Plasminogen Activator Inhibitor-1 Protein Research Articles

Deletion of adipocyte Sine Oculis Homeobox Homolog 1 prevents lipolysis and attenuates skin fibrosis.

Dermal fibrosis is a cardinal feature of systemic sclerosis (SSc) for which there are limited treatment strategies. This is in part due to our fragmented understanding of how dermal white adipose tissue (DWAT) contributes to skin fibrosis. We identified elevated sine oculis homeobox homolog 1 (SIX1) expression in SSc skin samples from the GENISOS and PRESS cohorts, the expression of which correlated with adipose-associated genes and molecular pathways. SIX1 localization studies identified increased signals in the DWAT area in SSc and in experimental models of skin fibrosis. Global and adipocyte specific Six1 deletion abrogated end-stage fibrotic gene expression and dermal adipocyte shrinkage induced by SQ bleomycin treatment. Further studies revealed a link between elevated SIX1 and increased expression of SERPINE1 and its protein PAI-1 which are known pro-fibrotic mediators. However, SIX1 deletion did not appear to affect cellular trans differentiation. Taken together these results point at SIX1 as a potential target for dermal fibrosis in SSc.

SERPINE1AS2 regulates intramuscular adipogenesis by inhibiting PAI1 protein expression

Antisense long non-coding RNAs (lncRNAs) played a crucial role in the precise regulation of essential biological processes and were abundantly present in animals. Many of these antisense lncRNAs have been identified as key roles in adipose tissue accumulation in livestock, underscoring their vital role in the regulation of animal physiology. Nonetheless, the functional roles of these antisense lncRNAs in regulating adipogenesis and the specific molecular mechanisms these processes were still unclear, which was a significant gap in current scientific research.In this study, we identified and characterized SERPINE1AS2, a novel natural antisense lncRNA, was highly expressed in the fat tissues of adult cattle and calves. Its expression gradually increased during the differentiation of intramuscular adipocytes. Through functional studies, we observed that knockdown of SERPINE1AS2 inhibited the proliferation and adipogenesis of intramuscular adipocytes, while overexpression of SERPINE1AS2 produced the opposite effect. RNA sequencing (RNA-seq) analysis following SERPINE1AS2 knockdown revealed that differential expression genes (DEGs) were significantly enriched in key signaling pathways, notably the MAPK, Wnt, and mTOR signaling pathways. Furthermore, SERPINE1AS2 interacted with Plasminogen Activator Inhibitor-1 (PAI1), forming RNA dimers through complementary base pairing and consequently influencing PAI1 expression. Interestingly, studies on PAI1 suggested that reduced expression facilitated adipogenesis and the downregulation of PAI1 alleviated the inhibitory effect of reduced SERPINE1AS2 on adipogenesis. In summary, this study suggested that SERPINE1AS2 played a crucial role in the adipogenesis of bovine intramuscular adipocytes by modulating the expression of PAI1. SERPINE1AS2 also regulated adipogenesis by engaging in the MAPK, Wnt, and mTOR signaling pathways. Our results suggested that SERPINE1AS2 had a complex regulatory mechanism on adipogenesis in intramuscular adipocytes.

Investigate the potential impact of Hemagglutinin from the H1N1 strain on severe pneumonia

The most prevalent glycoprotein on the influenza virus envelope is called hemagglutinin (HA), yet little is known about its involvement in the pathophysiology and etiology of severe influenza pneumonia. Here, after stimulating human bronchial epithelial cells (16-HBE) and mice with HA of H1N1 for 12 h, we investigated the proliferation, migration, inflammatory cytokines expression, and apoptosis in 16-HBE and the pathological damage in mouse lung tissue. The expression of inflammatory cytokines plasminogen activator inhibitor 1(PAI-1), urokinase‐type (uPA) and tissue‐type (tPA) plasminogen activators, and apoptosis were all enhanced by HA, which also prevented the proliferation and migration of bronchial epithelial cells. HA enhanced up-regulated PAI-1, uPA, and tPA protein expression within mouse lung tissue and caused lung injury. In conclusion, HA alone, but not the whole H1N1 virus, induces lung tissue injury by inhibiting cell proliferation and migration, while promoting the expression of inflammatory cytokines and apoptosis.

