SERPINE2 promotes liver cancer metastasis by inhibiting c-Cbl-mediated EGFR ubiquitination and degradation.

BACKGROUND: Liver cancer is a malignancy with high morbidity and mortality rates. Serpin family E member 2 (SERPINE2) has been reported to play a key role in the metastasis of many tumors. In this study, we aimed to investigate the potential mechanism of SERPINE2 in liver cancer metastasis. METHODS: The Cancer Genome Atlas database (TCGA), including DNA methylation and transcriptome sequencing data, was utilized to identify the crucial oncogene associated with DNA methylation and cancer progression in liver cancer. Data from the TCGA and RNA sequencing for 94 pairs of liver cancer tissues were used to explore the correlation between SERPINE2 expression and clinical parameters of patients. DNA methylation sequencing was used to detect the DNA methylation levels in liver cancer tissues and cells. RNA sequencing, cytokine assays, immunoprecipitation (IP) and mass spectrometry (MS) assays, protein stability assays, and ubiquitination assays were performed to explore the regulatory mechanism of SERPINE2 in liver cancer metastasis. Patient-derived xenografts and tumor organoid models were established to determine the role of SERPINE2 in the treatment of liver cancer using sorafenib. RESULTS: Based on the public database screening, SERPINE2 was identified as a tumor promoter regulated by DNA methylation. SERPINE2 expression was significantly higher in liver cancer tissues and was associated with the dismal prognosis in patients with liver cancer. SERPINE2 promoted liver cancer metastasis by enhancing cell pseudopodia formation, cell adhesion, cancer-associated fibroblast activation, extracellular matrix remodeling, and angiogenesis. IP/MS assays confirmed that SERPINE2 activated epidermal growth factor receptor (EGFR) and its downstream signaling pathways by interacting with EGFR. Mechanistically, SERPINE2 inhibited EGFR ubiquitination and maintained its protein stability by competing with the E3 ubiquitin ligase, c-Cbl. Additionally, EGFR was activated in liver cancer cells after sorafenib treatment, and SERPINE2 knockdown-induced EGFR downregulation significantly enhanced the therapeutic efficacy of sorafenib against liver cancer. Furthermore, we found that SERPINE2 knockdown also had a sensitizing effect on lenvatinib treatment. CONCLUSIONS: SERPINE2 promoted liver cancer metastasis by preventing EGFR degradation via c-Cbl-mediated ubiquitination, suggesting that inhibition of the SERPINE2-EGFR axis may be a potential target for liver cancer treatment.

Growth differentiation factor myostatin regulates epithelial-mesenchymal transition genes and enhances invasion by increasing serine protease inhibitors E1 and E2 in human trophoblast cells.

Placental insufficiency disorders, including preeclampsia and intrauterine growth restriction, are major obstetric complications that can have devastating effects on both the mother and the fetus. These syndromes have underlying poor placental trophoblast cell invasion into uterine tissues. Placental invasion is controlled by many hormones and growth factors. Myostatin (MSTN) is a transforming growth factor-ß superfamily member recognized for its important role in muscle growth control. MSTN has also been shown to be secreted and functioning in the placenta, and its serum and/or placental levels were found to be upregulated in preeclampsia and intrauterine growth restriction. Considering that the mechanistic role of MSTN in placentation remains poorly understood, we hypothesized that MSTN uses ALK4/5-SMAD2/3/4 signaling to increase human trophoblast invasion through a group of epithelial-mesenchymal transition genes including SERPINE2, PAI-1, and SOX4. mRNA sequencing of control and MSTN-treated primary human trophoblast cells (n = 5) yielded a total of 610 differentially expressed genes (false discovery rate <0.05) of which 380 genes were upregulated and 230 were downregulated. These differentially expressed genes were highly enriched in epithelial-mesenchymal transition genes, and a subset including SERPINE2, PAI-1, and SOX4 was investigated for its role in MSTN-induced trophoblast cell invasion. We found that MSTN induced upregulation of SERPINE2 via ALK4/5-SMAD2/3/4 signaling; however, SMAD2 was not involved in MSTN-induced PAI-1 upregulation. SOX4 was involved in MSTN-induced upregulation of SERPINE2, but not PAI-1. Collectively, this study discovers novel molecular mechanisms of MSTN-induced human trophoblast cell invasion and provides insight into the functional consequences of its dysregulation in placental insufficiency disorders.

