Past, Present, and Future Perspectives of Plasminogen Activator Inhibitor 1 (PAI-1).

Plasminogen activator inhibitor 1 (PAI-1), a SERPIN inhibitor, is primarily known for its regulation of fibrinolysis. However, it is now known that this inhibitor functions and contributes to many (patho)physiological processes including inflammation, wound healing, cell adhesion, and tumor progression.This review discusses the past, present, and future roles of PAI-1, with a particular focus on the discovery of this inhibitor in the 1970s and subsequent characterization in health and disease. Throughout the past few decades diverse functions of this serpin have unraveled and it is now considered an important player in many disease processes. PAI-1 is expressed by numerous cell types, including megakaryocytes and platelets, adipocytes, endothelial cells, hepatocytes, and smooth muscle cells. In the circulation PAI-1 exists in two pools, within plasma itself and in platelet α-granules. Platelet PAI-1 is secreted following activation with retention of the inhibitor on the activated platelet membrane. Furthermore, these anucleate cells contain PAI-1 messenger ribonucleic acid to allow de novo synthesis.Outside of the traditional role of PAI-1 in fibrinolysis, this serpin has also been identified to play important roles in metabolic syndrome, obesity, diabetes, and most recently, acute respiratory distress syndrome, including coronavirus disease 2019 disease. This review highlights the complexity of PAI-1 and the requirement to ascertain a better understanding on how this complex serpin functions in (patho)physiological processes.

Demonstration of Three Distinct High-Molecular-Weight Complexes between Plasminogen Activator Inhibitor Type 1 and Tissue-Type Plasminogen Activator.

BACKGROUND: Details of the molecular interaction between tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type-1 (PAI-1) remain unknown. METHODS AND RESULTS: Three distinct forms of high-molecular-weight complexes are demonstrated. Two of the forms were detected by mass spectrometry. The high molecular mass detected by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was 107,029 Da, which corresponds to the sum of molecular masses of the intact tPA (65,320 Da) and the intact PAI-1 (42,416 Da). The lower molecular mass was 104,367 Da and is proposed to lack the C-terminal bait peptide of PAI-1 (calculated mass: 3,804 Da), which was detected as a 3,808 Da fragment. When the complex was analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), only a single band was observed. However, after treatment by SDS and Triton X-100, two distinct forms of the complex with different mobilities were shown by SDS-PAGE. The higher molecular weight band demonstrated specific tPA activity on fibrin autography, whereas the lower molecular weight band did not. Peptide sequence analysis of these two bands, however, unexpectedly revealed the existence of the C-terminal cleavage peptide in both bands and its amount was less in the upper band. In the upper band, the sequences corresponding to the regions at the interface between two molecules in its Michaelis intermediate were diminished. Thus, these two bands corresponded to distinct nonacyl-enzyme complexes, wherein only the upper band liberated free tPA under the conditions employed. CONCLUSION: These data suggest that under physiological conditions a fraction of the tPA-PAI-1 population exists as nonacylated-enzyme inhibitor complex.

Serpin peptidase inhibitor, clade E nexin group 1 promotes cellular proliferative capacities and malignant behaviors in glioblastoma through upregulating hairy and enhancer of split-1.

PURPOSE: Glioblastoma (GBM) remains one of the most fatal malignancy with limited available treatment. Serpin peptidase inhibitor, clade E nexin group 1 (SERPINE1) was found up-regulated in multiple cancers and play crucial roles in facilitating tumor progression and metastasis respectively. However, the role of SERPINE1 in glioblastoma was poorly understood. METHODS: We tested the hypothesis that SERPINE1 mediated malignant behaviors in GBM via regulating hairy and enhancer of split-1 (HES1). RESULTS: First, SERPINE1 is confirmed to be up-regulated in GBM, while further functional analysis demonstrated that SERPINE1 promoted cell proliferation, migration and invasion in GBM by performing the CCK-8 assay, colony formation assay, wound healing assay and transwell assay. Finally, it was proved that SERPINE1 achieved its pro-tumor functions in GBM via regulating the expression of HES1. CONCLUSIONS: Collectively, our results highlight the critical contribution of SERPINE1 in a series of malignant characteristics of GBM via regulating the expression of HES1, which shed new light on a new direction to develop a more effective therapeutic management of malignant tumors like GBM.

Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape.

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure-function relationships for PAI-1 and for the interpretation of human genomic sequence variants.

Oral Squamous Cell Carcinoma Contributes to Differentiation of Monocyte-Derived Tumor-Associated Macrophages via PAI-1 and IL-8 Production.

Tumor-associated macrophages (TAMs) promote cancer cell proliferation and metastasis, as well as anti-tumor immune suppression. Recent studies have shown that tumors enhance the recruitment and differentiation of TAMs, but the detailed mechanisms have not been clarified. We thus examined the influence of cancer cells on the differentiation of monocytes to TAM subsets, including CD163+, CD204+, and CD206+ cells, in oral squamous cell carcinoma (OSCC) using immunohistochemistry, flow cytometry, and a cytokine array. Furthermore, we investigated the effect of OSCC cells (HSC-2, SQUU-A, and SQUU-B cells) on the differentiation of purified CD14+ cells to TAM subsets. The localization patterns of CD163+, CD204+, and CD206+ in OSCC sections were quite different. The expression of CD206 on CD14+ cells was significantly increased after the co-culture with OSCC cell lines, while the expressions of CD163 and CD204 on CD14+ cells showed no change. High concentrations of plasminogen activator inhibitor-1 (PAI-1) and interleukin-8 (IL-8) were detected in the conditioned medium of OSCC cell lines. PAI-1 and IL-8 stimulated CD14+ cells to express CD206. Moreover, there were positive correlations among the numbers of CD206+, PAI-1+, and IL-8+ cells in OSCC sections. These results suggest that PAI-1 and IL-8 produced by OSCC contribute to the differentiation of monocytes to CD206+ TAMs.

Role of PAI-1 in hepatic steatosis and dyslipidemia.

Plasminogen activator inhibitor 1 (PAI-1) is a functional biomarker of the metabolic syndrome. Previous studies have demonstrated that PAI-1 is a mechanistic contributor to several elements of the syndrome, including obesity, hypertension and insulin resistance. Here we show that PAI-1 is also a critical regulator of hepatic lipid metabolism. RNA sequencing revealed that PAI-1 directly regulates the transcriptional expression of numerous genes involved in mammalian lipid homeostasis, including PCSK9 and FGF21. Pharmacologic or genetic reductions in plasma PAI-1 activity ameliorates hyperlipidemia in vivo. These experimental findings are complemented with the observation that genetic deficiency of PAI-1 is associated with reduced plasma PCSK9 levels in humans. Taken together, our findings identify PAI-1 as a novel contributor to mammalian lipid metabolism and provides a fundamental mechanistic insight into the pathogenesis of one of the most pervasive medical problems worldwide.

Palmitic acid induces inflammation in placental trophoblasts and impairs their migration toward smooth muscle cells through plasminogen activator inhibitor-1.

A critical component of early human placental development includes migration of extravillous trophoblasts (EVTs) into the decidua. EVTs migrate toward and displace vascular smooth muscle cells (SMCs) surrounding several uterine structures, including spiral arteries. Shallow trophoblast invasion features in several pregnancy complications including preeclampsia. Maternal obesity is a risk factor for placental dysfunction, suggesting that factors within an obese environment may impair early placental development. Herein, we tested the hypothesis that palmitic acid, a saturated fatty acid circulating at high levels in obese women, induces an inflammatory response in EVTs that hinders their capacity to migrate toward SMCs. We found that SMCs and SMC-conditioned media stimulated migration and invasion of an EVT-like cell line, HTR8/SVneo. Palmitic acid impaired EVT migration and invasion toward SMCs, and induced expression of several vasoactive and inflammatory mediators in EVTs, including endothelin, interleukin (IL)-6, IL-8 and PAI1. PAI1 was increased in plasma of women with early-onset preeclampsia, and PAI1-deficient EVTs were protected from the anti-migratory effects of palmitic acid. Using first trimester placental explants, palmitic acid exposure decreased EVT invasion through Matrigel. Our findings reveal that palmitic acid induces an inflammatory response in EVTs and attenuates their migration through a mechanism involving PAI1. High levels of palmitic acid in pathophysiological situations like obesity may impair early placental development and predispose to placental dysfunction.