Plasminogen activator inhibitor-1 serum levels in frontotemporal lobar degeneration.

Plasminogen activator inhibitor-1 (PAI-1) impedes brain plasmin synthesis. Reduced plasmin activity facilitates cumulation of amyloid beta (Aβ) in Alzheimer's disease (AD). Since plasmin also regulates the synaptic activity, it is possible that altered PAI-1 is present in other neurodegenerative disorders. We investigated whether PAI-1 and its counter-regulatory tissue plasminogen activator (tPA) are altered in serum of patients with dementia due to frontotemporal lobar degeneration (FTLD). Thirty five FTLD patients (21 in mild cognitive impairment stage (MCI) and 14 in dementia stage) and 10 cognitively healthy controls were recruited. Serum tPA and PAI-1 protein levels were measured by anova. Correlation between biochemical and demographic data were explored by measuring Pearson correlation coefficient. Serum PAI-1 levels were elevated in the FTLD dementia group as compared to FTLD MCI and controls. tPA serum levels and PAI-1/tPA ratio did not significantly differ among groups. There was a negative correlation between PAI-1 serum levels and disease severity measured by MMSE score. No correlations of tPA serum levels and PAI-1/tPA ratio with MMSE were found. Increased PAI-1 serum levels may serve as a marker of dementia in FTLD, suggesting that, besides Aβ pathway, the plasmin system may affect cognition through synaptic activity.

Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 complex as a serum biomarker for COVID-19.

Patients with coronavirus disease-2019 (COVID-19) have an increased risk of thrombosis and acute respiratory distress syndrome (ARDS). Thrombosis is often attributed to increases in plasminogen activator inhibitor-1 (PAI-1) and a shut-down of fibrinolysis (blood clot dissolution). Decreased urokinase-type plasminogen activator (uPA), a protease necessary for cell-associated plasmin generation, and increased tissue-type plasminogen activator (tPA) and PAI-1 levels have been reported in COVID-19 patients. Because these factors can occur in free and complexed forms with differences in their biological functions, we examined the predictive impact of uPA, tPA, and PAI-1 in their free forms and complexes as a biomarker for COVID-19 severity and the development of ARDS. In this retrospective study of 69 Japanese adults hospitalized with COVID-19 and 20 healthy donors, we found elevated free, non-complexed PAI-1 antigen, low circulating uPA, and uPA/PAI-1 but not tPA/PAI-1 complex levels to be associated with COVID-19 severity and ARDS development. This biomarker profile was typical for patients in the complicated phase. Lack of PAI-1 activity in circulation despite free, non-complexed PAI-1 protein and plasmin/α2anti-plasmin complex correlated with suPAR and sVCAM levels, markers indicating endothelial dysfunction. Furthermore, uPA/PAI-1 complex levels positively correlated with TNFα, a cytokine reported to trigger inflammatory cell death and tissue damage. Those levels also positively correlated with lymphopenia and the pro-inflammatory factors interleukin1β (IL1β), IL6, and C-reactive protein, markers associated with the anti-viral inflammatory response. These findings argue for using uPA and uPA/PAI-1 as novel biomarkers to detect patients at risk of developing severe COVID-19, including ARDS.

Involvement of plasminogen activator inhibitor-1 in p300/p53-mediated age-related atrial fibrosis.

Plasminogen activator inhibitor-1 (PAI-1), a key regulator of the fibrinolytic system, is also intimately involved in the fibrosis. Although PAI-1 may be involved in the occurrence of atrial fibrillation (AF) and thrombosis in the elderly, but whether it participated in aging-related atrial fibrosis and the detailed mechanism is still unclear. We compared the transcriptomics data of young (passage 4) versus senescent (passage 14) human atrial fibroblasts and found that PAI-1 was closely related to aging-related fibrosis. Aged mice and senescent human and mouse atrial fibroblasts underwent electrophysiological and biochemical studies. We found that p300, p53, and PAI-1 protein expressions were increased in the atrial tissue of aged mice and senescent human and mouse atrial fibroblasts. Curcumin or C646 (p300 inhibitor), or p300 knockdown inhibited the expression of PAI-1 contributing to reduced atrial fibroblasts senescence, atrial fibrosis, and the AF inducibility. Furthermore, p53 knockdown decreased the protein expression of PAI-1 and p21 in senescent human and mouse atrial fibroblasts. Our results suggest that p300/p53/PAI-1 signaling pathway participates in the mechanism of atrial fibrosis induced by aging, which provides new sights into the treatment of elderly AF.