Proteome-wide mendelian randomization identifies causal plasma proteins in venous thromboembolism development.

Genome-wide association studies (GWAS) have identified numerous risk loci for venous thromboembolism (VTE), but it is challenging to decipher the underlying mechanisms. We employed an integrative analytical pipeline to transform genetic associations to identify novel plasma proteins for VTE. Proteome-wide association studies (PWAS) were determined by functional summary-based imputation leveraging data from a genome-wide association analysis (14,429 VTE patients, 267,037 controls), blood proteomes (1348 cases), followed by Mendelian randomization, Bayesian colocalization, protein-protein interaction, and pathway enrichment analysis. Twenty genetically regulated circulating protein abundances (F2, F11, ABO, PLCG2, LRP4, PLEK, KLKB1, PROC, KNG1, THBS2, SERPINA1, RARRES2, CEL, GP6, SERPINE2, SERPINA10, OBP2B, EFEMP1, F5, and MSR1) were associated with VTE. Of these 13 proteins demonstrated Mendelian randomized correlations. Six proteins (F2, F11, PLEK, SERPINA1, RARRES2, and SERPINE2) had strong support in colocalization analysis. Utilizing multidimensional data, this study suggests PLEK, SERPINA1, and SERPINE2 as compelling proteins that may provide key hints for future research and possible diagnostic and therapeutic targets for VTE.

DNA Methylation of Window of Implantation Genes in Cervical Secretions Predicts Ongoing Pregnancy in Infertility Treatment.

Window of implantation (WOI) genes have been comprehensively identified at the single cell level. DNA methylation changes in cervical secretions are associated with in vitro fertilization embryo transfer (IVF-ET) outcomes. Using a machine learning (ML) approach, we aimed to determine which methylation changes in WOI genes from cervical secretions best predict ongoing pregnancy during embryo transfer. A total of 2708 promoter probes were extracted from mid-secretory phase cervical secretion methylomic profiles for 158 WOI genes, and 152 differentially methylated probes (DMPs) were selected. Fifteen DMPs in 14 genes (BMP2, CTSA, DEFB1, GRN, MTF1, SERPINE1, SERPINE2, SFRP1, STAT3, TAGLN2, TCF4, THBS1, ZBTB20, ZNF292) were identified as the most relevant to ongoing pregnancy status. These 15 DMPs yielded accuracy rates of 83.53%, 85.26%, 85.78%, and 76.44%, and areas under the receiver operating characteristic curves (AUCs) of 0.90, 0.91, 0.89, and 0.86 for prediction by random forest (RF), naïve Bayes (NB), support vector machine (SVM), and k-nearest neighbors (KNN), respectively. SERPINE1, SERPINE2, and TAGLN2 maintained their methylation difference trends in an independent set of cervical secretion samples, resulting in accuracy rates of 71.46%, 80.06%, 80.72%, and 80.68%, and AUCs of 0.79, 0.84, 0.83, and 0.82 for prediction by RF, NB, SVM, and KNN, respectively. Our findings demonstrate that methylation changes in WOI genes detected noninvasively from cervical secretions are potential markers for predicting IVF-ET outcomes. Further studies of cervical secretion of DNA methylation markers may provide a novel approach for precision embryo transfer.

Single-cell RNA-seq integrated with multi-omics reveals SERPINE2 as a target for metastasis in advanced renal cell carcinoma.

