabolism in Triple-Negative Breast Cancer.

Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression. IMPLICATIONS: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

Abstract 151: Plasminogen Activator Inhibitor-1 Stimulates Smooth Muscle Senescence and Atherosclerosis Formation

Cell senescence, which is characterized by loss of replicative capacity and secretion of inflammatory mediators, was recently shown to play a causal role in atherosclerosis. Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that regulates fibrinolysis, cell migration, and cell proliferation, promotes cell senescence. This study tested the hypotheses that 1) PAI-1 stimulates vascular smooth muscle cell (SMC) senescence and 2) pharmacologic PAI-1 inhibition attenuates senescence of SMCs and atherosclerotic plaques. Human coronary artery SMCs were grown in culture and treated with recombinant PAI-1 and PAI-039 (tiplaxtinin), a specific pharmacological inhibitor of PAI-1. SMCs were scored for the presence of senescence-associated β-galactosidase (SA-βgal), an enzyme whose expression is specifically upregulated in senescent cells. PAI-1 (10 μg/mL) significantly increased SMC senescence (28.3±1.2% senescent cells, n=6) compared to vehicle control (14.3±1.4%, n=7, p <0.05). PAI-039 (25 μM) significantly decreased PAI-1-induced SMC senescence (19.1±1.1%, n=5, p <0.05 vs PAI-1-treated cells). To examine the significance of our findings in vivo, we studied the effects of PAI-039 on cell senescence in aortas of ldlr -/- mice fed a western diet (WD) with or without PAI-039 (5 mg/g of diet). After twelve weeks of WD aortas were harvested and SA-βgal activity was measured in intact specimens with FDG substrate. SA-βgal activity was significantly less in ldrl -/- mice fed WD containing PAI-039 (366±64 FU/mg, n=7) compared to controls (591±82 FU/mg, n=7, p<0.05) . The decrease in senescence was associated with a statistically significant reduction in atherosclerosis formation, assessed by quantitative Oil Red O imaging. In summary, SMC senescence is stimulated by PAI-1 and down-regulated by PAI-039, a specific PAI-1 inhibitor. PAI-039 also significantly inhibited cell senescence in aortas of ldlr -/- mice fed WD. These results suggest that PAI-1 is an important mediator of SMC senescence and that pharmacologic targeting of PAI-1 is an effective strategy to inhibit vascular senescence and atherogenesis in vivo.

Plasminogen Activator Inhibitor-1 Reduces Tissue-Type Plasminogen Activator–Dependent Fibrinolysis and Intrahepatic Hemorrhage in Experimental Acetaminophen Overdose

Acetaminophen (APAP)-induced liver injury in mice is associated with activation of the coagulation cascade and deposition of fibrin in liver. Plasminogen activator inhibitor-1 (PAI-1) is an important physiological inhibitor of tissue-type plasminogen activator (tPA) and plays a critical role in fibrinolysis. PAI-1 expression is increased in both experimental APAP-induced liver injury and patients with acute liver failure. Prior studies have shown that PAI-1 prevents intrahepatic hemorrhage and mortality after APAP challenge, but the downstream mechanisms are not clear. We tested the hypothesis that PAI-1 limits liver-related morbidity after APAP challenge by reducing tPA-dependent fibrinolysis. Compared with APAP-challenged (300 mg/kg) wild-type mice, hepatic deposition of cross-linked fibrin was reduced, with intrahepatic congestion and hemorrhage increased in PAI-1-deficient mice 24 hours after APAP overdose. Administration of recombinant wild-type human PAI-1 reduced intrahepatic hemorrhage 24 hours after APAP challenge in PAI-1-/- mice, whereas a mutant PAI-1 lacking antiprotease function had no effect. Of interest, tPA deficiency alone did not affect APAP-induced liver damage. In contrast, fibrinolysis, intrahepatic congestion and hemorrhage, and mortality driven by PAI-1 deficiency were reduced in APAP-treated tPA-/-/PAI-1-/- double-knockout mice. The results identify PAI-1 as a critical regulator of intrahepatic fibrinolysis in experimental liver injury. Moreover, the results suggest that the balance between PAI-1 and tPA activity is an important determinant of liver pathology after APAP overdose.

Inhibition of PAI-1 Activity by Toddalolactone as a Mechanism for Promoting Blood Circulation and Removing Stasis by Chinese Herb Zanthoxylum nitidum var. tomentosum.