Moshen granule ameliorates membranous nephropathy by blocking intrarenal renin-angiotensin system signalling via the Wnt1/β-catenin pathway

BackgroundMembranous nephropathy (MN) is one of the cardinal causes of nephrotic syndrome in adults, but an adequate treatment regimen is lacking. PurposeWe assessed the effect of Moshen granule (MSG) on patients with MN and cationic bovine serum albumin (CBSA)-induced rats. We further identified the bioactive components of MSG and revealed the underlying molecular mechanism of its renoprotective effects. MethodsWe determined the effect of MSG on patients with MN and CBSA-induced rats and its components on podocyte injury in zymosan-activated serum (ZAS)-elicited podocytes and revealed their regulatory mechanism on the Wnt/β-catenin/renin-angiotensin system (RAS) signalling axis. ResultsMSG treatment improved renal function and reduced proteinuria in MN patients and significantly reduced proteinuria and preserved the protein expression of podocin, nephrin, podocalyxin and synaptopodin in CBSA-induced MN rats. Mechanistically, MSG treatment significantly inhibited the protein expression of angiotensinogen, angiotensin converting enzyme and angiotensin II type 1 receptor, which was accompanied by inhibition of the protein expression of Wnt1 and β-catenin and its downstream gene products, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in CBSA-induced MN rats. We further identified 81 compounds, including astragaloside IV (AGS), calycosin, barleriside A and geniposidic acid, that preserve the podocyte-specific protein expression in ZAS-induced podocytes. Among these four compounds, AGS exhibited the strongest inhibitory effects on podocyte protein expression. AGS treatment significantly inhibited the protein expression of RAS components and Wnt1 and β-catenin and its downstream gene products in ZAS-induced podocytes. In contrast, the inhibitory effect of AGS on podocyte-specific proteins, β-catenin downstream gene products and RAS components was partially abolished in ZAS-induced podocytes treated with ICG-001 and β-catenin siRNA. ConclusionThis study first demonstrates that AGS mitigates podocyte injury by inhibiting the activation of RAS signalling via the Wnt1/β-catenin pathway by both pharmacological and genetic methods. Therefore, AGS might be considered a new β-catenin inhibitor that inhibits the Wnt1/β-catenin pathway to retard MN in patients.

Glucocorticoid-Responsive Tissue Plasminogen Activator (tPA) and Its Inhibitor Plasminogen Activator Inhibitor-1 (PAI-1): Relevance in Stress-Related Psychiatric Disorders.

Stressful events trigger a set of complex biological responses which follow a bell-shaped pattern. Low-stress conditions have been shown to elicit beneficial effects, notably on synaptic plasticity together with an increase in cognitive processes. In contrast, overly intense stress can have deleterious behavioral effects leading to several stress-related pathologies such as anxiety, depression, substance use, obsessive-compulsive and stressor- and trauma-related disorders (e.g., post-traumatic stress disorder or PTSD in the case of traumatic events). Over a number of years, we have demonstrated that in response to stress, glucocorticoid hormones (GCs) in the hippocampus mediate a molecular shift in the balance between the expression of the tissue plasminogen activator (tPA) and its own inhibitor plasminogen activator inhibitor-1 (PAI-1) proteins. Interestingly, a shift in favor of PAI-1 was responsible for PTSD-like memory induction. In this review, after describing the biological system involving GCs, we highlight the key role of tPA/PAI-1 imbalance observed in preclinical and clinical studies associated with the emergence of stress-related pathological conditions. Thus, tPA/PAI-1 protein levels could be predictive biomarkers of the subsequent onset of stress-related disorders, and pharmacological modulation of their activity could be a potential new therapeutic approach for these debilitating conditions.

