A serpin shapes the extracellular environment to prevent influenza A virus maturation.

Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

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.

Homologous recombination repair gene-based risk model predicts prognosis and immune microenvironment for primary lung cancer after previous malignancies.

BACKGROUND: Homologous recombination repair (HRR) plays an important role in cancer development, drug resistance, and immune escape, but the role of HRR genes in primary lung cancer (PLC) after previous malignancies is unclear. METHODS: We used HRR-related score constrcted by HRR genes to classify patients into two groups and compared clinical progression, differential genes, and their functions between them. Then, we constructed a prognostic risk model based on HRR-related score and screened key differentially expressed genes. We evaluated the potential roles, mutational information, and immune correlations of key genes. Finally, we compared the long-term prognosis and immune correlations of different prognostic risk subgroups. RESULTS: We found that HRR-related score was associated with T-stage, immunotherapy sensitivity, and prognosis of PLC after previous malignancies. Differential genes between HRR-related low-score and high-score groups are mainly involved in DNA replication and repair processes, such as the cell cycle. We identified three key genes, ABO, SERPINE2, and MYC, by machine learning, and MYC had the highest amplification mutation frequency. We verified that the key gene-based prognostic model can better assess the prognosis of patients. The risk score of the prognostic model was associated with immune microenvironment and efficacy of immunotherapy. CONCLUSIONS: Overall, we identified three key genes ABO, SERPINE2, and MYC associated with HRR status in PLC after previous malignancies. The risk model based on key genes is associated with immune microenvironment and can well predict the prognosis for PLC after previous malignancies.

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.

PAI-1 production by reactive astrocytes drives tissue dysfibrinolysis in multiple sclerosis models.

BACKGROUND: In multiple sclerosis (MS), disturbance of the plasminogen activation system (PAS) and blood brain barrier (BBB) disruption are physiopathological processes that might lead to an abnormal fibrin(ogen) extravasation into the parenchyma. Fibrin(ogen) deposits, usually degraded by the PAS, promote an autoimmune response and subsequent demyelination. However, the PAS disruption is not well understood and not fully characterized in this disorder. METHODS: Here, we characterized the expression of PAS actors during different stages of two mouse models of MS (experimental autoimmune encephalomyelitis-EAE), in the central nervous system (CNS) by quantitative RT-PCR, immunohistofluorescence and fluorescent in situ hybridization (FISH). Thanks to constitutive PAI-1 knockout mice (PAI-1 KO) and an immunotherapy using a blocking PAI-1 antibody, we evaluated the role of PAI-1 in EAE models and its impact on physiopathological processes such as fibrin(ogen) deposits, lymphocyte infiltration and demyelination. RESULTS: We report a striking overexpression of PAI-1 in reactive astrocytes during symptomatic phases, in two EAE mouse models of MS. This increase is concomitant with lymphocyte infiltration and fibrin(ogen) deposits in CNS parenchyma. By genetic invalidation of PAI-1 in mice and immunotherapy using a blocking PAI-1 antibody, we demonstrate that abolition of PAI-1 reduces the severity of EAE and occurrence of relapses in two EAE models. These benefits are correlated with a decrease in fibrin(ogen) deposits, infiltration of T4 lymphocytes, reactive astrogliosis, demyelination and axonal damage. CONCLUSION: These results demonstrate that a deleterious overexpression of PAI-1 by reactive astrocytes leads to intra-parenchymal dysfibrinolysis in MS models and anti-PAI-1 strategies could be a new therapeutic perspective for MS.

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.

Transcripts associated with chronic lung allograft dysfunction in transbronchial biopsies of lung transplants.

Transplanted lungs suffer worse outcomes than other organ transplants with many developing chronic lung allograft dysfunction (CLAD), diagnosed by physiologic changes. Histology of transbronchial biopsies (TBB) yields little insight, and the molecular basis of CLAD is not defined. We hypothesized that gene expression in TBBs would reveal the nature of CLAD and distinguish CLAD from changes due simply to time posttransplant. Whole-genome mRNA profiling was performed with microarrays in 498 prospectively collected TBBs from the INTERLUNG study, 90 diagnosed as CLAD. Time was associated with increased expression of inflammation genes, for example, CD1E and immunoglobulins. After correcting for time, CLAD manifested not as inflammation but as parenchymal response-to-wounding, with increased expression of genes such as HIF1A, SERPINE2, and IGF1 that are increased in many injury and disease states and cancers, associated with development, angiogenesis, and epithelial response-to-wounding in pathway analysis. Fibrillar collagen genes were increased in CLAD, indicating matrix changes, and normal transcripts were decreased-dedifferentiation. Gene-based classifiers predicted CLAD with AUC 0.70 (no time-correction) and 0.87 (time-corrected). CLAD related gene sets and classifiers were strongly prognostic for graft failure and correlated with CLAD stage. Thus, in TBBs, molecular changes indicate that CLAD primarily reflects severe parenchymal injury-induced changes and dedifferentiation.

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.

Bone morphogenetic protein 2 upregulates SERPINE2 expression through noncanonical SMAD2/3 and p38 MAPK signaling pathways in human granulosa-lutein cells.

