Plasminogen Activator Inhibitor-1 Inhibitor Research Articles

Plasminogen activator inhibitor-1 promotes immune evasion in tumors by facilitating the expression of programmed cell death-ligand 1.

Increased levels of plasminogen activator inhibitor-1 (PAI-1) in tumors have been found to correlate with poor clinical outcomes in patients with cancer. Although abundant data support the involvement of PAI-1 in cancer progression, whether PAI-1 contributes to tumor immune surveillance remains unclear. The purposes of this study are to determine whether PAI-1 regulates the expression of immune checkpoint molecules to suppresses the immune response to cancer and demonstrate the potential of PAI-1 inhibition for cancer therapy. The effects of PAI-1 on the expression of the immune checkpoint molecule programmed cell death ligand 1 (PD-L1) were investigated in several human and murine tumor cell lines. In addition, we generated tumor-bearing mice and evaluated the effects of a PAI-1 inhibitor on tumor progression or on the tumor infiltration of cells involved in tumor immunity either alone or in combination with immune checkpoint inhibitors. PAI-1 induces PD-L1 expression through the JAK/STAT signaling pathway in several types of tumor cells and surrounding cells. Blockade of PAI-1 impedes PD-L1 induction in tumor cells, significantly reducing the abundance of immunosuppressive cells at the tumor site and increasing cytotoxic T-cell infiltration, ultimately leading to tumor regression. The anti-tumor effect elicited by the PAI-1 inhibitor is abolished in immunodeficient mice, suggesting that PAI-1 blockade induces tumor regression by stimulating the immune system. Moreover, combining a PAI-1 inhibitor with an immune checkpoint inhibitor significantly increases tumor regression. PAI-1 protects tumors from immune surveillance by increasing PD-L1 expression; hence, therapeutic PAI-1 blockade may prove valuable in treating malignant tumors.

Abstract 3277: PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer

Abstract [Purpose] The mesenchymal epithelial transition factor receptor (MET) is a potential therapeutic target in various cancers, including non-small cell lung cancer (NSCLC). In NSCLC, the activation of MET pathway is thought to occur through diverse mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. These tumors show oncogene addiction to MET and exhibit remarkable responses to MET-Tyrosine Kinase Inhibitors (TKIs). However, the long-term effectiveness of MET-TKIs is usually limited due to acquired drug resistance. The mechanisms underlying primary and acquired resistance to MET-TKIs in the management of NSCLC remain unclear. In this study, we investigated the mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. [Experimental Designs] Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. The factors contributing to the resistance mechanism were assessed by genomic and transcriptomic data, and the alterations hypothesized to confer resistance were validated. [Results] Multiple mechanisms underlay acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the drug resistance mechanism was mediated by the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1). Crizotinib resistance was overcome by combination therapy with PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial to mesenchymal transition (EMT) with the upregulation of antiapoptotic proteins. In the H1993-derived resistant cells, the phosphorylated forms of MEK and ERK were observed to be upregulated and MEK inhibitors could be a potential therapeutic strategy for overcoming the resistance. [Conclusions] In this study, we revealed the different mechanisms of drug resistance to MET-TKI treatment and presented some possible strategies to overcome the resistance. Citation Format: Tomoaki Higashihara, Yin Min Thu, Ken Suzawa, Keiichi Date, Atsushi Matsuoka, Daisuke Mizuno, Ryo Yoshichika, Naohiro Hayashi, Fumiaki Mukohara, Mao Yoshikawa, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. PAI-1 mediates resistance to MET-targeted therapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3277.

UPREGULATION OF SERPINE1/PAI-1 INDUCED BY IL-33 CONTRIBUTES TO INTESTINAL FIBROSIS IN A CROHN’S DISEASE-LIKE ILEITIS MODEL