P184 Identification of the PAI-1, as a potential non-invasive, fecal biomarker in the Inflammatory Bowel Diseases

Abstract Background Inflammatory bowel diseases (IBD) are defined by unregulated immune response leading to intestinal inflammation. Monitoring of the intestinal inflammation are mostly invasive, time-consuming and expensive. Therefore, a simple, rapid and non-invasive test, with ability to differentiate IBD from other gastrointestinal conditions and to monitor disease activity and therapeutic response, will have important clinical implications. Our previous cytokine profiling identified that plasminogen activator inhibitor 1 (PAI-1) is expressed in IBD patients with active disease, but not in control. Therefore, our aim was to assess the utility of PAI-1 in monitoring disease activity and therapeutic response of IBD patients in different biological samples. Methods Biopsy, serum and fecal samples were collected from more than 150 active IBD patients and controls without IBD. Inclusion criteria included change of therapy or initiation of new treatment for IBD patients. Immunofluorescence (IF) staining was used to observe the presence and localisation of cytokines. Total protein and mRNA were isolated from biopsies and fecal samples. Protein levels were measured by ELISA and gene expression by qRT-PCR. Results The IF staining of the biopsy samples revealed a significantly higher percentage of PAI-1 positive cells in the inflamed mucosa compared to the control. The relative mRNA expression of PAI-1 was significant higher in non-responders and untreated, inflamed IBD patients compared to controls and responders. On the other hand, we were not able to detect significant changes between responders and non-responders in gene expression of TNF-α, IL-1β and IL-6. Serum, mucosal and fecal PAI-1 protein levels were significantly higher in IBD patients compared to controls. Whereas no significant difference was observed between Crohn’s disease and ulcerative colitis patients. The PAI-1 level was significant increased in endoscopically and clinically active disease compared to inactive patients and controls. In responders, PAI-1 concentration significantly decreased after initiation of therapy compared to non-responders. Significantly upregulated fecal PAI-1 level was found in active IBD compared to patients with inactive IBD, tumor, adenoma, diverticulosis and control. In addition, the stability analysis of fecal PAI-1 showed that the protein was detectable up to one week at room temperature and 4ºC. Conclusion Our findings suggest that serum, mucosal and fecal PAI-1 expression correlates with disease activity in IBD patients, suggesting that it could be utilized to monitor disease activity and possibly therapy response as a non-invasive biomarker in IBD.

Assessment of serum levels of homocysteine, Ang 1-7, and plasminogen activator Inhibitor-1 protein in samples of coronavirus disease 2019 patients in Iraq

Assessment of serum levels of homocysteine, Ang 1-7, and plasminogen activator Inhibitor-1 protein in samples of coronavirus disease 2019 patients in Iraq

Latency-associated upregulation of SERBP1 is important for the recruitment of transcriptional repressors to the viral major immediate early promoter of human cytomegalovirus during latent carriage.

Suppression of human cytomegalovirus (HCMV) major immediate early gene (IE) expression from the viral major immediate early promoter (MIEP) is known to be crucial for the establishment and maintenance of HCMV latency in myeloid progenitor cells and their undifferentiated derivatives. This suppression of the MIEP during latent infection is known to result from epigenetic histone modification imparting a repressive chromatin structure around the MIEP in undifferentiated myeloid cells. In contrast, reactivation, resulting from, e.g., myeloid cell differentiation, is associated with activatory chromatin marks around the MIEP. Recently, recruitment of the transcriptional repressor SETDB1, via KAP1, to latent HCMV genomes was shown to be involved in latency-associated MIEP suppression in CD34+ progenitor cells. KAP1 is also known to associate with Chromodomain-helicase-DNA-binding protein 3 (CHD3) as part of the NuRD complex which can aid transcriptional silencing. We now show that the cellular protein Plasminogen activator inhibitor 1 RNA-binding protein (SERBP1), a known interactor of CHD3, is significantly upregulated during HCMV latency and that this protein is required for MIEP suppression during latent infection of myeloid cells. We further show that SERBP1 mediates CHD3 association with the MIEP as well as KAP1 association with viral genomic DNA. We suggest that SERBP1 functions as a scaffold protein to recruit transcriptional repressors to the latent viral genome and to mediate transcriptional silencing of the MIEP during latent carriage.