Traditional Chinese medicine has been used to treat a variety of human diseases for many centuries. Zanthoxylum nitidum var. tomentosum is used as an adjuvant to promote blood circulation and remove stasis. However, the mechanisms of improving circulation and other biological activities of Z. nitidum var. tomentosum are still unclear. Plasminogen activator inhibitor-1 (PAI-1) regulates the plasminogen activation system through inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA). PAI-1 has been linked to fibrin deposition that evolves into organ fibrosis and atherosclerosis. In the present study, we showed that ethanol extract prepared from Z. nitidum var. tomentosum exhibited PAI-1 inhibitory activity, and identified toddalolactone as the main active component that inhibited the activity of recombinant human PAI-1 with IC50 value of 37.31 ± 3.23 μM, as determined by chromogenic assay, and the effect was further confirmed by clot lysis assay. In vitro study showed that toddalolactone inhibited the binding between PAI-1 and uPA, and therefore prevented the formation of the PAI-1/uPA complex. Intraperitoneal injection of toddalolactone in mice significantly prolonged tail bleeding and reduced arterial thrombus weight in a FeCl3-induced thrombosis model. In addition, the hydroxyproline level in the plasma and the degree of liver fibrosis in mice were decreased after intraperitoneal injection of toddalolactone in CCl4-induced mouse liver fibrosis model. Taken together, PAI-1 inhibition exerted by toddalolactone may represent a novel molecular mechanism by which Z. nitidum var. tomentosum manifests its effect in the treatment of thrombosis and fibrosis.

Abstract 3061: Tumor-derived PAI-1 promotes macrophage M2 polarization by stimulating an autocrine IL-6 /STAT3 pathway

Abstract Macrophages can exhibit a spectrum of activation states ranging from a pro-inflammatory and antitumorigenic M1 (CD80+; IL-12high; iNOShigh) to a pro-tumorigenic M2 [CD163+; IL-10high; CD206+; Arg+ (mouse)] phenotype. Plasminogen activator inhibitor-1 (PAI-1) is overexpressed in many cancers and although it acts as a protease inhibitor it is paradoxically correlated with poor outcome. PAI-1 exerts its pro-tumorigenic role via pro-angiogenic and anti-apoptotic functions. Increasing evidence points towards the importance of PAI-1 in inflammation and tumorigenesis, but its influence on the immune component of the tumor microenvironment has not yet been investigated. Here we report a novel observation that PAI-1 is a regulator of macrophage polarization. We observe that in PAI-1 KO mice xenotransplanted with 3 human cancer cell lines (HT1080, A549, HCT116) in which PAI-1 was knocked down (KD), tumors were less infiltrated with macrophages with lower Arg expression compared to WT mice implanted with PAI-1 pos. tumors. We demonstrate that recombinant PAI-1 through its uPA interactive domain increased the expression of M2 polarization markers (CD163, IL-10) and decreased or did not affect the expression of M1 polarization markers (iNOS, CD80) in human peripheral blood monocytes. Further investigation demonstrated that treatment of monocytes with PAI-1 induced a rapid (2 h.) increase in IL-6 mRNA and secretion (4 h.) of the protein that was followed by phosphorylation (8-24 h.) of signal transduction and activation of transcription 3 (STAT3) in monocytes. Further linking STAT3 activation to PAI-1, we observed a decreased pSTAT3 in monocytes co-cultured with HT1080 cells upon PAI-1 downregulation. We found that STAT3 activation was downstream of IL-6 and responsible for M2 polarization, as the blockage of the IL-6 receptor with a function blocking antibody (tocilizumab) or inhibition of STAT3 activation by a JAK2/STAT3 inhibitor (ruxolitinib) prevented M2 polarization of monocytes by PAI-1. The contribution of PAI-1 to M2 polarization of macrophages was tested in PAI-1 KO mice implanted with human tumors in which PAI-1 expression was controlled by doxycycline. These experiments demonstrated that induction of PAI-1 in established tumors increased the presence of tumor associated macrophages (TAM) (F4/80+) and decreased the presence of M1 (iNOS+) TAM. Further supporting the presence of an autocrine PAI-1/IL-6 pathway in macrophage polarization in human cancers, a meta-analysis of gene expression array data indicated strong correlations between PAI-1 and IL-6 expression in breast (P=<0.05) and colon (P<0.01) cancers and between PAI-1 and CD163 in colon cancer (P<0.0001). The data thus identify a new pro-tumorigenic function for PAI-1 in the communication between tumor cells and macrophages where tumor-derived PAI-1 activates an autocrine IL-6/STAT3 pathway that promotes M2 polarization. Citation Format: Marta H. Kubala, Veronica R. Placencio, Yves A. DeClerck. Tumor-derived PAI-1 promotes macrophage M2 polarization by stimulating an autocrine IL-6 /STAT3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3061. doi:10.1158/1538-7445.AM2017-3061