Abstract IL-33 is a pleiotropic cytokine known to possess dichotomous roles during gut health and disease. We previously described that IL-33 promotes epithelial restitution/repair in otherwise healthy (C57BL/6) mice that have been challenged with dextran sodium sulphate (DSS) to induce acute colitis, with the end result of efficient resolution of inflammation. However, in SAMP1/YitFc (SAMP) mice that spontaneously develop Crohn’s disease (CD)-like ileitis, elevated IL-33 levels persist as disease progresses, perpetuating chronic gut inflammation and severe fibrosis. While IL-33 has been implicated in the development of inflammation-associated fibrosis, the precise mechanism(s) by which this occurs remains unclear. The aim of this study was to determine IL-33-dependent events leading to intestinal fibrosis in ileitis-prone SAMP mice. Our results show IHC co-localization of IL-33 with fibrotic lesions, specifically in cells morphologically-consistent with both macrophages and subepithelial myofibroblasts (SEMFs). Bulk RNA-Seq analysis of the human SEMF cell line, CCD-18Co, stimulated +/- IL-33 identifies one of the highest-expressing transcripts as SERPINE1, encoding for plasminogen activator inhibitor-1 (PAI-1), which has previously been reported as highly-enriched in IBD patients with active disease that do not respond to anti-TNF therapy. Analysis of publicly-available scRNA-Seq data confirms the increased expression of SERPINE1 in IBD patients that localizes to mesenchymal cell populations. Of note, robust upregulation of SERPINE1 is detected in activated fibroblasts from involved vs. non-involved areas of CD patients. Furthermore, spatial transcriptomics of SAMP ilea reveal a progressive and concomitant increase in both Il33 and Serpine1, with strong clusterization compared to healthy AKR controls, particularly during later time points when fibrosis is evident. Currently, results are pending on the treatment of SAMP mice with the PAI-1 inhibitor, MDI-2268, to determine its direct effect(s) on the development of inflammation-associated intestinal fibrosis. Taken together, these findings suggest IL-33-dependent regulation of Serpine1/PAI-1 that promotes intestinal fibrosis, commonly observed in CD patients, and may provide a novel target to treat IBD patients with fibrostenoic disease.

P072 LDN-071, a novel first-in-class amino acid-based PAI-1 inhibitor, significantly reduces the severity of experimental colitis in mice

Abstract Background Inflammatory Bowel Diseases (IBD) are chronic inflammatory disorders characterised by alterations of acute relapses and remission. The past decades have brought substantial advances in the pharmacological management of IBD by wide range of medicines. Although, the sustained efficacy of the currently available anti-inflammatory therapies is still far from optimal. Therefore, the development of novel therapies represents a major unmet clinical need in this field. Recently the enrichment of the coagulation pathway genes and among these, Plasminogen activator inhibitor 1 (PAI-1) was described in IBD, whereas PAI-1 emerged as a link between the epithelium and inflammation. Moreover, the genetic deletion of PAI-1 impaired the severity of experimental colitis in mice. Based on these, our aim was to develop and test novel PAI-1 inhibitors for the treatment of IBD. Methods Novel, amino acid-based PAI-1 inhibitors were synthetized by A3SMO® platform. The inhibitory potential and cytotoxicity effects of the peptides were determined by in vitro assays. Based on the in vitro results LDN-071 was selected for further analysis. Experimental colitis was induced in mice with 3% DSS and after the induction of colitis 10 mg/bwkg LDN-071 was administered per os for four consecutive days. The changes of body weight, spleen weight and colon length were measured. Haematoxylin eosin (HE) and Periodic acid-Schiff (PAS) staining were performed for histology. ELISA and qRT-PCR measurements were applied to analyse the mucosal cytokine expressions. In vivo toxicity of LDN-071 was determined in mice. Results LDN-071 significantly inhibited PAI-1 in vitro without decreasing the cell viability in a concentration range of 1µM-1mM. The administration of 10 mg/bwkg LDN-071 in the DSS-treated mice prevented the body weight loss, the development of bloody diarrhoea, the decrease of colon length and increase of the spleen weight, which were displayed by the DSS+vehicle-treated animals. The histological analysis of the colon revealed that DSS treatment induced a severe transmural inflammation, ulceration, loss of crypt structures and goblet cell depletion. Importantly, all of these parameters were significantly impaired by the administration of 10 mg/bwkg LDN-071. Additionally, mucosal gene and protein expressions of TNF-α, IL-1β and IL-6 were significantly impaired by PAI-1 inhibition. Importantly, no signs of toxicity were LDN-071 observed in mice exposed to 100 mg/bwkg LDN-071 for 7 days. Conclusion Our results showed that LDN-071, a novel amino acid-based inhibitor of PAI-1. It significantly reduces the severity of experimental colitis in mice. We propose that the inhibition of PAI-1 could be a potential novel therapeutic strategy in IBD.

Endothelial H2S-AMPK dysfunction upregulates the angiocrine factor PAI-1 and contributes to lung fibrosis

Dysfunction of the vascular angiocrine system is critically involved in regenerative defects and fibrosis of injured organs. Previous studies have identified various angiocrine factors and found that risk factors such as aging and metabolic disorders can disturb the vascular angiocrine system in fibrotic organs. One existing key gap is what sense the fibrotic risk to modulate the vascular angiocrine system in organ fibrosis. Here, using human and mouse data, we discovered that the metabolic pathway hydrogen sulfide (H2S)-AMP-activated protein kinase (AMPK) is a sensor of fibrotic stress and serves as a key mechanism upregulating the angiocrine factor plasminogen activator inhibitor-1 (PAI-1) in endothelial cells to participate in lung fibrosis. Activation of the metabolic sensor AMPK was inhibited in endothelial cells of fibrotic lungs, and AMPK inactivation was correlated with enriched fibrotic signature and reduced lung functions in humans. The inactivation of endothelial AMPK accelerated lung fibrosis in mice, while the activation of endothelial AMPK with metformin alleviated lung fibrosis. In fibrotic lungs, endothelial AMPK inactivation led to YAP activation and overexpression of the angiocrine factor PAI-1, which was positively correlated with the fibrotic signature in human fibrotic lungs and inhibition of PAI-1 with Tiplaxtinin mitigated lung fibrosis. Further study identified that the deficiency of the antioxidative gas metabolite H2S accounted for the inactivation of AMPK and activation of YAP-PAI-1 signaling in endothelial cells of fibrotic lungs. H2S deficiency was involved in human lung fibrosis and H2S supplement reversed mouse lung fibrosis in an endothelial AMPK-dependent manner. These findings provide new insight into the mechanism underlying the deregulation of the vascular angiocrine system in fibrotic organs.

