Plasminogen Activator Inhibitor-1 Expression Levels Research Articles

PAI1 Regulates Cell Morphology and Migration Markers in Trastuzumab-Resistant HER2-Positive Breast Cancer Cells.

HER2-positive breast cancer is a significant cause of mortality. Overcoming trastuzumab resistance requires a deeper understanding of its molecular mechanisms to develop effective therapies. This study investigates the role of plasminogen activator inhibitor-1 (PAI1) in migration and drug resistance in trastuzumab-resistant HER2-positive breast cancer. Trastuzumab resistance poses a significant challenge in clinical management due to its association with aggressive disease behaviour and limited treatment options. This study focuses on PAI1, a key player in the TGF-β signalling pathway, which is implicated in cancer progression and metastasis. Trastuzumab-resistant cell lines (SKBR3 and HCC1954) demonstrated markedly elevated PAI1 expression levels, up to 40-fold compared to parental lines. This elevation was accompanied by increased expression of migration markers such as Col4a1, Fibronectin, ICAM1, Timp2, and Vimentin. Through overexpression and silencing experiments, we observed that modulating PAI1 levels significantly impacts cell morphology, transitioning cells from an epithelial to mesenchymal phenotype. Importantly, combining trastuzumab with aleplasinin, a PAI1 inhibitor, synergistically reduced PAI1 expression in both parental and resistant cell lines. This suggests a potential therapeutic strategy to overcome trastuzumab resistance. These findings emphasise PAI1 as a critical mediator of migration and therapeutic response in HER2-positive breast cancer, offering insights into novel treatment approaches targeting PAI1 to improve clinical outcomes in drug resistance.

Effect of aminoguanidine on plasminogen activator inhibitor-1 and receptor of advanced glycation endproduct in the liver of streptozotocin-induced diabetic rats

Objectives: Advanced glycation end products (AGEs) play an important role in the development and progression of diabetic complications. The receptor for AGE (RAGE) is the ligand-binding site of AGE that initiates and accelerates the atherosclerotic process. Plasminogen activator inhibitor-1 (PAI-1) is a prothrombotic factor that has been proposed as a biological marker for prognostic assessment, monitoring of microvascular and macrovascular complications in diabetes. The purpose of this study is to investigate the effects of aminoguanidine on RAGE and PAI-1 expression levels in the liver of streptozotocin-induced diabetic rats.
 Methods: Diabetes was induced in rats by intraperitoneal injection of streptozocin (STZ, 50 mg/kg). On day 3, diabetic rats were administered 50, 100, and 200 mg/kg/day of aminoguanidine. The expression of PAI-1 and RAGE in the liver tissue was evaluated using real-time PCR.
 Results: PAI-1 and RAGE gene expression levels were higher in the liver of the diabetic rats compared to the control group. Aminoguanidine at 50, 100, and 200 mg/kg decreased PAI-1 and RAGE gene expression in the liver (p<0.001 at all doses). However, these genes were downregulated only at a dose of 200 mg/kg in healthy rats (p<0.0001). In addition, hepatic AGE protein levels were significantly decreased following treatment of the diabetic rats with aminoguanidine (p<0.001). There was also a significant correlation between AGE protein concentration and the expression of PAI-1 and RAGE.
 Conclusion: In summary, the data of the present study suggest that aminoguanidine reduced the expression of PAI-1 and RAGE in the liver of the diabetic rats.

PAI-1 is a potential transcriptional silencer that supports bladder cancer cell activity

The extracellular activity of Plasminogen activator inhibitor-1 (PAI-1) is well described, acting as an inhibitor of tissue plasminogen activator and urokinase-type plasminogen activator, impacting fibrinolysis. Recent studies have revealed a pro-tumorigenic role of PAI-1 in human cancers, via the regulation of angiogenesis and tumor cell survival. In this study, immunohistochemical staining of 939 human bladder cancer specimens showed that PAI-1 expression levels correlated with tumor grade, tumor stage and overall survival. The typical subcellular localization of PAI-1 is cytoplasmic, but in approximately a quarter of the cases, PAI-1 was observed to be localized to both the tumor cell cytoplasm and the nucleus. To investigate the potential function of nuclear PAI-1 in tumor biology we applied chromatin immunoprecipitation (ChIP)-sequencing, gene expression profiling, and rapid immunoprecipitation mass spectrometry to a pair of bladder cancer cell lines. ChIP-sequencing revealed that PAI-1 can bind DNA at distal intergenic regions, suggesting a role as a transcriptional coregulator. The downregulation of PAI-1 in bladder cancer cell lines caused the upregulation of numerous genes, and the integration of ChIP-sequence and RNA-sequence data identified 57 candidate genes subject to PAI-1 regulation. Taken together, the data suggest that nuclear PAI-1 can influence gene expression programs and support malignancy.