Shenkang injection improves chronic kidney disease by inhibiting multiple renin-angiotensin system genes by blocking the Wnt/β-catenin signalling pathway.

Chronic kidney disease (CKD) is a major worldwide public health problem. The increase in the number of patients with CKD and end-stage kidney disease requesting renal dialysis or transplantation will progress to epidemic proportions in the next several decades. Although blocking the renin-angiotensin system (RAS) has been used as a first-line standard therapy in patients with hypertension and CKD, patients still progress towards end-stage kidney disease, which might be closely associated with compensatory renin expression subsequent to RAS blockade through a homeostatic mechanism. The Wnt/β-catenin signalling pathway is the master upstream regulator that controls multiple intrarenal RAS genes. As Wnt/β-catenin regulates multiple RAS genes, we inferred that this pathway might also be implicated in blood pressure control. Therefore, discovering new medications to synchronously target multiple RAS genes is necessary and essential for the effective treatment of patients with CKD. We hypothesized that Shenkang injection (SKI), which is widely used to treat CKD patients, might ameliorate CKD by inhibiting the activation of multiple RAS genes via the Wnt/β-catenin signalling pathway. To test this hypothesis, we used adenine-induced CKD rats and angiotensin II (AngII)-induced HK-2 and NRK-49F cells. Treatment with SKI inhibited renal function decline, hypertension and renal fibrosis. Mechanistically, SKI abrogated the increased protein expression of multiple RAS elements, including angiotensin-converting enzyme and angiotensin II type 1 receptor, as well as Wnt1, β-catenin and downstream target genes, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in adenine-induced rats, which was verified in AngII-induced HK-2 and NRK-49F cells. Similarly, our results further indicated that treatment with rhein isolated from SKI attenuated renal function decline and epithelial-to-mesenchymal transition and repressed RAS activation and the hyperactive Wnt/β-catenin signalling pathway in both adenine-induced rats and AngII-induced HK-2 and NRK-49F cells. This study first revealed that SKI repressed epithelial-to-mesenchymal transition by synchronously targeting multiple RAS elements by blocking the hyperactive Wnt/β-catenin signalling pathway.

Connexin 43 overexpression induces lung cancer angiogenesis in vitro following phosphorylation at Ser279 in its C-terminus.

Blocking angiogenesis can inhibit tumor growth and metastasis. However, the mechanism underlying regulation of lung cancer angiogenesis remains unclear. The gap junction protein connexin 43 (Cx43) is implicated in angiogenesis. The aim of the present study was to determine the role of Cx43 in angiogenesis in vitro and its signaling pathways. Human pulmonary microvascular endothelial cells were transfected with Cx43-targeting siRNA or Cx43-overexpressing recombinant plasmid vector. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to determine Cx43, zonula occludens-1 (ZO-1), E-cadherin, β-catenin, von Willebrand factor (vWF), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression levels, respectively. Tyr265, Ser279, Ser368, and Ser373 phosphorylation levels in the C-terminus of Cx43 and intracellular and membranal Cx43 contents were determined using western blotting. Additionally, immunofluorescence, tube formation, Cell Counting Kit-8, and Transwell migration assays were performed. The results revealed that compared with that in the control samples, Cx43, ZO-1, E-cadherin, β-catenin, vWF, and PAI-1 mRNA and protein expression were significantly increased in the Cx43 overexpression group and significantly decreased in the Cx43-knockdown group. Moreover, the phosphorylation level of Ser279 as well as cell proliferation and migration rates were markedly increased in the Cx43 overexpression group, and tube formation revealed that the potential of angiogenesis was also increased. Conversely, in the Cx43-knockdown group, the phosphorylation level of Ser279 and cell proliferation and migration rates were reduced, and the potential of angiogenesis was greatly impaired. Under Cx43 overexpression, membranal Cx43 content was significantly increased, whereas under Cx43 knockdown, it was significantly reduced. Therefore, Cx43 overexpression could induce pulmonary angiogenesis in vitro by promoting cell proliferation and migration and activating ZO-1, E-cadherin, β-catenin, vWF, and PAI-1. This may be achieved by promoting phosphorylation and activation of the intracellular signal site Ser279 at the C-terminus of Cx43.