PAI-1, the Plasminogen System, and Skeletal Muscle.

The plasminogen system is a critical proteolytic system responsible for the remodeling of the extracellular matrix (ECM). The master regulator of the plasminogen system, plasminogen activator inhibitor-1 (PAI-1), has been implicated for its role in exacerbating various disease states not only through the accumulation of ECM (i.e., fibrosis) but also its role in altering cell fate/behaviour. Examination of PAI-1 has extended through various tissues and cell-types with recent investigations showing its presence in skeletal muscle. In skeletal muscle, the role of this protein has been implicated throughout the regeneration process, and in skeletal muscle pathologies (muscular dystrophy, diabetes, and aging-driven pathology). Needless to say, the complete function of this protein in skeletal muscle has yet to be fully elucidated. Given the importance of skeletal muscle in maintaining overall health and quality of life, it is critical to understand the alterations-particularly in PAI-1-that occur to negatively impact this organ. Thus, we provide a comprehensive review of the importance of PAI-1 in skeletal muscle health and function. We aim to shed light on the relevance of this protein in skeletal muscle and propose potential therapeutic approaches to aid in the maintenance of skeletal muscle health.

PAI1 is a Marker of Bad Prognosis in Rectal Cancer but Predicts a Better Response to Treatment with PIM Inhibitor AZD1208.

Colorectal cancer (CRC) is the third most common cancer worldwide. The standard treatment in locally advanced rectal cancer is preoperative radiation alone or in combination with chemotherapy, followed by adjuvant chemotherapy. Rectal cancer is highly lethal, with only 20% of patients showing a complete remission (by RECIST) after standard treatment, although they commonly show local or systemic relapse likely due to its late detection and high chemotherapy resistance, among other reasons. Here, we explored the role of PAI1 (Serpin E1) in rectal cancer through the analyses of public patient databases, our own cohort of locally advanced rectal cancer patients and a panel of CRC cell lines. We showed that PAI1 expression is upregulated in rectal tumors, which is associated with decreased overall survival and increased metastasis and invasion in advanced rectal tumors. Accordingly, PAI1 expression is correlated with the expression of (Epithelial-to-Mesenchymal Transition) EMT-associated genes and genes encoding drug targets, including the tyrosine kinases PDGFRb, PDGFRa and FYN, the serine/threonine kinase PIM1 and BRAF. In addition, we demonstrate that cells expressing PAI1 protein are more sensitive to the PIM inhibitor AZD1208, suggesting that PAI1 could be used to predict response to treatment with PIM inhibitors and to complement radiotherapy in rectal tumors.

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

OBJECTIVE: 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. CONCLUSIONS: 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.

The prognostic value of the urokinase-plasminogen activator system (uPA) in bladder cancer patients treated with radical cystectomy (RC).

PURPOSE: Urokinase-plasminogen activator (uPA), its receptor (uPAR), and the plasmin-activator inhibitor type 1 (PAI-1) have been associated with oncologic outcomes in various malignancies and could help identify bladder cancer (BC) patients treated with radical cystectomy (RC) who are likely to benefit from intensification of therapy to prevent disease progression. Our aim was to assess the value of uPA, uPAR, and PAI-1 for prognosticating survival outcomes of patients treated with RC for BC. MATERIALS AND METHODS: Tumor specimens from 272 consecutive patients treated with RC for advanced BC were assessed with immunohistochemical staining for uPA, uPAR, and PAI-1. Overexpression was assessed by pathological image analysis. Kaplan-Meier estimates and multivariable Cox-regression were used to analyze survival. Harrell's C-index was used to assess for clinical impact of the uPA system. RESULTS: uPA, uPAR, and PAI-1 were overexpressed in 48.2%, 51.1%, and 52.2% of patients, respectively. uPA overexpression was associated with lymphovascular invasion (P = 0.034) and nodal status (P = 0.013); PAI-1 overexpression was associated with primary muscle-invasive BC (P = 0.015) and lymphovascular invasion (P = 0.024). uPA, uPAR, and the number of overexpressed markers were all 3 significantly associated with shorter overall recurrence-free-, distant recurrence-free-, and cancer-specific survival. In multivariable analyses, uPA overexpression remained associated with shorter recurrence-free survival (hazard ratio [HR] = 1.79; P = 0.036) in the entire cohort, in patients without lymph node metastasis (HR = 1.98; P = 0.018) and those with nonorgan-confined disease (HR = 1.98; P = 0.022). uPAR overexpression was associated with shorter recurrence-free survival in patients without lymph node metastasis (HR = 2.01; P = 0.021) and those with organ-confined disease (HR = 4.11; P = 0.037). CONCLUSION: Members of the uPA system are associated with features of biologically aggressive BC and oncologic outcomes. However, their value beyond currently available information remains limited.