Precision targeting of the plasminogen activator inhibitor-1 mechanism increases efficacy of fibrinolytic therapy in empyema.

Plasminogen activator inhibitor‐1 (PAI‐1) is an endogenous irreversible inhibitor of tissue‐type (tPA) and urokinase (uPA) plasminogen activators. PAI‐1‐targeted fibrinolytic therapy (PAI‐1‐TFT) is designed to decrease the therapeutic dose of tPA and uPA, attenuating the risk of bleeding and other complications. Docking site peptide (DSP) mimics the part of the PAI‐1 reactive center loop that interacts with plasminogen activators, thereby affecting the PAI‐1 mechanism. We used DSP for PAI‐1‐TFT in two rabbit models: chemically induced pleural injury and Streptococcus pneumoniae induced empyema. These models feature different levels of inflammation and PAI‐1 expression. PAI‐1‐TFT with DSP (2.0 mg/kg) converted ineffective doses of single chain (sc) tPA (72.5 µg/kg) and scuPA (62.5 µg/kg) into effective ones in chemically induced pleural injury. DSP (2.0 mg/kg) was ineffective in S. pneumoniae empyema, where the level of PAI‐1 is an order of magnitude higher. DSP dose escalation to 8.0 mg/kg resulted in effective PAI‐1‐TFT with 0.25 mg/kg sctPA (1/8th of the effective dose of sctPA alone) in empyema. There was no increase in the efficacy of scuPA. PAI‐1‐TFT with DSP increases the efficacy of fibrinolytic therapy up to 8‐fold in chemically induced (sctPA and scuPA) and infectious (sctPA) pleural injury in rabbits. PAI‐1 is a valid molecular target in our model of S. pneumoniae empyema in rabbits, which closely recapitulates key characteristics of empyema in humans. Low‐dose PAI‐1‐TFT is a novel interventional strategy that offers the potential to improve fibrinolytic therapy for empyema in clinical practice.

PAI-1 derived from cancer-associated fibroblasts in esophageal squamous cell carcinoma promotes the invasion of cancer cells and the migration of macrophages

AbstractCancer-associated fibroblasts (CAFs) contribute to the progression of various cancers. Previously, we reported the significance of CAFs in esophageal squamous cell carcinoma (ESCC); however, the functions of CAFs in the ESCC microenvironment remain unknown. To investigate CAFs' function, we established an indirect coculture assay between human bone marrow-derived mesenchymal stem cells (MSCs) and ESCC cells. Cocultured MSCs expressed more fibroblast activation protein, one of the markers of CAFs, compared with monocultured MSCs. Therefore, we defined cocultured MSCs as CAF-like cells. To identify molecules associated with the ESCC progression in CAFs, we conducted a cDNA microarray analysis on monocultured MSCs and CAF-like cells to compare their gene expression profiles. We found that SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was more abundant in CAF-like cells than in monocultured MSCs, and the PAI-1 derived from CAF-like cells induced the abilities of migration and invasion in both ESCC cells and macrophages by the Akt and Erk1/2 signaling pathways via the low-density lipoprotein receptor-related protein 1 (LRP1), which is a PAI-1 receptor. Based on immunohistochemistry assays of ESCC tissues, higher expression levels of PAI-1 and LRP1 were correlated with poor prognosis in ESCC patients. These results suggest that the PAI-1/LRP1 axis contributes to the progression of ESCC, making it a potential target for ESCC therapy.The authors show that plasminogen activator inhibitor-1 (PAI-1) derived from cancer-associated fibroblasts promotes the invasion of esophageal squamous cell carcinoma (ESCC) cells and the migration of macrophages via lipoprotein receptor-related protein 1 (LRP1). High expression of PAI-1 and/or LRP1 is associated with poor prognosis in patients with ESCC, and the PAI-1/LRP1 axis could be a target of anticancer therapy.

Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat type II alveolar epithelial cells

To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS). AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC50). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 μmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). According to the inhibition rate curve, the IC50 of berberine on RLE-6TN cells was 81.16 μmol/L. Therefore, 20, 50 and 80 μmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 μmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 μmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2-ΔΔCt): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2-ΔΔCt): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2-ΔΔCt): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (μg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (μg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (μg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01]. Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).

Expression and clinical significance of thrombospondin-1 and plasminogen activator inhibitor-1 in patients with mesangial proliferative glomerulonephritis.

As a common pathological type of glomerular disease in China, mesangial proliferative glomerulonephritis is related to plasminogen activator inhibitor-1 (PAI-1) and thrombospondin-1 (TSP-1). Here, this study aims to investigate the expression and clinical significance of TSP-1 and PAI-1 in patients with mesangial proliferative glomerulonephritis. Renal tissue specimens from 46 patients with mesangial proliferative glomerulonephritis admitted to this hospital were selected as the subjects, and 8 specimens of renal tissue from autopsy were used as controls. The expression levels of TSP-1 and PAI-1 were detected by immunohistochemistry and analyzed. The 24-hour urine protein, triglyceride, and total cholesterol levels of patients with severe mesangial hyperplasia were significantly higher than those of patients with mild and moderate mesangial hyperplasia, and serum albumin was lower than that of patients with mild and moderate mesangial hyperplasia (P<0.05). The 24-hour urine protein level of patients with moderate mesangial hyperplasia was higher than that of patients with mild mesangial hyperplasia while the albumin level was lower (P<0.05), but there was no significant difference in triglyceride and total cholesterol (P>0.05). There was no significant difference in creatinine clearance (Ccr) between the three groups (P>0.05).The 24-hour urine protein and urine alpha-1-microglobulin (A1M) levels in patients with renal interstitial disease were higher than those in patients without renal interstitial disease, while their Ccr level was lower (P<0.05). TSP-1 and PAI-1 were not positively expressed in the glomeruli and renal tubules of specimens of the control group. However, in mesangial hyperplasia patients, the expression of TSP-1 and PAI-1 in mesangial hyperplasia with varying degrees and in different renal tubular damage were as follows: mild degree < moderate degree < severe degree (P<0.05). The pathological changes of mesangial proliferative glomerulonephritis are related to 24- hour urine protein, triglyceride, total cholesterol level, urine A1M, and Ccr level. The expression of TSP1 and PAI-1 in the mesenchyme of glomerular and renal tubules significantly increased with the severity of the disease, suggesting that TSP-1 and PAI-1 play an important role in the occurrence and development of mesangial proliferative glomerulonephritis.

Sevoflurane leads to learning and memory dysfunction via breaking the balance of tPA/PAI-1

Exposure to general anesthesia in early childhood may lead to adverse effects on adolescent neurocognition. This study investigated the effects of multiple inhalations of sevoflurane on long-term learning and memory in developing rats, and explored the mechanistic role of the tissue plasminogen activator (tPA)/plasminogen activator inhibitor-1 (PAI-1) fibrinolysis system and its regulatory relationship with the brain derived neurotrophic factor (BDNF) by activation of tropomysin related kinase B (TrkB). After rats were inhaled with sevoflurane for 2 h/d for three days, the expression levels of tPA, PAI-1, BDNF, its precursor(proBDNF), TrkB and phosphorylation of TrkB (p-TrkB) were detected at different time points. After 28 d, Morris water maze was used to examine learning and memory function; Golgi staining was used to investigate synaptic plasticity and synaptic-related proteins, such as Synapsin I(SYN1), growth associated protein 43(GAP-43), and postsynaptic density protein 95(PSD-95). Rats were given exogenous tPA and an inhibitor of PAI-1, TM5275. The results showed multiple inhalation of sevoflurane led to learning and memory dysfunction, downregulated the expression of the synaptic-related proteins, decreased dendritic spine density in the hippocampus, increased the expression level of proBDNF and PAI-1, and reduced expression of BDNF, tPA, and p-TrkB. Interestingly, tPA or TM5275 partially reversed the learning and memory dysfunction and the reduction of synaptic plasticity induced by sevoflurane exposure. Furthermore, they blocked the upregulation of proBDNF and PAI-1 protein expression and increased the expression of BDNF, tPA, and p-TrkB. The protective effect of tPA or TM5275 on rats following multiple sevoflurane inhalation was blocked by a TrkB inhibitor. Multiple inhalation of sevoflurane in rats inhibited the cleavage of proBDNF by disrupting the balance of the tPA/PAI-1 fibrinolysis system. This blocked the activation of the downstream TrkB signaling pathway and reduced hippocampal synaptic plasticity, leading to long-term learning and memory dysfunction. Therefore, Sevoflurane exposure could lead to learning and memory dysfunction by inhibiting BDNF cleavage via breaking the balance of tPA/PAI-1.