Recombinant Human Plasminogen Activator Inhibitor-1 Accelerates Odontoblastic Differentiation of Human Stem Cells from Apical Papilla.

Dental caries, the most prevalent oral disease in dental patients, involves the phases of demineralization and destruction of tooth hard tissues like enamel, dentin, and cementum. Dentin is a major component of the root and is also the innermost layer that protects the tooth nerve, exposure of which results in pain. In this study, we used human stem cells from apical papilla (hSCAP), which are early progenitor cells, to examine the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on odontogenic differentiation in vitro and in vivo. We demonstrated that rhPAI-1 promoted the proliferation and odontogenic differentiation of hSCAP and increased the expression levels of odontoblast-associated markers. We also observed that rhPAI-1 upregulated the expression of Smad4, nuclear factor I-C (NFI-C), Runx2, and osterix (OSX) during odontogenic differentiation. Notably, transplantation of rhPAI-1-treated hSCAP effectively induced odontoblastic differentiation and dentinal formation. And the differentiated odontoblast-like cells showed numerous odontoblast processes inserted in dentin tubules and arranged collagen fibers. Furthermore, odontoblast-associated markers were more highly expressed in the rhPAI-1-induced differentiated odontoblast-like cells compared with the control group. These markers were also more highly expressed in the newly formed dentin-like tissue of the rhPAI-1-treated group compared with the control group. Consistent with our in vitro results, the expression levels of Smad4, NFI-C, and OSX were also increased in the rhPAI-1-treated group compared with the control group. Taken together, these results suggest that rhPAI-1 promotes odontoblast differentiation and dentin formation of hSCAP, and Smad4/NFI-C/OSX may play critical roles in the rhPAI-1-induced odontogenic differentiation. Thus, dental stem cells from apical papilla combined with rhPAI-1 could lead to dentin regeneration in clinical implications.

Plasminogen activator inhibitor-1 increases migration of monocytes to the tumor and skews their differentiation towards M2 macrophage phenotype (TUM6P.1002)

Abstract Macrophages exhibiting M2 (pro-tumorigenic) phenotype have been linked to cancer progression. Plasminogen activator inhibitor-1 (PAI-1) is overexpressed by a variety of tumors and indicates poor clinical outcome. This pro-tumorigenic role has been linked to pro-angiogenic and anti-apoptotic functions. Here we demonstrate that PAI-1 is an orchestrator of monocyte migration towards tumor cells and of their M2 polarization. We observed that PAI-1 KO mice xenotransplanted with HT1080 PAI-1 KD cells exhibited tumors less infiltrated with macrophages compared to WT mice (p-value: 1.27E-09). In vitro, recombinant PAI-1 (40 nM) increased monocyte migration by an average 4-fold and increased the expression of CD163 (a M2 marker) by 10-fold (Median Fluorescence Intensity 3 to 30). PAI-1 induced monocyte production of IL-6 and IL-10 to 4000 pg/mL and 600 pg/mL, respectively, and activated STAT3 in the cells. Supporting the critical role for IL-6 and STAT3 in PAI-1 effect on M2 polarization in monocytes, we show that both blocking the IL-6 receptor (tocilizumab) and STAT3 activation (ruxolitinib) in monocytes treated with PAI-1 blocked CD163 expression. Our data point to a potential new pro-tumorigenic activity of PAI-1 via an effect on monocyte migration and polarization.