Transcriptomic Analysis of Cellular Pathways in Healing Flexor Tendons of Plasminogen Activator Inhibitor 1 (PAI-1/Serpine1) Null Mice.

Injuries to flexor tendons can be complicated by fibrotic adhesions, which severely impair the function of the hand. Plasminogen activator inhibitor 1 (PAI-1/SERPINE1), a master suppressor of fibrinolysis and protease activity, is associated with adhesions. Here, we used next-generation RNA sequencing (RNA-Seq) to assess genome-wide differences in messenger RNA expression due to PAI-1 deficiency after zone II flexor tendon injury. We used the ingenuity pathway analysis to characterize molecular pathways and biological drivers associated with differentially expressed genes (DEG). Analysis of hundreds of overlapping and DEG in PAI-1 knockout (KO) and wild-type mice (C57Bl/6J) during tendon healing revealed common and distinct biological processes. Pathway analysis identified cell proliferation, survival, and senescence, as well as chronic inflammation as potential drivers of fibrotic healing and adhesions in injured tendons. Importantly, we identified the activation of PTEN signaling and the inhibition of FOXO1-associated biological processes as unique transcriptional signatures of the healing tendon in the PAI-1/Serpine1 KO mice. Further, transcriptomic differences due to the genetic deletion of PAI-1 were mechanistically linked to PI3K/Akt/mTOR, PKC, and MAPK signaling cascades. These transcriptional observations provide novel insights into the biological roles of PAI-1 in tendon healing and could identify therapeutic targets to achieve scar-free regenerative healing of tendons. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:43-58, 2020.

Expression of urokinase-type plasminogen activator system in non-metastatic prostate cancer.

PURPOSE: To investigate the prognostic role of expression of urokinase-type plasminogen activator system members, such as urokinase-type activator (uPA), uPA-receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in patients treated with radical prostatectomy (RP) for prostate cancer (PCa). METHODS: Immunohistochemical staining for uPA system was performed on a tissue microarray of specimens from 3121 patients who underwent RP. Cox regression analyses were performed to investigate the association of overexpression of these markers alone or in combination with biochemical recurrence (BCR). Decision curve analysis was used to assess the clinical impact of these markers. RESULTS: uPA, uPAR, and PAI-1 were overexpressed in 1012 (32.4%), 1271 (40.7%), and 1311 (42%) patients, respectively. uPA overexpression was associated with all clinicopathologic characteristics of biologically aggressive PCa. On multivariable analysis, uPA, uPAR, and PAI-1 overexpression were all three associated with BCR (HR: 1.75, p < 0.01, HR: 1.22, p = 0.01 and HR: 1.20, p = 0.03, respectively). Moreover, the probability of BCR increased incrementally with increasing cumulative number of overexpressed markers. Decision curve analysis showed that addition of uPA, uPAR, and PAI-1 resulted in a net benefit compared to a base model comparing standard clinicopathologic features across the entire threshold probability range. In subgroup analyses, overexpression of all three markers remained associated with BCR in patients with favorable pathologic characteristics. CONCLUSION: Overexpression of uPA, uPAR, and PAI-1 in PCa tissue were each associated with worse BCR. Additionally, overexpression of all three markers is informative even in patients with favorable pathologic characteristics potentially helping clinical decision-making regarding adjuvant therapy and/or intensified follow-up.