Fetuin-B regulates vascular plaque rupture via TGF-β receptor-mediated Smad pathway in vascular smooth muscle cells.

Fetuin-B is a serum protein linked to the regulation of physiological or pathophysiological events such as fertility, energy metabolism, and liver disease. Recently, fetuin-B has been reported to be involved in the modulation of the rupture of atherosclerotic plaques associated with acute myocardial infarction. However, the exact mechanism involved in the modulation of atherosclerotic plaque rupture event by fetuin-B is not fully elucidated yet. In the present study, we investigated whether fetuin-B could influence atherosclerotic plaque rupture through vascular smooth muscle cells (VSMCs). Immunoprecipitation assay using membrane proteins from VSMCs revealed that fetuin-B tightly bound to transforming growth factor-β receptor (TGF-βR). Fetuin-B treatment elevated TGF-βR signals (e.g., phosphorylation of Smad2 and Smad3) in VSMCs. Fetuin-B also stimulated nuclear translocation of phosphorylated Smads. Phosphorylation of Smad and its nuclear translocation by treatment with fetuin-B were inhibited in VSMCs by treatment with SB431542, a selective inhibitor of TGF-βR. Fetuin-B enhanced expression levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in VSMCs through its epigenetic modification including recruitments of both histone deacetylase 1 and RNA polymerase II. These epigenetic alterations in VSMCs were also inhibited by treatment with SB431542. In vivo administration of fetuin-B protein increased expression levels of PAI-1 and MMP-2 in the vascular plaque. However, these increases in expression were inhibited by the administration of SB43154. These results indicate that fetuin-B may modulate vascular plaque rupture by promoting expression of PAI-1 and MMP-2 in VSMCs via TGF-βR-mediated Smad pathway.

Role of plasminogen activator inhibitor-1 in methotrexate-induced epithelial-mesenchymal transition in alveolar epithelial A549 cells

There is increasing evidence that epithelial-mesenchymal transition (EMT) contributes to the development of organ fibrosis. We demonstrated that methotrexate (MTX) clearly induced EMT through the transforming growth factor (TGF)-β-related signaling pathway in human alveolar epithelial cell line, A549. However, critical factors associated with MTX-induced EMT have not yet been identified. In our study, we attempted to identify factors playing a crucial role in MTX-induced EMT in A549 cells. We focused on plasminogen activator inhibitor-1 (PAI-1) as the possible target for the prevention of MTX-induced EMT-related lung injury. Comprehensive gene expression analysis by microarray revealed that mRNA expression level of PAI-1 was clearly increased by MTX treatment. In addition, using several cloned A549 cells, we found a good correlation between MTX-induced increase in mRNA expression levels of α-smooth muscle actin (SMA), a representative EMT marker, and PAI-1. Furthermore, MTX upregulated mRNA and protein expression levels of PAI-1 in A549 cells; this upregulation was canceled by co-treatment with SB431542, a TGF-β-related signaling pathway inhibitor. Notably, tiplaxtinin, a PAI-1 inhibitor, and knockdown of urokinase-type plasminogen activator receptor (uPAR) prevented MTX-induced EMT in A549 cells. These findings indicate that MTX may induce EMT via upregulation of PAI-1 expression and interaction of PAI-1 with uPAR in A549 cells.