The relationship between Dietary approaches to stop hypertension diet adherence and inflammatory factors and insulin resistance in overweight and obese women: A cross-sectional study

BackgroundAlthough there is abundant evidence for an association between dietary pattern, weight, and other related factors, such as homeostatic model assessment for insulin resistance (HOMA-IR) and inflammatory markers; there is limited information pertaining to levels of plasminogen activator inhibitor-1 (PAI-1) and Monocyte Chemoattractant Protein −1 (MCP-1). Therefore, this study sought to examine the association between adherence to the dietary approaches to stop hypertension (DASH) diet and levels of inflammatory factors PAI-1, MCP-1, and HOMA-IR. MethodsThis cross-sectional study was performed on 305 obese and overweight women. The typical food intake of individuals was assessed using the 147 items semi-quantitative food frequency questionnaire (FFQ). Body components were measured for all participants. Insulin resistance was estimated by homeostasis model assessment (HOMA), and biochemical parameters were examined. ResultsNo significant relationship was observed between the DASH diet and MCP-1 (P-trend = 0.70), PAI-1 (P-trend = 0.92), or HOMA-IR (P-trend = 0.08) in the crude model. However, there was a significant inverse relationship between the DASH diet and HOMA-IR (P-trend = 0.03) after adjusting for age, BMI, and physical activity. ConclusionThis study showed that higher adherence to DASH pattern is inversely correlated with HOMA-IR in overweight and obese women.

Visceral fat-specific regulation of plasminogen activator inhibitor-1 in aged septic mice.

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are documented in patients with sepsis and levels positively correlate with disease severity and mortality. Our previous work demonstrated that visceral adipose tissues (VAT) are a major source of PAI-1, especially in the aged (murine endotoxemia), that circulating PAI-1 protein levels match the trajectory of PAI-1 transcript levels in VAT (clinical sepsis), and that PAI-1 in both VAT and plasma are positively associated with acute kidney injury (AKI) in septic patients. In the current study utilizing preclinical sepsis models, PAI-1 tissue distribution was examined and cellular sources, as well as mechanisms mediating PAI-1 induction in VAT, were identified. In aged mice with sepsis, PAI-1 gene expression was significantly higher in VAT than in other major organs. VAT PAI-1 gene expression correlated with PAI-1 protein levels in both VAT and plasma. Moreover, VAT and plasma levels of PAI-1 were positively associated with AKI markers, modeling our previous clinical data. Using explant cultures of VAT, we determined that PAI-1 is secreted robustly in response to recombinant transforming growth factor β (TGFβ) and tumor necrosis factor α (TNFα) treatment; however, neutralization was effective only for TNFαindicating that TGFβ is not an endogenous modulator of PAI-1. Within VAT, TNFα was localized to neutrophils and macrophages. PAI-1 protein levels were fourfold higher in stromal vascular fraction (SVF) cells compared with mature adipocytes, and among SVF cells, both immune and nonimmune compartments expressed PAI-1 in a similar fashion. PAI-1 was localized predominantly to macrophages within the immune compartment and preadipocytes and endothelial cells within the nonimmune compartment. Collectively, these results indicate that induction and secretion of PAI-1 from VAT is facilitated by a complex interaction among immune and nonimmune cells. As circulating PAI-1 contributes to AKI in sepsis, understanding PAI-1 regulation in VAT could yield novel strategies for reducing systemic consequences of PAI-1 overproduction.