A randomized double-blind placebo-controlled trial of an inhibitor of plasminogen activator inhibitor-1 (TM5614) in mild to moderate COVID-19

An inhibitor of plasminogen activator inhibitor (PAI)-1, TM5614, inhibited thrombosis, inflammation, and fibrosis in several experimental mouse models. To evaluate the efficacy and safety of TM5614 in human COVID-19 pneumonia, phase IIa and IIb trials were conducted. In an open-label, single-arm trial, 26 Japanese COVID-19 patients with mild to moderate pneumonia were treated with 120–180 mg of TM5614 daily, and all were discharged without any notable side effects. Then, a randomized, double-blind, placebo-controlled trial was conducted in Japanese COVID-19 patients with mild to moderate pneumonia. The number of study participants was set to be 50 in each arm. Even after extension of the enrollment period, the number of study participants did not reach the initially intended sample size, and 75 patients were enrolled in the study. The total oxygenation scale from Day 1 to Day 14 as the primary endpoint was 1.5 in the TM5614 group vs 4.0 in the placebo group (p = 0.22), and the number of days of oxygen administration required as the secondary endpoint was 2.0 days in the TM5614 group vs 3.5 days in the placebo group (p = 0.34). Further studies will be necessary to verify the efficacy of PAI-1 inhibition for the treatment of COVID-19 pneumonia.Clinical trial registration: Two studies were conducted: a prospective, multicenter, open-label phase II study at https://jrct.niph.go.jp (jRCT2021200018) (First registration date 18/08/2020) and a prospective, multicenter, randomized, double-blind, placebo-controlled, phase II study at https://jrct.niph.go.jp (jRCT2021210006) (First registration date 28/05/2021).

PAI-1 mediates acquired resistance to MET-targeted therapy in non-small cell lung cancer.

Mechanisms underlying primary and acquired resistance to MET tyrosine kinase inhibitors (TKIs) in managing non-small cell lung cancer remain unclear. In this study, we investigated the possible mechanisms acquired for crizotinib in MET-amplified lung carcinoma cell lines. Two MET-amplified lung cancer cell lines, EBC-1 and H1993, were established for acquired resistance to MET-TKI crizotinib and were functionally elucidated. Genomic and transcriptomic data were used to assess the factors contributing to the resistance mechanism, and the alterations hypothesized to confer resistance were validated. Multiple mechanisms underlie acquired resistance to crizotinib in MET-amplified lung cancer cell lines. In EBC-1-derived resistant cells, the overexpression of SERPINE1, the gene encoding plasminogen activator inhibitor-1 (PAI-1), mediated the drug resistance mechanism. Crizotinib resistance was addressed by combination therapy with a PAI-1 inhibitor and PAI-1 knockdown. Another mechanism of resistance in different subline cells of EBC-1 was evaluated as epithelial-to-mesenchymal transition with the upregulation of antiapoptotic proteins. In H1993-derived resistant cells, MEK inhibitors could be a potential therapeutic strategy for overcoming resistance with downstream mitogen-activated protein kinase pathway activation. In this study, we revealed the different mechanisms of acquired resistance to the MET inhibitor crizotinib with potential therapeutic application in patients with MET-amplified lung carcinoma.

Specific inhibition on PAI-1 reduces the dose of Alteplase for ischemic stroke treatment