The urokinase-type plasminogen activator and inhibitors in resectable lung adenocarcinoma

BackgroundThe urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients. MethodsUPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules’ relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA. ResultsUPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P < 0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χ2 = 8.372, P = 0.004). Multivariate analyses showed TNM stage III was an independent adverse prognostic factor (hazard ratio = 3.736, 95 % confidence interval = 1.097–12.72, P = 0.035). Kaplan-Meier method revealed that uPA, PAI-1 and PAI-2 expression levels were not related to the OS for 84 resectable lung adenocarcinoma patients. According to TCGA data, PAI-1 expression level was identified as a potential adverse predictor for prognosis of resectable lung adenocarcinoma (Gehan-Breslow-Wilcoxon test, P = 0.025). ConclusionsOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.

Effect of electroacupuncture on renal fibrosis in spontaneously hypertension rats and its related mechanisms

To observe the effect of electroacupuncture (EA) on blood pressure, renal fibrosis and expression of tissue inhibitors of metalloproteinase-1 (TIMP-1), plasminogen activator inhibitor 1 (PAI-1), and alpha smooth muscle actin (α-SMA) in spontaneous hypertension rats (SHR), so as to explore its mechanisms underlying improving hypertensive renal damage. Forty male SHR (15 weeks in age) were randomly divided into 5 groups: model, medication (Losartan), Shenshu, Geshu, and Shenshu＋Geshu groups(n=8 rats in each group), and the same age-old male 8 Wistar-Kyoto (WKY) rats were used as the normal control group. Rats of the medication group were treated by gavage of Losartan potassium solution (3 mg/mL, 30 mg·kg-1·d-1, once a day for 12 weeks), and those of the 3 EA groups treated by EA stimulation of bilateral "Shenshu" (BL23), "Geshu"(BL17) or both BL23 and BL17 (2 Hz/100 Hz, 1 mA, 15 min each time, once every other day for 12 weeks). The systolic blood pressure of the tail artery was measured before, and 4, 8 and 12 weeks after the intervention. The expression of TIMP-1, PAI-1 and α-SMA proteins of the right kidney tissue was measured by immunohistochemistry. Histopathological changes of the right renal tissue were observed under light microscope after H.E. stain. The blood pressure was significantly higher in the mo-del group than those in the normal control group (P<0.01), and considerably decreased at the 4th , 8th, and 12th week of the interventions in the medication and 3 EA groups (P<0.01). The expression levels of renal TIMP-1, PAI-1 and α-SMA proteins were notably higher in the model group than those in the normal control group and considerably decreased at the 12th week of the interventions in the medication and 3 EA groups than in the model group (P<0.01). H.E. staining of the renal tissue showed disordered arrangement of the renal cells, congestion and dilation of capillaries with thickened vascular wall, renal tubule atrophy and lumen stenosis with some necrosis of renal tubules, protein tubule and cell tubules, increase of some glomerular mesangial matrix and hyperplasia of fibrous tissue in the model group, which was re-latively milder in the medication and 3 EA groups. EA of BL23 and BL17 can reduce the blood pressure in SHR, which may be related to its function in down-regulating expression of TIMP-1, PAI-1 and α-SMA proteins.

MiR145-5p inhibits proliferation of PMVECs via PAI-1 in experimental hepatopulmonary syndrome rat pulmonary microvascular hyperplasia.