Tumor growth, metastasis and therapeutic response are believed to be regulated by the tumor and its microenvironment (TME) in advanced renal cell carcinoma (RCC). However, the mechanisms underlying genomic, transcriptomic and epigenetic alternations in RCC progression have not been completely defined. In this study, single-cell RNA-sequencing (scRNA-seq) data were obtained from eight tissue samples of RCC patients, including two matched pairs of primary and metastatic sites (lymph nodes), along with Hi-C, transposable accessible chromatin by high-throughput (ATAC-seq) and RNA-sequencing (RNA-seq) between RCC (Caki-1) and human renal tubular epithelial cell line (HK-2). The identified target was verified in clinical tissue samples (microarray of 407 RCC patients, TMA-30 and TMA-2020), whose function was further validated by in vitro and in vivo experiments through knockdown or overexpression. We profiled transcriptomes of 30514 malignant cells, and 14762 non-malignant cells. Comprehensive multi-omics analysis revealed that malignant cells and TME played a key role in RCC. The expression programs of stromal cells and immune cells were consistent among the samples, whereas malignant cells expressed distinct programs associated with hypoxia, cell cycle, epithelial differentiation, and two different metastasis patterns. Comparison of the hierarchical structure showed that SERPINE2 was related to these NNMF expression programs, and at the same time targeted the switched compartment. SERPINE2 was highly expressed in RCC tissues and lowly expressed in para-tumor tissues or HK-2 cell line. SERPINE2 knockdown markedly suppressed RCC cell growth and invasion, while SERPINE2 overexpression dramatically promoted RCC cell metastasis both in vitro and in vivo. In addition, SERPINE2 could activate the epithelial-mesenchymal transition pathway. The above findings demonstrated that the role of distinct expression patterns of malignant cells and TME played a distinct role in RCC progression. SERPINE2 was identified as a potential therapeutic target for inhibiting metastasis in advanced RCC.

LHX2 Enhances the Malignant Phenotype of Esophageal Squamous Cell Carcinoma by Upregulating the Expression of SERPINE2.

LHX2 dysregulations have been found to present in cancers, but the function of LHX2 in esophageal squamous cell carcinoma (ESCC) remains unknown. Here, we report that LHX2 was upregulated in ESCC tissues in comparison to the LHX2 levels in adjacent normal tissues. Loss- and gain-of-function experiments demonstrated that the knockdown of LHX2 markedly inhibited ESCC cells' proliferation, migration, invasion, tumor growth and metastasis, whereas the overexpression of LHX2 had the opposite effects. A mechanistic investigation revealed that LHX2 bound to the promoter of SERPINE2 gene and transcriptionally regulated the expression of SERPINE2. Collectively, LHX2 facilitates ESCC tumor progression, and it could be a potential therapeutic target for ESCC.

SERPINE2/PN-1 regulates the DNA damage response and radioresistance by activating ATM in lung cancer.

Although the DNA damage response (DDR) is associated with the radioresistance characteristics of lung cancer cells, the specific regulators and underlying mechanisms of the DDR are unclear. Here, we identified the serine proteinase inhibitor clade E member 2 (SERPINE2) as a modulator of radiosensitivity and the DDR in lung cancer. Cells exhibiting radioresistance after ionizing radiation show upregulation of SERPINE2, and SERPINE2 knockdown improves tumor radiosensitivity in vitro and in vivo. Functionally, SERPINE2 deficiency causes a reduction in homologous recombination repair, rapid recovery of cell cycle checkpoints, and suppression of migration and invasion. Mechanistically, SERPINE2 knockdown inhibits the accumulation of p-ATM and the downstream repair protein RAD51 during DNA repair, and RAD51 can restore DNA damage and radioresistance phenotypes in lung cancer cells. Furthermore, SERPINE2 can directly interact with MRE11 and ATM to facilitate its phosphorylation in HR-mediated DSB repair. In addition, high SERPINE2 expression correlates with dismal prognosis in lung adenocarcinoma patients, and a high serum SERPINE2 concentration predicts a poor response to radiotherapy in non-small cell lung cancer patients. In summary, these findings indicate a novel regulatory mechanism by which SERPINE2 modulates the DDR and radioresistance in lung cancer.

Transcription factor SOX4 facilitates BMP2-regulated gene expression during invasive trophoblast differentiation.

The interplay between growth factors, signaling pathways and transcription factors during placental development is key to controlling trophoblast differentiation. Bone morphogenetic protein 2 (BMP2) has been implicated in trophoblast invasion and spiral artery remodeling during early placental development. However, the molecular mechanisms by which these are accomplished have not been fully elucidated, particularly for transcriptional regulation of key transcription factors. Here, we identified SOX4 as a direct target gene induced by BMP2 in first-trimester placental trophoblasts. Analysis of single-cell RNA-seq data from first-trimester placentas and decidua tissues revealed that SOX4 expression is mainly localized in extravillous trophoblast and decidual stromal cells. Moreover, gain- and loss-of-function approaches demonstrated that SOX4 exerts a pro-invasive role in human trophoblasts, and this effect contributes to BMP2-enhanced trophoblast invasion. Importantly, we found that SOX4 was required for BMP2-induced regulation of a subset of genes associated with cell migration and extracellular matrix organization. We also show that SOX4-dependent regulation of the BMP2 target SERPINE2 occurs via binding of SOX4 to regulatory elements such as enhancers, thereby promoting BMP2-induced trophoblast invasion. In conclusion, these findings uncover a novel mechanism involving SOX4 that shapes the BMP2-regulated transcriptional network during invasive trophoblast development.