PAI-1 contributes to homocysteine-induced cellular senescence.

Cellular Senescence is associated with organismal aging and related pathologies. Previously, we reported that plasminogen activator inhibitor-1 (PAI-1) is an essential mediator of senescence and a potential therapeutic target for preventing aging-related pathologies. In this study, we investigate the efficacies of PAI-1 inhibitors in both in vitro and in vivo models of homocysteine (Hcy)-induced cardiovascular aging. Elevated Hcy, a known risk factor of cardiovascular diseases, induces endothelial senescence as evidenced by increased senescence-associated ß-Gal positivity (SA-ß-Gal), flattened cellular morphology, and cylindrical appearance of cellular nuclei. Importantly, inhibition of PAI-1 by small molecule inhibitors reduces the number of SA-ß-Gal positive cells, normalizes cellular morphology and nuclear shape. Furthermore, while Hcy induces the levels of senescence regulators PAI-1, p16, p53 and integrin ß3, and suppresses catalase expression, treatment with PAI-1 inhibitors blocks the Hcy-induced stimulation of senescence cadres, and reverses the Hcy-induced suppression of catalase, indicating that PAI-1 specific small molecule inhibitors are efficient to prevent Hcy-induced cellular senescence. Our in vivo study shows that the levels of integrin ß3, a recently identified potential regulator of cellular senescence, and its interaction with PAI-1 are significantly elevated in Hcy-treated heart tissues. In contrast, Hcy suppresses antioxidant gene regulator Nrf2 expression in hearts. However, co-treatment with PAI-1 inhibitor completely blocks the stimulation of Hcy-induced induction of integrin ß3 and reverses Nrf2 expression. Collectively these in vitro and in vivo studies indicate that pharmacological inhibition of PAI-1 improves endothelial and cardiac health by suppressing the pro-senescence effects of hyperhomocysteinemia through suppression of Hcy-induced master regulators of cellular senescence PAI-1 and integrin ß3. Therefore, PAI-1 inhibitors are promising drugs for amelioration of hyperhomocysteinemia-induced vascular aging and aging-related disease.

Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI).

Objective- Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes sequestration of FXIa (activated factor XI) activity. This study aims to determine the role of endothelial cells (ECs) in the regulation of the intrinsic pathway of coagulation. Approach and Results- Using a chromogenic assay, we observed that human umbilical veins ECs selectively blocked FXIa yet supported kallikrein and FXIIa activity. Western blotting and mass spectrometry analyses revealed that FXIa formed a complex with endothelial PAI-1 (plasminogen activator inhibitor-1). Blocking endothelial PAI-1 increased the cleavage of a chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation in plasma. Western blot and immunofluorescence analyses showed that FXIa-PAI-1 complexes were either released into the media or trafficked to the early and late endosomes and lysosomes of ECs. When baboons were challenged with Staphylococcus aureus to induce a prothrombotic phenotype, an increase in circulating FXIa-PAI-1 complex levels was detected by ELISA within 2 to 8 hours postchallenge. Conclusions- PAI-1 forms a complex with FXIa on ECs, blocking its activity and inducing the clearance and degradation of FXIa. Circulating FXIa-PAI-1 complexes were detected in a baboon model of S. aureus sepsis. Although ECs support kallikrein and FXIIa activity, inhibition of FXIa by ECs may promote the clearance of intravascular FXIa. Visual Overview- An online visual overview is available for this article.

Plasminogen activator, tissue type regulates germinal vesicle breakdown and cumulus expansion of bovine cumulus-oocyte complex in vitro†.