Abstract 188: Selective Inhibition of Vascular Smooth Muscle Cell Migration by Targeting Plasminogen Activator Inhibitor-1

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of mammalian plasminogen activators and an important regulator of cell migration. We have shown that tiplaxtinin, a small molecule, specific inhibitor of PAI-1, inhibits intimal hyperplasia in a murine vein graft model. However, little is known about the effects of pharmacological inhibition of PAI-1 on vascular cell migration under physiologically relevant conditions. Methods: We studied the effects of tiplaxtinin on migration of smooth muscle cells (SMCs) and endothelial cells (ECs). Results: Tiplaxtinin significantly inhibited migration of murine SMCs through 3-dimensional (3-D) collagen matrix in a concentration-dependent manner. Tiplaxtinin did not inhibit SMC proliferation, and it did not inhibit migration of PAI-1-deficient SMCs, suggesting that tiplaxtinin’s effect on SMCs was non-toxic and PAI-1-dependent. The anti-migratory effect of tiplaxtinin on SMCs was preserved in collagen 3-D matrix containing vitronectin and other extracellular matrix molecules, further supporting the physiological significance of the effect. In contrast to SMCs, tiplaxtinin did not inhibit migration of human aortic ECs in vitro or murine ECs in vivo, the latter assessed in a murine carotid injury model. To study the basis for the differential effect of tiplaxtinin on SMCs vs. ECs, we compared expression of LDL receptor-related protein 1 (LRP1), a motogenic receptor for PAI-1, between cell types by RT-PCR and found that LRP1 gene expression was significantly lower in ECs than in SMCs. Furthermore, recombinant PAI-1 stimulated the migration of wild-type mouse embryonic fibroblasts (MEFs), but not LRP1-deficient MEFs. Conclusions: Tiplaxtinin, a pharmacological inhibitor of PAI-1, inhibits SMC migration under physiological conditions, while having no inhibitory effect on EC migration. The differential effect of PAI-1 inhibition on SMCs vs. ECs appears to be mediated by LRP1 and may be of clinical significance, as it is advantageous to prevent intimal hyperplasia by inhibiting SMC migration without inhibiting EC migration, which is key to preserving an intact, anti-thrombotic vascular endothelium.

Recombinant Human Plasminogen Activator Inhibitor-1 Promotes Cementogenic Differentiation of Human Periodontal Ligament Stem Cells.

The periodontium, consisting of gingiva, periodontal ligament (PDL), cementum, and alveolar bone, is necessary for the maintenance of tooth function. Specifically, the regenerative abilities of cementum with inserted PDL are important for the prevention of tooth loss. Periodontal ligament stem cells (PDLSCs), which are located in the connective tissue PDL between the cementum and alveolar bone, are an attractive candidate for hard tissue formation. We investigated the effects of recombinant human plasminogen activator inhibitor-1 (rhPAI-1) on cementogenic differentiation of human PDLSCs (hPDLSCs) in vitro and in vivo. Untreated and rhPAI-1-treated hPDLSCs mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and dentin matrix were transplanted subcutaneously into the dorsal surface of immunocompromised mice to assess their capacity for hard tissue formation at 8 and 10 weeks posttransplantation. rhPAI-1 accelerated mineral nodule formation and increased the mRNA expression of cementoblast-associated markers in hPDLSCs. We also observed that rhPAI-1 upregulated the levels of osterix (OSX) and cementum protein 1 (CEMP1) through Smad2/3 and p38 pathways, whereas specific inhibitors of Smad3 and p38 inhibited the enhancement of mineralization of hPDLSCs by rhPAI-1. Furthermore, transplantation of hPDLSCs with rhPAI-1 showed a great ability to promote cementogenic differentiation. Notably, rhPAI-1 induced hPDLSCs to regenerate cementum-like tissue with PDL fibers inserted into newly formed cementum-like tissue. These results suggest that rhPAI-1 may play a key role in cementogenic differentiation of hPDLSCs. rhPAI-1 with hPDLSCs may be a good candidate for future clinical applications in periodontal tissue regeneration and possibly in tooth root bioengineering.

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.

Targeted Inhibition of PAI-1 Activity Impairs Epithelial Migration and Wound Closure Following Cutaneous Injury.