Direct Blood Pressure-Independent Anti-Fibrotic Effects by the Selective Nonsteroidal Mineralocorticoid Receptor Antagonist Finerenone in Progressive Models of Kidney Fibrosis.

INTRODUCTION: The nonsteroidal mineralocorticoid receptor (MR) antagonist finerenone and sodium-glucose cotransporter-2 (SGLT2) inhibitors have demonstrated clinical benefits in chronic kidney disease patients with type 2 diabetes. Precise molecular mechanisms responsible for these benefits are incompletely understood. Here, we investigated potential direct anti-fibrotic effects and mechanisms of nonsteroidal MR antagonism by finerenone or SGLT2 inhibition by empagliflozin in 2 relevant mouse kidney fibrosis models: unilateral ureter obstruction and sub-chronic ischemia reperfusion injury. METHODS: Kidney fibrosis was induced in mice via unilateral ureteral obstruction or ischemia. In a series of experiments, mice were treated orally with the MR antagonist finerenone (3 or 10 mg/kg), the SGLT2 inhibitor empagliflozin (10 or 30 mg/kg), or in a direct comparison of both drugs. Interstitial myofibroblast accumulation was quantified via alpha-smooth muscle actin and interstitial collagen deposition via Sirius Red/Fast Green staining in both models. Secondary analyses included the assessment of inflammatory cells, kidney mRNA expression of fibrotic markers as well as functional parameters (serum creatinine and albuminuria) in the ischemic model. Blood pressure was measured via telemetry in healthy conscious compound-treated animals. RESULTS: Finerenone dose-dependently decreased pathological myofibroblast accumulation and collagen deposition with no effects on systemic blood pressure and inflammatory markers in the tested dose range. Reduced kidney fibrosis was paralleled by reduced kidney plasminogen activator inhibitor-1 (PAI-1) and naked cuticle 2 (NKD2) expression in finerenone-treated mice. In contrast, treatment with empagliflozin strongly increased urinary glucose excretion in both models and reduced ischemia-induced albuminuria but had no effects on kidney myofibroblasts or collagen deposition. DISCUSSION/CONCLUSION: Finerenone has direct anti-fibrotic properties resulting in reduced myofibroblast and collagen deposition accompanied by a reduction in renal PAI-1 and NKD2 expression in mouse models of progressive kidney fibrosis at blood pressure-independent dosages.

p16-3MR: A Novel Model to Study Cellular Senescence in Cigarette Smoke-Induced Lung Injuries.

Cellular senescence and lung aging are associated with the pathogenesis of chronic obstructive pulmonary disease (COPD). COPD progresses with aging, and chronic smoking is the key susceptibility factor in lung pathological changes concurrent with mitochondrial dysfunction and biological aging. However, these processes involving cigarette smoke (CS)-mediated lung cellular senescence are difficult to distinguish. One of the impediments to studying cellular senescence in relation to age-related lung pathologies is the lack of a suitable in vivo model. In view of this, we provide evidence that supports the suitability of p16-3MR mice to studying cellular senescence in CS-mediated and age-related lung pathologies. p16-3MR mice have a trimodal reporter fused to the promoter of the p16INK4a gene that enables detection, isolation, and selective elimination of senescent cells, thus making them a suitable model to study cellular senescence. To determine their suitability in CS-mediated lung pathologies, we exposed young (12-14 months) and old (17-20 months) p16-3MR mice to 30 day CS exposure and studied the expression of senescent genes (p16, p21, and p53) and SASP-associated markers (MMP9, MMP12, PAI-1, and FN-1) in air- and CS-exposed mouse lungs. Our results showed that this model could detect cellular senescence using luminescence and isolate cells undergoing senescence with the help of tissue fluorescence in CS-exposed young and old mice. Our results from the expression of senescence markers and SASP-associated genes in CS-exposed young and old p16-3MR mice were comparable with increased lung cellular senescence and SASP in COPD. We further showed alteration in the; (i) tissue luminescence and fluorescence, (ii) mRNA and protein expressions of senescent markers and SASP genes, and (iii) SA-ß-gal activity in CS-exposed young and old p16-3MR mice as compared to their air controls. Overall, we showed that p16-3MR is a competent model for studying the cellular senescence in CS-induced pathologies. Hence, the p16-3MR reporter mouse model may be used as a novel tool for understanding the pathobiology of cellular senescence and other underlying mechanisms involved in COPD and fibrosis.