Effect of Nitinol on Metabolic and Coagulation Activity of Endothelial Cells Culture

We studied the effect of nitinol, the most prevalent material for endovascular stents, on metabolic and coagulation activity of a primary culture of human umbilical vein endothelial cells (HUVEC). Metabolic activity was evaluated using MTS-test and by the level of stable NO metabolites in the conditioned medium, coagulation activity was assessed by activity of von Willebrand factor (vWF) and levels of plasminogen activator inhibitor-1 (PAI-1) and soluble endothelial protein C receptors (sEPCR). Exposure to nitinol reduced metabolic activity of the cell culture by 11.1% in comparison with the control (p<0.001). Although absolute activity of vWF and absolute level of sEPCR were elevated, incubation with nitinol did not lead to a statistically significant elevation of these parameters in comparison with the control, which can indicate the absence of substantial hypercoagulation effects of nitinol.

Involvement of plasminogen activator inhibitor-1 and its related molecules in atrial fibrosis in patients with atrial fibrillation.

Atrial fibrillation is the most common form of cardiac arrhythmia. Atrial fibrosis is a significant feature of atrial fibrillation though its mechanism is not well understood. We searched the Gene Expression Omnibus database to compare mRNA expression patterns between atrial fibrillation and sinus rhythm samples; one hundred and forty eight differentially expressed genes were identified. Most of these genes were significantly enriched in the extracellular matrix organization process and collagen-activated tyrosine kinase receptor signaling pathway. To screen hub genes involved in atrial fibrosis, we constructed a protein-protein interaction network and found that three hub genes (SERPINE1/plasminogen activator inhibitor-1/PAI-1, TIMP Metallopeptidase Inhibitor 3/TIMP3 and decorin/DCN) play vital roles in atrial fibrosis, especially plasminogen activator inhibitor-1. Elevated plasminogen activator inhibitor-1 expression was positively correlated with the p53 signaling pathway. Plasminogen activator inhibitor-1 and p53 protein expression levels were verified in patients with sinus rhythm and atrial fibrillation by Western blot analysis. Compared with the sinus rhythm controls, p53 and plasminogen activator inhibitor-1 protein expressions were upregulated in the atrial tissues of patients with atrial fibrillation. p53 was also found to regulate plasminogen activator inhibitor-1 based on the results of cellular and molecular experiments. Thus, the p53/plasminogen activator inhibitor-1 signaling axis may participate in the pathophysiological processes of atrial fibrillation, and plasminogen activator inhibitor-1 may serve as a new therapeutic biomarker in atrial fibrillation.

Divergent Regulation of Alveolar Type 2 Cell and Fibroblast Apoptosis by Plasminogen Activator Inhibitor 1 in Lung Fibrosis

Increased apoptosis sensitivity of alveolar type 2 (ATII) cells and increased apoptosis resistance of (myo)fibroblasts, the apoptosis paradox, contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF). The mechanism underlying the apoptosis paradox in IPF lungs, however, is unclear. Aging is the greatest risk factor for IPF. In this study, we show, for the first time, that ATII cells from old mice are more sensitive, whereas fibroblasts from old mice are more resistant, to apoptotic challenges, compared with the corresponding cells from young mice. The expression of plasminogen activator inhibitor 1 (PAI-1), an important profibrogenic mediator, was significantly increased in both ATII cells and lung fibroblasts from aged mice. In vitro studies using PAI-1 siRNA and active PAI-1 protein indicated that PAI-1 promoted ATII cell apoptosis but protected fibroblasts from apoptosis, likely through dichotomous regulation of p53 expression. Deletion of PAI-1 in adult mice led to a reduction in p53, p21, and Bax protein expression, as well as apoptosis sensitivity in ATII cells, and their increase in the lung fibroblasts, as indicated by in vivo studies. This increase was associated with an attenuation of lung fibrosis after bleomycin challenge. Since PAI-1 is up-regulated in both ATII cells and fibroblasts in IPF, the results suggest that increased PAI-1 may underlie the apoptosis paradox of ATII cells and fibroblasts in IPF lungs.

Elevated postischemic tissue injury and leukocyte-endothelial adhesive interactions in mice with global deficiency in caveolin-2: role of PAI-1.

Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.

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).