ABSTRACTHepatopulmonary syndrome (HPS) is a triad of advanced liver disease, intrapulmonary vasodilatation and arterial hypoxemia. Increasing evidence shows that HPS is associated with pulmonary microvascular hyperplasia. The aim of this work was to investigate the underlying mechanism of miR-145 in regulating the proliferation of pulmonary microvascular endothelial cells (PMVECs) and angiogenesis in HPS via plasminogen activator inhibitor-1 (PAI-1). To test this, morphology score and number of pulmonary microvascular were assessed in lung tissues from rats with HPS by Hematoxylin and Eosin (H&E) staining. Expression levels of PAI-1 were assessed in lung tissues from HPS rats, as well as in PMVECs treated with HPS rat serum. We also selected the putative microRNA binding site on PAI-1 by bioinformatics analysis. Then, miR145-3p and miR145-5p expression levels in the lungs and PMVECs of rats were detected by qRT-PCR because miR145-5p is a microRNA binding site on PAI-1. In addition, the effects of miR-145-5p regulation on PAI-1 were examined by upregulation and downregulation of miR-145-5p and specific lentivirus transfection was used to overexpress and knockdown PAI-1 to assess PAI-1 function on PMVECs proliferation. Our data showed that levels of PAI-1 expression in lung tissue of rats increased significantly when rats were treated with common bile duct ligation. We found that levels of miR-145-5p were frequently downregulated in HPS tissues and cell lines, and overexpression of miR-145-5p dramatically inhibited PMVECs proliferation. We further verified PAI-1 as a novel and direct target of miR-145-5p in HPS. MiR-145-5p inhibits PAI-1 synthesis and the expression changes of PAI-1 directly affect the proliferation of PMVECs. We concluded that miR-145-5p negatively regulates PMVEC proliferation through PAI-1 expression. In addition, overexpression of miR-145-5p may prove beneficial as a therapeutic strategy for HPS treatment.

MiR-34a exerts as a key regulator in the dedifferentiation of osteosarcoma via PAI-1\u2013Sox2 axis

Osteosarcoma (OS) is a malignant bone cancer with severe chromosomal abnormalities and genetic aberrations. Our previous work reported the dedifferentiation of OS, which is related to poor prognosis. However, the molecular mechanism that regulates OS dedifferentiation is still a subject of exploration. Emerging evidence has suggested that microRNAs (miRNAs) are associated with the pathogenesis of OS and could potentially be developed for use as diagnostic biomarkers and therapeutic strategies. In the present study, we intended to illustrate the role of miR-34a in the dedifferentiation of OS. Upregulation of miR-34a was observed while OS cells were induced into stem-like phenotype. Notably, inhibition of miR-34a could promote the reprogramming transition of OS. Further exploration on the downstream network of miR-34a identified that blocking plasminogen activator inhibitor-1 (PAI-1) expression could restrain OS dedifferentiation into cancer stem-like cells by downregulating SRY-related-HMG box (Sox) 2. We also showed that Sox2 overexpression rescued the suppression phenotype driven by PAI-1 inhibition. Conversely, PAI-1 inhibitor (PAI-039) could suppress the upregulation of Sox2 expression caused by miR-34a inhibition. Be applying bone extracellular matrix (BEM)-OS models, we demonstrated the phenotypic heterogeneity of OS cells, consistent with a strong concordance between PAI-1 and Sox2 expression levels. Taken together, our findings proved miR-34a to be a bona fide suppressor involved in the regulation of OS dedifferentiation. Targeting miR-34a or its direct target PAI-1 could offer new strategies for OS treatment.

SK-216, a Novel Inhibitor of Plasminogen Activator Inhibitor-1, Suppresses Lung Metastasis of Human Osteosarcoma.

Lung metastasis constitutes the leading cause of the death in patients with osteosarcoma. We have previously reported that plasminogen activator inhibitor-1 (PAI-1) regulates the invasion and lung metastasis of osteosarcoma cells in a mouse model and as well as in clinical samples. In the present study, we examined the anti-metastatic effect of SK-216, a small compound PAI-1 inhibitor, in human 143B osteosarcoma cells. An in vitro study showed that SK-216 treatment suppressed invasion activity by inhibiting PAI-1 expression in 143B cells, but had no influence on their proliferation or migration. 143B cells treated with SK-216 exhibited reduced matrix metalloproteinase-13 (MMP-13) secretion in a dose-dependent manner. Moreover, intraperitoneal injection of SK-216 into mouse models resulted in downregulation of PAI-1 expression levels in the primary tumors and showed suppression of lung metastases without influencing the proliferative activity of the tumor cells in the primary lesions. These results indicate that SK-216, a PAI-1 inhibitor, may serve as a novel drug to prevent lung metastasis in human osteosarcoma.