SERPINE2 is an oral cancer-promoting factor that induces angiogenesis and lymphangiogenesis.

BACKGROUND: LEM domain containing 1 (LEMD1) is a novel factor involved in the development of oral squamous cell carcinoma (OSCC). We previously performed a microarray analysis and found that serpin peptidase inhibitor, clade E, member 2 (SERPINE2) is an LEMD1-related signal. SERPINE2 is an extracellular serine proteinase inhibitor with secretory capacity. Although SERPINE2 displays tumor-promoting properties in many cancers, some reports indicate that SRPINE2 also has a tumor-suppressing function. Therefore, there are many unclear points about its role in cancer. In this study, we investigated SERPINE2 expression in OSCC. METHODS: The gene expression and secretion levels of SERPINE2 were examined in 42 frozen specimens of OSCC, and SERPINE2 immunostaining was investigated in 167 cases of OSCC. Furthermore, the effect of SERPINE2 on angiogenesis and lymphangiogenesis was analyzed using OSCC cells and endothelial cells. RESULTS: In the frozen specimens, the gene expression (P < 0.0001) and secretion levels (P < 0.0001) of SERPINE2 were higher in OSCC than in the normal oral mucosa. According to the immunohistochemical analysis, SERPINE2 expression was correlated with the depth of invasion (P = 0.0163), nodal metastasis (P = 0.0085), microvessel density (P < 0.0001), and lymphovessel density (P < 0.0001). Additionally, univariate and multivariate analyses indicated that the SERPINE2 expression level was an independent poor prognostic factor for OSCC. In vitro studies using OSCC cells revealed that SERPINE2 promotes angiogenesis and lymphangiogenesis. CONCLUSION: These results suggest that SRPX2 might be a useful tumor marker for OSCC.

SERPINE2 feedback regulates EGF/EGFR signaling in human papillary thyroid carcinoma cells.

Thyroid cancer (TC) is the most prevalent malignant tumor in the endocrine system. Serpin peptidase inhibitor clade E member 2 (SERPINE2) is closely associated with tumor metastasis. The aim of the present study was to investigate whether SERPINE2 forms a feedback loop with epidermal growth factor (EGF)/EGF receptor (EGFR) that regulates cellular processes in human papillary thyroid carcinoma (TPC­1) cells. Reverse transcription­quantitative PCR and western blotting were utilized to analyze the expression of SERPINE2. Cell proliferation ability was detected with a cell proliferation and cytotoxicity assay kit (MTT) and by clone formation assay. The proliferation markers, including proliferating cell nuclear antigen and Ki­67, were also investigated to analyze the proliferative activity of TPC­1 cells. Besides, cell migration and invasion were analyzed by wound healing and Transwell assays, respectively, while cell apoptosis was analyzed by TUNEL staining. The results showed that SERPINE2 expression was increased in TPC cells, and SERPINE2 and EGF/EGFR regulated each other. Furthermore, SERPINE2 overexpression and silencing regulated TPC cell proliferation, migration, invasion and apoptosis. Besides, an EGFR inhibitor blocked the effects of SERPINE2 overexpression on the aforementioned biological processes. Therefore, the present study confirmed that SERPINE2 formed a positive feedback with EGF/EGFR to regulate the proliferation, invasion and migration of TPC cells, possibly providing novel insights into potential therapeutic targets of papillary TC.

Integrated analysis of transcriptome and proteome to explore the genes related to steroid-induced femoral head necrosis.