Administration of recombinant tPA (rtPA, or trade name Alteplase®) is an FDA-approved therapy for acute ischemic stroke (AIS), but poses the risk of hemorrhagic complications. Recombinant tPA can be rapidly inactivated by the endogenous inhibitor, plasminogen activator inhibitor 1 (PAI-1). In this work, we study a novel treatment approach that combines a PAI-1 inhibitor, PAItrap4, with a reduced dose of rtPA to address the hemorrhagic concern of rtPA. PAItrap4 is a highly specific and very potent protein-based inhibitor of PAI-1, comprising of a variant of uPA serine protease domain, human serum albumin, and a cyclic RGD peptide. PAItrap4 efficiently targets and inhibits PAI-1 on activated platelets, and also possesses a long half-life in vivo. Our results demonstrate that PAItrap4 effectively counteracts the inhibitory effects of PAI-1 on rtPA, preserving rtPA activity based on amidolytic and clot lysis assays. In an in vivo murine stroke model, PAItrap4, together with low-dose rtPA, enhances the blood perfusion in the stroke-affected areas, reduces infarct size, and promotes neurological recovery in mice. Importantly, such treatment does not increase the amount of cerebral hemorrhage, thus reducing the risk of cerebral hemorrhage. In addition, PAItrap4 does not compromise the normal blood coagulation function in mice, demonstrating its safety as a therapeutic agent. These findings highlight this combination therapy as a promising alternative for the treatment of ischemic stroke, offering improved safety and efficacy.

Vasoinhibin's Apoptotic, Inflammatory, and Fibrinolytic Actions Are in a Motif Different From Its Antiangiogenic HGR Motif.

Vasoinhibin, a proteolytic fragment of the hormone prolactin, inhibits blood vessel growth (angiogenesis) and permeability, stimulates the apoptosis and inflammation of endothelial cells, and promotes fibrinolysis. The antiangiogenic and antivasopermeability properties of vasoinhibin were recently traced to the HGR motif located in residues 46 to 48 (H46-G47-R48), allowing the development of potent, orally active, HGR-containing vasoinhibin analogues for therapeutic use against angiogenesis-dependent diseases. However, whether the HGR motif is also responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin has not been addressed. Here, we report that HGR-containing analogues are devoid of these properties. Instead, the incubation of human umbilical vein endothelial cells with oligopeptides containing the sequence HNLSSEM, corresponding to residues 30 to 36 of vasoinhibin, induced apoptosis, nuclear translocation of NF-κB, expression of genes encoding leukocyte adhesion molecules (VCAM1 and ICAM1) and proinflammatory cytokines (IL1B, IL6, and TNF), and adhesion of peripheral blood leukocytes. Also, intravenous or intra-articular injection of HNLSSEM-containing oligopeptides induced the expression of Vcam1, Icam1, Il1b, Il6, and Tnf in the lung, liver, kidney, eye, and joints of mice and, like vasoinhibin, these oligopeptides promoted the lysis of plasma fibrin clots by binding to plasminogen activator inhibitor-1 (PAI-1). Moreover, the inhibition of PAI-1, urokinase plasminogen activator receptor, or NF-κB prevented the apoptotic and inflammatory actions. In conclusion, the functional properties of vasoinhibin are segregated into 2 different structural determinants. Because apoptotic, inflammatory, and fibrinolytic actions may be undesirable for antiangiogenic therapy, HGR-containing vasoinhibin analogues stand as selective and safe agents for targeting pathological angiogenesis.

Arid5a/IL-6/PAI-1 Signaling Is Involved in the Pathogenesis of Lipopolysaccharide-Induced Kidney Injury.

A systemic inflammatory response leads to widespread organ dysfunction, such as kidney dysfunction. Plasminogen activator inhibitor-1 (PAI-1) is involved in the pathogenesis of inflammatory kidney injury; however, the regulatory mechanism of PAI-1 in injured kidneys remains unclear. PAI-1 is induced by interleukin (IL)-6 in patients with sepsis. In addition, the stabilization of IL-6 is regulated by the adenine-thymine-rich interactive domain-containing protein 5a (Arid5a). Therefore, the aim of the present study was to examine the involvement of Arid5a/IL-6/PAI-1 signaling in lipopolysaccharide (LPS)-induced inflammatory kidney injury. LPS treatment to C57BL/6J mice upregulated Pai-1 mRNA in the kidneys. Enzyme-linked immunosorbent assay (ELISA) revealed that PAI-1 expression was induced in the culture supernatants of LPS-treated human umbilical vein endothelial cells, but not in those of LPS-treated human kidney 2 (HK-2) cells, a tubular cell line. Combined with single-cell analysis, endothelial cells were found to be responsible for PAI-1 elevation in LPS-treated kidneys. Administration of TM5441, a PAI-1 inhibitor, reduced the urinary albumin/creatinine ratio, concomitant with downregulation of Il-6 and Arid5a mRNA expressions. IL-6 treatment in LPS model mice further upregulated Pai-1 mRNA expression compared with LPS alone, accompanied by renal impairment. Furthermore, the expression of Il-6 and Pai-1 mRNA was lower in Arid5a knockout mice than in wild-type mice after LPS treatment. Taken together, the vicious cycle of Arid5a/IL-6/PAI-1 signaling is involved in LPS-induced kidney injury.