Objective: Aberrant plasminogen activator inhibitor-1 (PAI-1) expression and activity have been implicated in bleeding disorders, multiorgan fibrosis, and wound healing anomalies. This study details the physiological consequences of targeted PAI-1 functional inhibition on cutaneous injury repair. Approach: Dorsal skin wounds from FVB/NJ mice, created with a 4 mm biopsy punch, were treated topically with the small-molecule PAI-1 antagonist tiplaxtinin (or vehicle control) for 5 days and then analyzed for markers of wound repair. Results: Compared to controls, tiplaxtinin-treated wounds displayed dramatic decreases in wound closure and re-epithelialization. PAI-1 immunoreactivity was evident at the migratory front in all injury sites indicating these effects were due to PAI-1 functional blockade and not PAI-1 expression changes. Stimulated HaCaT keratinocyte migration in response to recombinant PAI-1 in vitro was similarly attenuated by tiplaxtinin. While tiplaxtinin had no effect on keratinocyte proliferation, cell cycle progression, or apoptosis, it effectively reduced collagen deposition, the number of Ki-67+ fibroblasts, and incidence of differentiated myofibroblasts (i.e., smooth muscle α-actin immunoreactive cells), but not fibroblast apoptosis. Innovation: The role for PAI-1 in hemostasis and fibrinolysis is established; involvement of PAI-1 in cutaneous wound healing, however, remains unclear. This study tests the effect of a small-molecule PAI-1 inhibitor in a murine model of skin wound repair. Conclusion: Loss of PAI-1 activity significantly impaired wound closure. Re-epithelialization and fibroblast recruitment/differentiation were both reduced in tiplaxtinin-treated mice. Therapies directed at manipulation of PAI-1 expression and/or activity may have applicability as a treatment option for chronic wounds and scarring disorders.

Molecular Mechanisms of Pro-Survival and Differentiating Function of Bone Marrow-Derived Adipocytes On Acute Monoblastic Leukemia Cells.

AbstractAbstract 2582Survival of adult patients with acute monocytic leukemia remains dismal. We proposed the hypothesis that bone marrow (BM) adipocytes abundant in aging marrow microenvironment play a role in chemoresistance of AML cells. We have reported that BM adipocytes promote monocytic differentiation, activation, and survival of monoblastic leukemia cells (ASH, 2012).In this study, we examined the molecular mechanism of BM adipocytes causing survival and differentiation of monoblastic leukemia cells. Monoblastic leukemia cell line U937 and primary samples from acute monoblastic leukemia patients (n=6) were co-cultured with BM-derived marrow stromal cells (MSC) and MSC-differentiated adipocytes. Under limiting serum conditions, MSCs and adipocytes inhibited spontaneous cell death of U937 cells with decrease in subG1 fractions, more prominently in co-cultures with adipocytes than with MSCs (% subG1 fraction; cultured alone 23.9+9.0, co-cultured with MSC 15.7+1.9%, co-cultured with adipocytes 11.9+1.2%, p=0.04). Adipocytes but not MSC moderately reduced spontaneous apoptosis in primary leukemic blasts (n=6, p=0.02).We next focused on adipocytokines, lepin and plasminogen activator inhibitor 1(PAI-1), both implicated in cancer progression, as potential mediators of the protective effects by adipocytes. mRNA levels of leptin, leptin receptor and PAI-1 were up-regulated in MSC-derived adipocytes during adipocytic differentiation (relative expression to GAPDH, leptin: MSC 0, adipocyte 123.3+35.0; leptin receptor: MSC 0.16+0.05, adipocyte 4.47+0.37; PAI-1: MSC 314.9+46.5, adipocyte 3766.1+656.2). High level of secreted leptin was confirmed by ELISA in adipocyte conditioning medium (MSC 23.1+2.9, adipocyte 110.0+4.6 pg/mL). In serum-starved conditions, human recombinant leptin (5ng/mL) stimulated cell growth of monoblastic U937 cells known to express leptin receptor (35.8+7.9% increase of viable cell number at 48 hours, p=0.02) and inhibited apoptosis (%subG1 fraction; control 26.2+3.9, leptin16.4+4.1%, p=0.04).Human recombinant PAI-1 inhibited spontaneous and ara-C-induced apoptosis in U937 cells (11.2+1.1% and 15.1+1.7% decrease of Annexin V posivitity p=0.01). These results suggest that BM adipocytes produced leptin and PAI-1contribute to support myeloid leukemia cells survival within BM microenvironment.We next focused on molecular changes in U937 cells co-cultured with adipocytes. We found up-regulation of monocyte/macrophage differentiation gene PPARg and of PPARg target genes, CD36 and FABP4, by quantitative RT-PCR (relative expression to GAPDH, control vs co-culture with adipocytes: PPARg: 8.8+0.9 v.s.18.8+2.6, CD36; 105.0+11.8v.s.425.7+35.6; FABP4: 0.27+0.03v.s.153.7+12.8). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. PPARγ is known to promote monocytic differentiation and uptake of oxidized cholesterol through the induction of scavenger receptor CD36, a protein marker of mature monocytes/macrophages. The up-regulation of cell surface CD36 expression by co-culture with adipocytes was indeed found in U937 cells and in 4 of the 6 primary AML samples by flow cytometory (increase in CD36 MFI: U937; 230.28, Pt samples; 46.4+20.2). These data suggest that BM adipocytes promote monocytic differentiation of monoblastic leukemia cells.In summary, our findings indicate that secretion of leptin and PAI-1 by BM adipocytes supports leukemic cell survival, while PAI-1 primarily and directly inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of monocytic leukemia cells to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Protumorigenic activity of plasminogen activator inhibitor-1 through an antiapoptotic function.