PURPOSE: Femoral head necrosis (FHN) is a common disease of hip. However, the pathogenesis of FHN is not well understood. This study attempted to explore the potentially important genes and proteins involved in FHN. METHODS: We integrated the transcriptomic and proteomic methods to quantitatively screen the differentially expressed genes (DEGs) and proteins (DEPs) between Control and FHN groups. Gene ontology (GO) terms and KEGG pathway enrichment analysis were used to assess the roles of DEGs and DEPs. qRT-PCR and western blot were performed to verify the key genes/proteins in FHN. CCK-8 assay was performed to measure cell viability. The protein expression of Bax and Bcl-2 were used to evaluate cell apoptosis. RESULTS: Transcriptome and proteome studies indicated 758 DEGs and 1097 DEPs between Control and FHN groups, respectively. Cell division, extracellular exosome, and serine-type endopeptidase activity were the most common terms in biological process (BP), cellular component (CC), and molecular function (MF) enrichment, respectively. DEPs were mainly enriched in cellular process, cell, and binding for BP, CC, and MF categories, respectively. DEGs were mainly involved in PI3K-Akt pathway and DEPs were mainly focused in glycolysis/gluconeogenesis pathway. Notably, 14 down-regulated and 22 up-regulated genes/proteins were detected at both the transcript and protein level. LRG1, SERPINE2, STMN1, COL14A1, SLC37A2, and MMP2 were determined as the key genes/proteins in FHN. SERPINE2/STMN1 overexpression increased viability and decreased apoptosis of dexamethasone-treated MC3T3-E1 cells. CONCLUSIONS: Our study investigated some pivotal regulatory genes/proteins in the pathogenesis of FHN, providing novel insight into the genes/proteins involved in FHN.

An expanded analysis framework for multivariate GWAS connects inflammatory biomarkers to functional variants and disease.

Multivariate methods are known to increase the statistical power to detect associations in the case of shared genetic basis between phenotypes. They have, however, lacked essential analytic tools to follow-up and understand the biology underlying these associations. We developed a novel computational workflow for multivariate GWAS follow-up analyses, including fine-mapping and identification of the subset of traits driving associations (driver traits). Many follow-up tools require univariate regression coefficients which are lacking from multivariate results. Our method overcomes this problem by using Canonical Correlation Analysis to turn each multivariate association into its optimal univariate Linear Combination Phenotype (LCP). This enables an LCP-GWAS, which in turn generates the statistics required for follow-up analyses. We implemented our method on 12 highly correlated inflammatory biomarkers in a Finnish population-based study. Altogether, we identified 11 associations, four of which (F5, ABO, C1orf140 and PDGFRB) were not detected by biomarker-specific analyses. Fine-mapping identified 19 signals within the 11 loci and driver trait analysis determined the traits contributing to the associations. A phenome-wide association study on the 19 representative variants from the signals in 176,899 individuals from the FinnGen study revealed 53 disease associations (p < 1 × 10-4). Several reported pQTLs in the 11 loci provided orthogonal evidence for the biologically relevant functions of the representative variants. Our novel multivariate analysis workflow provides a powerful addition to standard univariate GWAS analyses by enabling multivariate GWAS follow-up and thus promoting the advancement of powerful multivariate methods in genomics.

High expression level of serpin peptidase inhibitor clade E member 2 is associated with poor prognosis in lung adenocarcinoma.

BACKGROUND: Recent studies have revealed that serpin peptidase inhibitor clade E member 2 (SERPINE2) is associated with tumorigenesis. However, SERPINE2 expression and its role in lung adenocarcinomas are still unknown. METHODS: The expression levels of SERPINE2 in 74 consecutively resected lung adenocarcinomas were analyzed by using immunostaining. Inhibition of SERPINE2 expression by small interfering RNA (siRNA) was detected by quantitative PCR. Cell number assays and cell apoptosis assays were performed to clarify the cell-autonomous function of SERPINE2 in A549 and PC9 lung cancer cells. RESULTS: The overall survival of patients with high SERPINE2 expression was significantly worse than that of patients with low SERPINE2 expression (P = 0.0172). Multivariate analysis revealed that SERPINE2 expression was an independent factor associated with poor prognosis (P = 0.03237). The interference of SERPINE2 decreased cell number and increased apoptosis in A549 and PC9 cells CONCLUSION: These results suggest that SERPINE2 can be used as a novel prognostic marker of lung adenocarcinoma.

tPA Deficiency Underlies Neurovascular Coupling Dysfunction by Amyloid-ß.