Antioxidant and anti-inflammatory activities of alpha lipoic acid protect against indomethacin-induced gastric ulcer in rats

Little is known about the role of tumor necrosis factor-alpha (TNF-α), plasminogen activator inhibitor-1 (PAI-1), and inducible nitric oxide synthase (iNOS) in the gastric ulcer and the effect of alpha lipoic acid (ALA) in their modulation. Hence, this experimental study was designed to assess the possible protective effect of ALA against indomethacin (IND)-induced gastric ulcer in rats, as well as to determine the possible underlying mechanisms with a special focus on TNF-α, PAI-1, and iNOS. Adult male rats (n = 28) were divided into four equal groups: the control group received distilled water, the vehicle group received 0.5% carboxymethylcellulose, the ulcer group received a single oral dose of IND (50 mg/kg) and the ALA-treated group received ALA (100 mg/kg) orally for 3 days before ulcer induction. Four hours after IND administration, all rats were sacrificed. The ulcer index, and gastric tissue homogenate contents of total antioxidant capacity (TAC), malondialdehyde (MDA), TNF-α, and PAI-1 were evaluated. Immunohistochemical evaluation of iNOS protein expression and histopathological examination of gastric tissue were investigated. The results revealed that ALA pretreatment significantly decreased the ulcer index, the gastric levels of MDA, TNF-α, PAI-1, and iNOS protein expression while increased the gastric levels of TAC as well as improved the histopathological appearance of gastric tissues. In conclusion, ALA ameliorated the IND-induced gastric ulceration. This could be attributed to its antioxidant and anti-inflammatory activities via suppression of TNF-α-induced elevation of both PAI-1 level and iNOS expression in the gastric tissue.

Role of plasminogen activator inhibitor-1 gene polymorphisms in osteoporosis: A study in Chinese post-menopausal women

Osteoporosis is a chronic multifactorial disease characterized by deterioration of bone mass and is vulnerable to bone fracture. Plasminogen activator inhibitor-1 (PAI-1) is an important molecule for maintenance of optimum bone mass. Several single-nucleotide polymorphisms (SNPs) in PAI-1 have been reported to alter PAI-1 expression and/or the translational level. In this report, we explored the possible role of common PAI-1 gene polymorphisms on predisposition to osteoporosis in a Chinese cohort. A total of 364 post-menopausal Chinese women diagnosed of having osteoporosis and 350 healthy females hailing from similar areas were enrolled in this study. Five common SNPs (−844G &gt; A, −6754G/5G, +43G &gt; A, +9785G &gt; A and +11053T &gt; G) were genotyped by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Relative expression of PAI-1 mRNA and plasma PAI-1 levels were quantified by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Prevalence of homozygous mutant (5G/5G) and minor allele (5G) of PAI-1 (−675 4G/5G) polymorphism was significantly more frequent in patients than in healthy controls (5G/5G: P &lt; 0.0001, odds ratio (OR) = 3.18; 5G: P &lt; 0.0001, OR = 1.65). Both plasma PAI-1 and relative mRNA expression levels were significantly lower in patients compared to healthy controls. Interestingly, the quantity of plasma PAI-1 and mRNA expression was correlated with PAI-1 (−675 4G/5G) polymorphism: subjects with 4G/4G genotype had elevated PAI-1 in comparison to homozygous mutant, and displayed lower quantity of PAI-1 protein and mRNA values. PAI-1 (−675 4G/5G) mutant is associated with susceptibility to development of osteoporosis in post-menopausal Chinese women. Furthermore, this variant in the promoter region alters plasma protein levels and relative expression of PAI-1.

Rivaroxaban attenuates thrombosis by targeting the NF-κB signaling pathway in a rat model of deep venous thrombus