Abstract 14009: Plasminogen Activator Inhibitor-1 (PAI-1) is an Essential Driver of Cardiovascular Aging in Mice

Introduction: PAI-1 is the protein product of the gene Serpine1 . PAI-1 is a key contributor to cellular senescence in humans and mice, and elevated PAI-1 predicts the development of aging-related disorders including Type 2 diabetes and hypertension. GrimAge, a composite measure of epigenetic age, includes a DNA methylation estimator of plasma PAI-1 levels that independently predicts the development of cardiovascular (CV) morbidity including hypertension and time to heart failure. While many mechanistic contributions of PAI-1 to senescence and aging are well documented, its fundamental role in CV aging has not been fully investigated. Hypothesis: PAI-1 overexpression in mice promotes CV aging, while PAI-1 deficiency attenuates this phenotype in the presence of a stressor. Methods: To assess the effect of PAI-1 overexpression on CV aging, mice transgenic for a stable variant of human PAI-1 (n=14 transgenics, n=8 wild-type littermates) were characterized at 2 months and 7 months. To assess the effect of PAI-1 deficiency, mice heterozygous for a Serpine1 frame-shifting loss-of-function mutation (n=9) and wild-type littermates (n=10) were assessed; these mice underwent cardiovascular stress using N(ω)-nitro-L-arginine methyl ester (L-NAME) (1mg/L) for 8 weeks. Cardiovascular phenotyping included treadmill, pulse-wave velocity (PWV), echocardiography, and blood pressure (BP) measurement at the beginning and end of study. Histology was performed on aortas using Masson’s Trichome and analyzed for fibrosis quantification. Results: PAI-1 transgenic mice exhibited worse diastolic (E/e’) and vascular function (PWV, systolic and diastolic BP) compared to their wild-type counterparts. Histology showed more extensive peri-aortic fibrosis. In contrast, PAI-1 loss-of-function heterozygotes were broadly resistant to the vascular aging effects of L-NAME compared to wild-type mice. Molecular profiling of vascular tissues collected from animals is underway and includes bulk aorta RNAseq and measures of epigenetic age. Conclusion: PAI-1 is necessary and sufficient for the development of a CV aging phenotype in mice while PAI-1 deficiency attenuates stress-induced CV aging. Inhibition of PAI-1 will likely be tested for preventing CV aging in humans.

Pharmacological inhibition of plasminogen activator inhibitor-1 prevents memory deficits and reduces neuropathology in APP/PS1 mice.

Extracellular proteolytic activity plays an important role in memory formation and the preservation of cognitive function. Previous studies have shown increased levels of plasminogen activator inhibitor-1 (PAI-1) in the brain of mouse models of Alzheimer's disease (AD) and plasma of AD patients, associated with memory and cognitive decline; however, the exact function of PAI-1 in AD onset and progression is largely unclear. In this study, we evaluated a novel PAI-1 inhibitor, TM5A15, on its ability to prevent or reverse memory deficits and decrease Aβ levels and plaque deposition in APP/PS1 mice. We administered TM5A15 mixed in a chow diet to 3-month and 9-month-old APP/PS1 mice before and after neuropathological changes were distinguishable. We then evaluated the effects of TM5A15 on memory function and neuropathology at 9 months and 18 months of age. In the younger mice, 6 months of TM5A15 treatment protected against recognition and short-term working memory impairment. TM5A15 also decreased oligomer levels and amyloid plaques, and increased mBDNF expression in APP/PS1 mice at 9 months of age. In aged mice, 9 months of TM5A15 treatment did not significantly improve memory function nor decrease amyloid plaques. However, TM5A15 treatment showed a trend in decreasing oligomer levels in APP/PS1 mice at 18 months of age. Our results suggest that PAI-1 inhibition could improve memory function and reduce the accumulation of amyloid levels in APP/PS1 mice. Such effects are more prominent when TM5A15 is administered before advanced AD pathology and memory deficits occur.

Chuanxiong Rhizoma extracts prevent cholestatic liver injury by targeting H3K9ac-mediated and cholangiocyte-derived secretory protein PAI-1 and FN.