The amyloid-ß (Aß) peptide, a key pathogenic factor in Alzheimer's disease, attenuates the increase in cerebral blood flow (CBF) evoked by neural activity (functional hyperemia), a vital homeostatic response in which NMDA receptors (NMDARs) play a role through nitric oxide, and the CBF increase produced by endothelial factors. Tissue plasminogen activator (tPA), which is reduced in Alzheimer's disease and in mouse models of Aß accumulation, is required for the full expression of the NMDAR-dependent component of functional hyperemia. Therefore, we investigated whether tPA is involved in the neurovascular dysfunction of Aß. tPA activity was reduced, and the tPA inhibitor plasminogen inhibitor-1 (PAI-1) was increased in male mice expressing the Swedish mutation of the amyloid precursor protein (tg2576). Counteracting the tPA reduction with exogenous tPA or with pharmacological inhibition or genetic deletion of PAI-1 completely reversed the attenuation of the CBF increase evoked by whisker stimulation but did not ameliorate the response to the endothelium-dependent vasodilator acetylcholine. The tPA deficit attenuated functional hyperemia by suppressing NMDAR-dependent nitric oxide production during neural activity. Pharmacological inhibition of PAI-1 increased tPA activity, prevented neurovascular uncoupling, and ameliorated cognition in 11- to 12-month-old tg2576 mice, effects associated with a reduction of cerebral amyloid angiopathy but not amyloid plaques. The data unveil a selective role of the tPA in the suppression of functional hyperemia induced by Aß and in the mechanisms of cerebral amyloid angiopathy, and support the possibility that modulation of the PAI-1-tPA pathway may be beneficial in diseases associated with amyloid accumulation.SIGNIFICANCE STATEMENT Amyloid-ß (Aß) peptides have profound neurovascular effects that may contribute to cognitive impairment in Alzheimer's disease. We found that Aß attenuates the increases in blood flow evoked by neural activation through a reduction in tissue plasminogen activator (tPA) caused by upregulation of its endogenous inhibitor plasminogen inhibitor-1 (PAI-1). tPA deficiency prevents NMDA receptors from triggering nitric oxide production, thereby attenuating the flow increase evoked by neural activity. PAI-1 inhibition restores tPA activity, rescues neurovascular coupling, reduces amyloid deposition around blood vessels, and improves cognition in a mouse model of Aß accumulation. The findings demonstrate a previously unappreciated role of tPA in Aß-related neurovascular dysfunction and in vascular amyloid deposition. Restoration of tPA activity could be of therapeutic value in diseases associated with amyloid accumulation.

Interacting hepatic PAI-1/tPA gene regulatory pathways influence impaired fibrinolysis severity in obesity.

Fibrinolysis is initiated by tissue-type plasminogen activator (tPA) and inhibited by plasminogen activator inhibitor 1 (PAI-1). In obese humans, plasma PAI-1 and tPA proteins are increased, but PAI-1 dominates, leading to reduced fibrinolysis and thrombosis. To understand tPA-PAI-1 regulation in obesity, we focused on hepatocytes, a functionally important source of tPA and PAI-1 that sense obesity-induced metabolic stress. We showed that obese mice, like humans, had reduced fibrinolysis and increased plasma PAI-1 and tPA, due largely to their increased hepatocyte expression. A decrease in the PAI-1 (SERPINE1) gene corepressor Rev-Erbα increased PAI-1, which then increased the tPA gene PLAT via a PAI-1/LRP1/PKA/p-CREB1 pathway. This pathway was partially counterbalanced by increased DACH1, a PLAT-negative regulator. We focused on the PAI-1/PLAT pathway, which mitigates the reduction in fibrinolysis in obesity. Thus, silencing hepatocyte PAI-1, CREB1, or tPA in obese mice lowered plasma tPA and further impaired fibrinolysis. The PAI-1/PLAT pathway was present in primary human hepatocytes, and associations among PAI-1, tPA, and PLAT in livers from obese and lean humans were consistent with these findings. Knowledge of PAI-1 and tPA regulation in hepatocytes in obesity may suggest therapeutic strategies for improving fibrinolysis and lowering the risk of thrombosis in this setting.

Development and characterization of single-domain antibodies neutralizing protease nexin-1 as tools to increase thrombin generation.