Plasminogen activator, tissue type (PLAT) and its inhibitor serpin family E member 1 (SERPINE1) cooperatively regulate PLAT activity in various reproductive processes. However, it is unknown whether this includes bovine oocyte maturation. We addressed this question in the present study by evaluating PLAT and SERPINE1 protein localization in immature cumulus-oocyte complexes (COCs), as well as PLAT mRNA and protein expression in cultured COCs after 0, 8, 16, and 24 h of in vitro maturation (IVM). We also examined the effects of PLAT and SERPINE1 on germinal vesicle breakdown (GVBD) and oocyte cyclic 3' 5' adenosine monophosphate (cAMP) levels, cumulus expansion index, and expansion-related gene expression in oocytes derived from bovine COCs cultured for 4, 8, and 12 h and in COCs cultured for 16 h. Both PLAT and SERPINE1 localized in cumulus cells but only the latter was detected in oocytes. PLAT and SERPINE1 transcript levels increased during IVM; however, from 8 to 16 h, the levels of PLAT remained stable whereas those of SERPINE1 increased, resulting in a decline in PLAT concentration. Additionally, PLAT delayed GVBD, increased oocyte cAMP levels, and blocked cumulus expansion and associated gene expression, which was reversed by SERPINE1 supplemented. Thus, PLAT delays bovine oocyte GVBD by enhancing oocyte cAMP levels during the first 8 h of IVM; suppression of PLAT activity via accumulation of SERPINE1 in COCs results in cumulus expansion from 8 to 16 h of IVM. These findings provide novel insights into the molecular mechanisms underlying in vitro bovine oocyte maturation.

Investigations on the Role of the Fibrinolytic Pathway on Outflow Facility Regulation.

Purpose: To understand the role and further dissect pathways downstream of tissue plasminogen activator (tPA) and the fibrinolytic pathway in modulating outflow facility. Methods: Outflow facility of tissue plasminogen activator (Plat) knockout (KO) mice was determined and compared to that of wild-type (WT) littermates. Gene expression of urokinase plasminogen activator (Plau), plasminogen activator inhibitor (Pai-1), plasminogen (Plg), and matrix metalloproteinases (Mmp-2, -9, and -13) was measured in angle tissues. Expression of the same genes and outflow facility were measured in KO and WT mice treated with triamcinolone acetonide (TA). Amiloride was used to inhibit urokinase plasminogen activator (uPA) in Plat KO mice, and outflow facility was measured. Results: Plat deletion resulted in outflow facility reduction and decreased Mmp-9 expression in angle tissues. Plasminogen expression was undetectable in both KO and WT mice. TA led to further reduction in outflow facility and decreases in expression of Plau and Mmp-13 in plat KO mice. Amiloride inhibition of uPA activity prevented the TA-induced outflow facility reduction in Plat KO mice. Conclusions: tPA deficiency reduced outflow facility in mice and was associated with reduced MMP expression. The mechanism of action of tPA is unlikely to involve plasminogen activation. tPA is not the only mediator of TA-induced outflow facility change, as TA caused reduction in outflow facility of Plat KO mice. uPA did not substitute for tPA in outflow facility regulation but abrogated the effect of TA in the absence of tPA, suggesting a complex role of components of the fibrinolytic system in outflow regulation.

Molecular biomarkers of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO), a complication of Graves' disease (GD), is typified by orbital inflammation, ocular tissue expansion and remodeling and, ultimately, fibrosis. Orbital fibroblasts are key effectors of GO pathogenesis exhibiting exaggerated inflammatory and fibroproliferative responses to cytokines released by infiltrating immune cells. Activated orbital fibroblasts also produce inflammatory mediators that contribute to disease progression, facilitate the orbital trafficking of monocytes and macrophages, promote differentiation of matrix-producing myofibroblasts and stimulate accumulation of a hyaluronan-rich stroma, which leads to orbital tissue edema and fibrosis. Proteomic and transcriptome profiling of the genomic response of ocular and non-ocular fibroblasts to INF-γ and TGF-ß1 focused on identification of translationally-relevant therapeutic candidates. Induction of plasminogen activator inhibitor-1 (PAI-1, SERPINE1), a clade E member of the serine protease inhibitor (SERPIN) gene family and a prominent regulator of the pericellular proteolytic microenvironment, was one of the most highly up-regulated proteins in INF-γ- or TGF-ß1-stimulated GO fibroblasts as well as in severe active GD compared to patients without thyroid disease. PAI-1 has multifunctional roles in inflammatory and fibrotic processes that impact tissue remodeling, immune cell trafficking and survival as well as signaling through several receptor systems. This review focuses on the pathophysiology of the GO fibroblast and possible targets for effective drug therapy.