Plasminogen activator inhibitor-1 (PAI-1) is a protease inhibitor but is paradoxically associated with poor outcomes in cancer patients. However, the mechanisms of its effects on tumor cells have not been explored. Endogenous PAI-1 in human tumor cell lines (HT-1080, A549, HCT-116, and MDA-MB-231) was suppressed by small interfering RNAs (siRNAs) and PAI-039, a small molecule inhibitor of PAI-1, and the effects on apoptosis were examined. Tumorigenicity of PAI-1 knockdown (KD) tumor cells was examined in immunodeficient PAI-1 wild-type and knockout (KO) mice (9-15 per group), and event-free survival was analyzed by the Kaplan-Meier method. The effect of PAI-1 suppression on HT-1080 xenotransplanted tumors was evaluated for cell proliferation, apoptosis, and angiogenesis. All statistical tests were two-sided. Genetic and pharmacological inhibition of PAI-1 in the four tumor cell lines increased spontaneous apoptosis (mean fold increase relative to control: HT-1080, siRNA#1, mean = 4.0, 95% CI = 2.6 to 5.3, P < .001; siRNA#2, mean = 2.6, 95% CI = 2.4 to 2.9, P < .001, Student t test), which was blocked in the presence of recombinant PAI-1, a caspase-8 inhibitor, or Fas/FasL neutralizing antibodies and was partially attenuated by a plasmin inhibitor-aprotinin. PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells had decreased tumorigenesis and prolonged survival compared with control mice (P = .002, log-rank test), and their tumors exhibited decreased cell proliferation and angiogenesis and increased apoptosis. Furthermore, five of 15 PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells never developed tumors. These data suggest that PAI-1 exerts a protective effect against tumor cell apoptosis by a mechanism that, in part, involves plasmin activation and inhibition of Fas/Fas-L-mediated apoptosis and may be a promising therapeutic target.

BioKnife, a uPA Activity-dependent Oncolytic Sendai Virus, Eliminates Pleural Spread of Malignant Mesothelioma via Simultaneous Stimulation of uPA Expression

Malignant pleural mesothelioma (MPM) is highly intractable and readily spreads throughout the surface of the pleural cavity, and these cells have been shown to express urokinase-type plasminogen activator receptor (uPAR). We here examined the potential of our new and powerful recombinant Sendai virus (rSeV), which shows uPAR-specific cell-to-cell fusion activity (rSeV/dMFct14 (uPA2), named "BioKnife"), for tumor cell killing in two independent orthotopic xenograft models of human. Multicycle treatment using BioKnife resulted in the efficient rescue of these models, in association with tumor-specific fusion and apoptosis. Such an effect was also seen on both MSTO-211H and H226 cells in vitro; however, we confirmed that the latter expressed uPAR but not uPA. Of interest, infection with BioKnife strongly facilitated the uPA release from H226 cells, and this effect was completely abolished by use of either pyrrolidine dithiocarbamate (PDTC) or BioKnife expressing the C-terminus-deleted dominant negative inhibitor for retinoic acid-inducible gene-I (RIG-IC), indicating that BioKnife-dependent expression of uPA was mediated by the RIG-I/nuclear factor-κB (NF-κB) axis, detecting RNA viral genome replication. Therefore, these results suggest a proof of concept that the tumor cell-killing mechanism via BioKnife may have significant potential to treat patients with MPM that is characterized by frequent uPAR expression in a clinical setting.