Chuanxiong Rhizoma (CX, the dried rhizome of Ligusticum wallichii Franch.), a well-known traditional Chinese medicine, is clinically used for treating cardiovascular, cerebrovascular and hepatobiliary diseases. Cholestatic liver damage is one of the chronic liver diseases with limited effective therapeutic strategies. Currently, little is known about the mechanism links between CX-induced anti-cholestatic action and intercellular communication between cholangiocytes and hepatic stellate cells (HSCs). The study aimed to evaluate the hepatoprotective activity of different CX extracts including the aqueous, alkaloid, phenolic acid and phthalide extracts of CX (CXAE, CXAL, CXPA and CXPHL) and investigate the intercellular communication-related mechanisms by which the most effective extracts work on cholestatic liver injury. The active compounds of different CX extracts were identified by UPLC-MS/MS. A cholestatic liver injury mouse model induced by bile duct ligation (BDL), and transforming growth factor-β (TGF-β)-treated human intrahepatic biliary epithelial cholangiocytes (HIBECs) and HSC cell line (LX-2 cells) were used for in vivo and in vitro studies. Histological and other biological techniques were also applied. The results indicated that CXAE, CXAL and CXPHL significantly reduced ductular reaction (DR) and improved liver fibrosis in the BDL mice. Meanwhile, both CXAE and CXPHL suppressed DR in injured HIBECs and reduced collagen contraction force and the expression of fibrosis biomarkers in LX-2 cells treated with TGF-β. CXPHL suppressed the transcription and transfer of plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) from the 'DR-like' cholangiocytes to activated HSCs. Mechanistically, the inhibition of PAI-1 and FN by CXPHL was attributed to the untight combination of the acetyltransferase KAT2A and SMAD3, followdd by the suppression of histone 3 lysine 9 acetylation (H3K9ac)-mediated transcription in cholangiocytes. In conclusion, CXPHL exerts stronger anti-cholestatic activity in vivo and in vitro than other CX extracts, and its protective effect on the intracellular communication between cholangiocytes and HSCs is achieved by reducing KAT2A/H3K9ac-mediated transcription and release of PAI-1 and FN.

Inhibition Effect of Physalis angulata Leaf Extract on Viability, Collagen Type I, and Tissue Inhibitor of Metalloproteinase 1 (TIMP-1) but Not Plasminogen Activator Inhibitor-1 (PAI-1) of Keloid Fibroblast Culture.

Keloids are excessive fibroproliferative diseases that are caused by abnormal wound healing. The anti-proliferative activity of Physalis angulata compounds has potential as a keloid therapeutic agent. This study aimed to observe the effects of P. angulata on fibroblast viability and collagen type I, tissue inhibitor of metalloproteinase 1 (TIMP-1), and plasminogen activator inhibitor 1 (PAI-1) levels in human keloid fibroblasts. We conducted an experimental study of P. angulata ethanol extract on three primary human keloid fibroblast 3 passage cultures with four replications. Fibroblast viability was measured using the MTT assay after incubation with 3, 5, and 10 µg/mL P. angulata. Concentrations of P. angulata used to observe effects on TIMP-1, PAI-1, and collagen type I levels were 10%, 20%, 30%, and 40% of inhibitory concentration 50 (IC50). The levels of collagen type I, TIMP-1, and PAI-1 were measured by ELISA. Mean comparisons between multiple treatment groups were analyzed using one-way ANOVA followed by post-hoc analysis. The 10 µg/mL P. angulata group had significantly lower fibroblast viability than the control group (p<0.05) with an IC50 6.3 µg/mL. The collagen type I level of 10% IC50 (0.63 µg/mL) P. angulata group was lower than control (12.910 vs 47.866 ng/mL) (p=0.042). Level of TIMP-1 in 40% IC50 (2.51 µg/mL) P. angulata group was lower than control (5.350 vs 9.972 ng/mL) (p=0.043). There was no significant difference in the PAI-1 levels. This study showed the inhibitory effect of 10 µg/mL P. angulata extract on keloid fibroblast viability, with an IC50 of 6.3 µg/mL. This study also showed collagen type-1 and TIMP-1 inhibition, but not PAI-1 inhibition, after P. angulate treatment.

Suppression of antitumor cytokine IL‑24 by PRG4 and PAI‑1 may promote myxoid liposarcoma cell survival.

Suppression of the antitumor cytokine interleukin-24 (IL-24) is critical for the survival of myxoid liposarcoma (MLS) cells. It has been previously demonstrated by the authors that an MLS-specific chimeric oncoprotein, translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP), supresses IL24 mRNA expression via induction of proteoglycan 4 (PRG4) to sustain MLS cell proliferation. However, IL-24 has also been revealed to be suppressed by the ubiquitin-proteasome system in human ovarian and lung cancer cells. Therefore, the aim of the present study was to elucidate the mechanism of IL-24 suppression in MLS cells. The results revealed that the proteasome inhibitor, MG-132, induced cell death in MLS cells in vitro; this effect was reduced following IL-24 knockdown. This indicated that proteasomal degradation of IL-24 may be an important process for MLS cell survival. In addition, it was also previously revealed by the authors that knockdown of plasminogen activator inhibitor-1 (PAI-1), a TLS-CHOP downstream molecule, suppressed the growth of MLS cells, thus instigating the investigation of the effect of PAI-1 on IL-24 expression in MLS cells. Double knockdown of PAI-1 and IL-24 negated the growth-suppressive effect of PAI-1 single knockdown in MLS cells. Interestingly, PAI-1 single knockdown did not increase the mRNA expression of IL24, but it did increase the protein abundance of IL-24, indicating that PAI-1 suppressed IL-24 expression by promoting its proteasomal degradation. Moreover, treatment of MLS cells with a PAI-1 inhibitor, TM5275, induced IL-24 protein expression and apoptosis. Collectively, the results of the present as well as previous studies indicated that IL-24 expression may be suppressed at the transcriptional level by PRG4 and at the protein level by PAI-1 in MLS cells. Accordingly, PAI-1 may represent an effective therapeutic target for MLS treatment.