BACKGROUND: Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin. OBJECTIVES: Here, we aimed to develop inhibitory single-domain antibodies (VHHs) that show specific binding to both human (hPN-1) and murine (mPN-1) PN-1. METHODS: PN-1-binding VHHs were isolated via phage-display using llama-derived or synthetic VHH-libraries. Following bacterial expression, purified VHHs were analyzed in binding and activity assays. RESULTS AND CONCLUSIONS: By using a llama-derived library, 2 PN-1 specific VHHs were obtained (KB-PN1-01 and KB-PN1-02). Despite their specificity, none displayed inhibitory activity toward hPN-1 or mPN-1. From the synthetic library, 4 VHHs (H12, B11, F06, A08) could be isolated that combined efficient binding to both hPN-1 and mPN-1 with negligible binding to PAI-1. Of these, B11, F06, and A08 were able to fully restore thrombin activity by blocking PN-1. As monovalent VHH, half-maximal inhibitory concentration values for hPN-1 were 50 ± 10, 290 ± 30, and 960 ± 390 nmol/L, for B11, F06, and A08, respectively, and 1580 ± 240, 560 ± 130, and 2880 ± 770 nmol/L for mPN-1. The inhibitory potential was improved 4- to 7-fold when bivalent VHHs were engineered. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. In conclusion, we report the generation of inhibitory anti-PN-1 antibodies using a specific approach to avoid cross-reactivity with the homologous Serpin PAI-1.

miR-34a targets PAI-1 to regulate urinary microalbumin and renal function in hypertensive mice.

BACKGROUND: The aim of the study is to investigate the effects of miR-34a targeted at PAI-1 on urinary microalbumin and renal function in hypertensive mice. METHODS: Twenty specific-pathogen-free (SPF) BPN/3J mice were selected in normal group, and 120 SPF BPH/2J mice were evenly divided into model group, negative control group, miR-34a mimic group, miR-34a inhibitor group, Si-PAI-1 group, and miR-34a inhibitor + Si-PAI-1 group. qRT-PCR was used to detect the expression of miR-34a and PAI-1 mRNA. The protein expressions of PAI-1, angiotensin-converting enzyme (ACE) and ACE2 were detected by Western blot. Serum levels of AngII and Ang1-7 were detected by ELISA. RESULTS: miR-34a negatively regulated the expression of PAI-1. Compared with the normal group, mice in the other groups had significantly lower body weight, increased systolic blood pressure and 24-h urinary microalbumin content, decreased miR-34a expression, superoxide dismutase (SOD) and nitric oxide (NO) content, and ACE2 protein expression, and increased PAI-1 expression, serum creatinine (Scr), blood urea nitrogen (BUN) malondialdehyde (MDA), AngII and Ang1-7 levels, and ACE protein expression (all P < 0.05). Compared with the model group, mice in the miR-34a mimic group and Si-PAI-1 group had no significant changes in body weight (all P > 0.05), while they had significantly lower systolic blood pressure and 24-h urinary microalbumin content, increased SOD and NO levels and ACE2 protein expression, and decreased PAI-1 expression, Scr, BUN, MDA, AngII and Ang1-7 levels, and ACE protein expression (all P < 0.05). Compared with the miR-34a inhibitor group, symptoms in miR-34a inhibitor + Si-PAI-1 group were significantly improved (all P < 0.05). CONCLUSIONS: miR-34a can inhibit the expression of PAI-1, thereby reducing urinary microalbumin content in hypertensive mice and protecting their renal function.

Integrated transcriptomic correlation network analysis identifies COPD molecular determinants.

Chronic obstructive pulmonary disease (COPD) is a complex and heterogeneous syndrome. Network-based analysis implemented by SWIM software can be exploited to identify key molecular switches - called "switch genes" - for the disease. Genes contributing to common biological processes or defining given cell types are usually co-regulated and co-expressed, forming expression network modules. Consistently, we found that the COPD correlation network built by SWIM consists of three well-characterized modules: one populated by switch genes, all up-regulated in COPD cases and related to the regulation of immune response, inflammatory response, and hypoxia (like TIMP1, HIF1A, SYK, LY96, BLNK and PRDX4); one populated by well-recognized immune signature genes, all up-regulated in COPD cases; one where the GWAS genes AGER and CAVIN1 are the most representative module genes, both down-regulated in COPD cases. Interestingly, 70% of AGER negative interactors are switch genes including PRDX4, whose activation strongly correlates with the activation of known COPD GWAS interactors SERPINE2, CD79A, and POUF2AF1. These results suggest that SWIM analysis can identify key network modules related to complex diseases like COPD.