Anticoagulant therapy is commonly used for the prevention and treatment of patients with deep venous thrombus. Evidence has shown that rivaroxaban is a potential oral anticoagulant drug for the acute treatment of venous thromboembolism. However, the rivaroxaban-mediated molecular mechanism involved in the progression of deep venous thrombosis has not been investigated. In the present study, we investigated the efficacy of rivaroxaban and the underlying signaling pathways in the prevention and treatment of rats with deep venous thrombosis. A rat model with deep vein thrombus formation was established and received treatment with rivaroxaban or PBS as control. The thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) were analyzed both in vitro and in vivo. The progression of thrombosis and stroke was evaluated after treatment with rivaroxaban or PBS. Nuclear factor-κB (NF-κB) signaling pathway in venous endothelial cells and in the rat model of deep venous thrombus was assessed. The therapeutic effects of rivaroxaban were evaluated as determined by changes in deep venous thrombosis in the rat model. Our results showed that rivaroxaban markedly inhibited TAFI and PAI-1 expression levels, neutrophils, tissue factor, neutrophil extracellular traps (NETs), myeloperoxidase and macrophages in venous endothelial cells and in the rat model of deep venous thrombus. Expression levels of ADP, PAIs, von Willebrand factor (vWF) and thromboxane were downregulated in vein endothelial cells and in serum from the experimental rats. Importantly, the incidences of inferior vena cava filter thrombus were protected by rivaroxaban during heparin-induced thrombolysis deep venous thrombosis in the rat model. We observed that activity of the NF-κB signaling pathway was inhibited by rivaroxaban in vein endothelial cells both in vitro and in vivo. Notably, immunohistology indicated that rivaroxaban attenuated deep venous thrombosis and the accumulation of inflammatory factors in the lesions in venous thrombus. Matrix metalloproteinase (MMP) expression and activity were downregulated in rivaroxaban-treated rats with deep venous thrombus. Rivaroxaban inhibited the elasticity of the extracellular matrix and collagen-elastin fibers. On the whole, these results indicate that rivaroxaban attenuates deep venous thrombus through MMP-9-mediated NF-κB signaling pathway.

Association of plasminogen activator inhibitor-1 and vitamin D receptor expression with the risk of keloid disease in a Chinese population

Keloid disease (KD) is a benign fibroproliferative scarring condition of unknown etiopathogenesis. Plasminogen activator inhibitor-1 (PAI-1) and vitamin D receptor (VDR) have been shown to play important roles in the progression of tissue fibrosis; therefore, both these genes are potential susceptibility genes for KD. We aimed to determine whether the gene expression levels of PAI-1 and VDR are altered in Chinese KD patients. We measured the expression of PAI and VDR in human peripheral blood lymphocytes in 236 patients with keloid and 219 age- and sex-matched healthy controls by quantitative real-time polymerase chain reaction. We found that PAI-1 expression in peripheral blood lymphocytes was significantly higher in patients with KD than in control individuals (p < 0.0001), while VDR expression was significantly lower in KD patients than in control individuals (p < 0.0001). High levels of PAI-1 and low levels of VDR expression were significantly associated with an increased risk for KD. PAI-1 and VDR might play important roles in keloid development. Gene expression levels of PAI-1 and VDR may, therefore, be used as potential markers for the prediction of keloid development after scarring.

Macrophage-derived exosomes induce inflammatory factors in endothelial cells under hypertensive conditions.

Hypertension is one of the most important cardiovascular risk factors and results in macrophage infiltration of blood vessels. However, how macrophages coordinate inflammatory responses with endothelial cells (ECs) remains unclear. In this study, we investigated whether exosomes upregulate the expression of inflammatory factors in ECs under hypertensive conditions. Hypertension was induced in rats by continuous infusion of angiotensin II (Ang II). Exosomes were purified from rat serum by density gradient and ultracentrifugation and used to stimulate human coronary artery ECs (HCAECs). Moreover, the interactions between HCAECs and exosomes from human THP-1-derived macrophages were analyzed. Administration of Ang II enhanced the expression of CD68, a macrophage marker, in rat hearts, suggesting enhanced infiltration of macrophages. In addition, the expression of intracellular adhesion molecule-1 (ICAM1) and plasminogen activator inhibitor-1 (PAI-1), a proinflammatory factor, was increased in hypertensive rat hearts compared with control rats. CD68 protein expression and an increase in the expression of some exosome markers were detected in exosomes from hypertensive rat serum. Moreover, the exosomes upregulated the expression levels of ICAM1 and PAI-1 in HCAECs. The level of miR-17, a negative regulator of ICAM1 expression, was markedly decreased in exosomes from hypertensive rat serum compared with exosomes from control rats. Interestingly, Ang II-stimulated THP-1-derived exosomes also enhanced the expression of ICAM1 and PAI-1 and contained reduced levels of miR-17 compared with exosomes from unstimulated cells. These results suggest that inflammation of ECs under hypertensive conditions is caused, at least in part, by macrophage-derived exosomes.