Dual deletion of guanylyl cyclase-A and p38 mitogen-activated protein kinase in podocytes with aldosterone administration causes glomerular intra-capillary thrombi

Natriuretic peptides exert not only blood-lowering but also kidney-protective effects through guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. Signaling through GC-A has been shown to protect podocytes from aldosterone-induced glomerular injury, and a p38 mitogen-activated protein kinase (MAPK) inhibitor reduced glomerular injury in aldosterone-infused podocyte-specific GC-A knockout mice. To explore the role of p38 MAPK in podocytes, we constructed podocyte-specific p38 MAPK and GC-A double knockout mice (pod-double knockout mice). Unexpectedly, aldosterone-infused and high salt-fed (B-ALDO)-treated pod-double knockout mice resulted in elevated serum creatinine, massive albuminuria, macrophage infiltration, foot process effacement, nephrin and podocin reduction, and additionally, intra-capillary fibrin thrombi, indicating endothelial injury. Microarray analysis showed increased plasminogen activator inhibitor-1 (PAI-1) in glomeruli of B-ALDO-treated pod-double knockout mice. In B-ALDO-treated pod-double knockout mice, PAI-1 increased in podocytes, and treatment with PAI-1 neutralizing antibody ameliorated intra-capillary thrombus formation. Invitro, deletion of p38 MAPK by the CRISPR/Cas9 system and knockdown of GC-A in human cultured podocytes upregulated PAI-1 and transforming growth factor- β1 (TGF-β1). When p38 MAPK knockout podocytes, transfected with a small interfering RNA to suppress GC-A, were co-cultured with glomerular endothelial cells in a transwell system, the expression of TGF-β1 was increased in glomerular endothelial cells. PAI-1 inhibition ameliorated both podocyte and endothelial injury in the transwell system signifying elevated PAI-1 in podocytes is a factor disrupting normal podocyte-endothelial crosstalk. Thus, our results indicate that genetic dual deletion of p38 MAPK and GC-A in podocytes accelerates both podocyte and endothelial injuries, suggesting these two molecules play indispensable roles in podocyte function.

Obesity paradox in acute myocardial infarction: Is plasminogen activator inhibitor 1 the culprit?

The obesity paradox continues to puzzle scientists. The unexpected observation of a higher incidence but more favorable outcomes in obese patients has been described in several cardiovascular and non-cardiovascular conditions. A number of possible causes of the paradox have been proposed. Plasminogen activator inhibitor 1 (PAI-1) has not yet been evaluated in this context. Our study group investigated the prognostic value of PAI-1 dynamics in the early stages of acute ST elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention. A higher rise in PAI-1 activity within 24 h of STEMI was associated with worse long-term outcomes. Within the study population, which involved 87 consecutive patients, we observed a certain pattern of PAI-1 dynamics that may be protective in obese patients. In these patients, initial PAI-1 activity levels were elevated, a fact in line with the higher incidence of acute coronary syndromes in the obese population. However, the increase in PAI-1 activity during first 24 h of STEMI was not as pronounced in obese patients. It is our hypothesis that this correlation may prove protective in this population. If confirmed in larger studies, this observation may provide a piece of the puzzle towards elucidating the origins of the obesity paradox, as well as further promote the development of PAI-1 inhibitors as a viable therapeutic option for STEMI patients.

Abstract 659: Plasminogen Activator Inhibitor-1 (PAI-1) Increases Mitochondrial Membrane Potential In Vascular Smooth Muscle Cells And Coronary Arteries

Background: Plasminogen activator inhibitor- 1 (PAI-1) expression is increased in cardiometabolic diseases associated with mitochondrial dysfunction and atherosclerosis, including obesity and diabetes mellitus. However, the role of PAI-1 in triggering mitochondrial dysfunction in the vasculature is poorly defined. Mitochondrial membrane potential (ΔΨm) is as a central bioenergetic parameter controlling reactive oxygen species (ROS) generation. We investigated the roles of PAI-1 in controlling Ψm in human coronary artery smooth muscle cells (HCASMCs) and murine coronary arteries, as well as ROS production in the arterial wall. Methods: HCASMCs were cultured in high glucose (25 mM), normal glucose (4.6 mM), and isosmotic control (mannitol 25 mM) conditions (with and without PAI-039, a specific PAI-1 inhibitor) for 1 hour. JC-1 (5 μM), a ratiometric marker of mitochondrial membrane potential, was added and red/green fluorescent imaging was used to quantify ΔΨm. To evaluate effects of PAI-1 on ΔΨm and mitochondrial ROS production in the vessel wall, coronary artery segments were isolated from wild-type (WT) and PAI-1-deficient mice, pressurized ex vivo (100 cmH 2 O), loaded with JC-1 (5μM) and Mitosox (5 μM), then subjected to quantitative fluorescence imaging. Results: Compared to vehicle control, PAI-039 significantly decreased ΔΨm in HCASMCs under all experimental conditions (normal glucose: 2.08±0.15 vs . 1.47±0.04 AU; high glucose: 2.49±0.05 vs . 1.83±0.15 AU; and isosmotic control: 2.04±0.09 vs . 1.52±0.07 AU, n=4 for all groups, P&lt;0.05 for all comparisons). The ΔΨm was significantly lower in coronary arteries from PAI-1-deficient mice vs. WT controls (0.85±0.03 vs . 1.09±0.09AU, n=6/group, P&lt;0.05). Coronary arteries from PAI-1 knockout mice showed a statistically insignificant trend toward decreased ROS production vs . WT controls (6.4± 1.44 vs . 10.2±1.31 AU, n=6/group, P=0.07). Conclusion: Pharmacologic inhibition of PAI-1 decreases ΔΨm in HCASMCs. Endogenous expression of PAI-1 increases ΔΨm in coronary arteries, which is accompanied by a trend towards increased ROS production. These findings indicate that PAI-1 regulates mitochondrial function in vascular SMCs and by such effects may increase ROS generation in the vasculature.

Abstract 1684: Plasminogen activator inhibitor-1 (PAI-1) promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis

Abstract Resistance to platinum-based chemotherapy is one of major causes of therapy failure and tumor recurrence in ovarian cancer patients. However, the molecular mechanism of platinum resistance is not fully understood. Here, we compared gene expression between cisplatin-sensitive and -resistant ovarian cancer cells using gene expression microarray analysis and found the significantly up-regulated expression of SERPINE1 encoding plasminogen activator inhibitor-1 (PAI-1), a serine protease regulating the fibrinolysis and extracelluar matrix, in cisplatin-resistant ovarian cancer cells. The correlation of high PAI-1 expression with low cisplatin sensitivity was confirmed in ovarian cancer cells. PAI-1 overexpression in ovarian cancer cells significantly decreased the sensitivity to cisplatin, whereas PAI-1 down-regulation by SERPINE1 siRNA or PAI-1 inhibitor increased cisplatin sensitivity. PAI-1 inhibited cisplatin-induced apoptosis in ovarian cancer cells by suppressing reactive oxygen species (ROS) production and DNA damage. PAI-1 also decreased cisplatin-induced ferroptosis through the upregulation of GPX4 levels. Furthermore, Kaplan-Meier survival analysis using TGGA data demonstrated that high PAI-1 expression was significantly associated with shorter platinum-free survival of patients with ovarian cancer. Collectively, these results illustrate that PAI-1 promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis, suggesting that targeting PAI-1 increases the sensitivity to cisplatin in ovarian cancer. Citation Format: Mi Jeong Kwon, Ha Yeong Chae, So Young Lee, Shin Ung Kang, Tae Woong Jung, Soo Youn Cho, Jinil Han, Hyojin Jeong, Chang Ohk Sung, Young Kee Shin. Plasminogen activator inhibitor-1 (PAI-1) promotes cisplatin resistance in ovarian cancer cells by inhibiting ROS-mediated apoptosis and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1684.

Circadian rhythm of plasminogen activator inhibitor-1 and cardiovascular complications in type 2 diabetes.

Cardiovascular complications are a common death cause in type 2 diabetes patients, as they are often combined. Plasminogen-activator Inhibitor 1 (PAI-1) participates in the development and progression of cardiovascular complications in diabetes. Insulin resistance increases PAI-1 production, and high PAI-1 levels lead to an environment conducive to thrombosis and earlier and more severe vascular disease. Current evidence also suggests that PAI-1 has a rhythmic profile of circadian fluctuations and acrophase in the morning within a single day, which might explain the high morning incidence of cardiovascular events. Thus, PAI-1 is a possible drug target. Although several PAI-1 inhibitors have been developed, none have yet been allowed for clinical use. Research on rhythm has also led to the concept of "chronotherapy", a rhythm-based drug regimen expected to improve the treatment of cardiovascular complications in diabetic patients. Herein, we searched several databases and reviewed relevant articles to describe the circadian rhythm characteristics and endogenous molecular mechanisms of PAI-1, its relationship with insulin resistance, the causes of cardiovascular complications caused by PAI-1, and the current development of PAI-1 inhibitors. We also summarized the possibility of using the circadian rhythm of PAI-1 to treat cardiovascular complications in diabetic patients.