Serine protease inhibitor-E2 (SERPINE2) is highly expressed in the granulosa cells of growing follicles and the dynamic changes in SERPINE2 expression are correlated with follicular development and ovulation in several mammals, including mice, cattle, sheep, and humans. Bone morphogenetic proteins (BMPs) and their functional receptors are extensively expressed in the ovary and play critical roles in the regulation of ovarian folliculogenesis and luteal function. To date, whether BMPs regulate the expression of SERPINE2 during human follicular development remains to be elucidated. The aim of this study was to investigate the effects of BMPs on the regulation of SERPINE2 expression (a major regulator of plasminogen activators [PA]) and the underlying mechanisms using primary and immortalized human granulosa-lutein (hGL) cells. Our results demonstrated that these BMPs (BMP2, BMP4, BMP6, BMP7, and BMP15) induced differential upregulation of SERPINE2 expression. In this regard, BMP2 is the major modulator that has the best cellular activity, which further decreased the production of urokinase PA and tissue PA in hGL cells. In addition to canonical SMAD1/5/8 signaling, BMP2 also activates noncanonical SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) signaling. Using two inhibition approaches (kinase receptor inhibitors and siRNA-mediated knockdown), we found that SMAD2/3-SMAD4 and p38 MAPK, but not SMAD1/5/8 signaling, was involved in the BMP2-induced upregulation of SERPINE2 expression via activin receptor-like kinase 3. These findings deepen our understanding of the differential effect of BMPs in regulating follicular function and provide new insights of the molecular mechanisms by which BMP2 regulates the expression of SERPINE2 in human granulosa cells.

Transcriptional profile of cumulus associated GJA1, PTX3, PRSS35, and SERPINE2 genes with oocytes and embryonic development in water buffalo.

BACKGROUND: In the present study, the potential of different groups of cumulus-oocyte complexes (COC's) for in vitro maturation (IVM) and embryonic development was assessed in two groups of COC's of water buffalo. Further, the expression pattern of cumulus-associated GJA1, PTX3, PRSS35, and SERPINE2 genes and their effects on embryonic development was analyzed. Slaughterhouse-derived buffalo COC's were graded into groups A and B. An equal number of 410 COC's were taken in both groups. IVM was carried out using Slaughterhouse-derived buffalo epididymis. A remarkable degree of cumulus expansion was noticed in group A (92.68%) as compared to group B (81.25%) oocytes. On in vitro fertilization (IVF) and embryo culture, group A produced a significantly higher rate of cleavage and blastocyst (92.682 ± 0.7179% and 42.682 ± 0.9683%) as compared to group B (85.365 ± 0.7608% and 31.707 ± 0.9688%). Also, the transcriptional analysis of cumulus-associated genes revealed significantly higher expression in group A as compared to group B. RESULTS: It was revealed that oocytes having good cumulus mass had a higher developmental potential. Based on differential gene expression of cumulus-associated genes, different quality of COC's, and the resultant embryos after IVF, it was concluded that these genes could be used as a marker for predicting the developmental competence of the oocytes. CONCLUSION: We concluded that morphologically good quality of COC's had a higher developmental competence, and also the differential expressions of cumulus-associated genes in cumulus cells and embryos. So, we can conclude that these genes could be used as marker genes for predicting the developmental competence of buffalo's oocytes.

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.

A Novel L-Phenylalanine Dipeptide Inhibits the Growth and Metastasis of Prostate Cancer Cells via Targeting DUSP1 and TNFSF9.

Prostate cancer (PCa) is a common malignant cancer of the urinary system. Drug therapy, chemotherapy, and radical prostatectomy are the primary treatment methods, but drug resistance and postoperative recurrence often occur. Therefore, seeking novel anti-tumor compounds with high efficiency and low toxicity from natural products can produce a new tumor treatment method. Matijin-Su [N-(N-benzoyl-L-phenylalanyl)-O-acetyl-L-phenylalanol, MTS] is a phenylalanine dipeptide monomer compound that is isolated from the Chinese ethnic medicine Matijin (Dichondra repens Forst.). Its derivatives exhibit various pharmacological activities, especially anti-tumor. Among them, the novel MTS derivative HXL131 has a significant inhibitory effect against prostate tumor growth and metastasis. This study is designed to investigate the effects of HXL131 on the growth and metastasis of human PCa cell lines PC3 and its molecular mechanism through in vitro experiments combined with proteomics, molecular docking, and gene silencing. The in vitro results showed that HXL131 concentration dependently inhibited PC3 cell proliferation, induced apoptosis, arrested cell cycle at the G2/M phase, and inhibited cell migration capacity. A proteomic analysis and a Western blot showed that HXL131 up-regulated the expression of proliferation, apoptosis, cell cycle, and migration-related proteins CYR61, TIMP1, SOD2, IL6, SERPINE2, DUSP1, TNFSF9, OSMR, TNFRSF10D, and TNFRSF12A. Molecular docking, a cellular thermal shift assay (CETSA), and gene silencing showed that HXL131 had a strong binding affinity with DUSP1 and TNFSF9, which are important target genes for inhibiting the growth and metastasis of PC3 cells. This study demonstrates that HXL131 exhibited excellent anti-prostate cancer activity and inhibited the growth and metastasis of prostate cancer cells by regulating the expression of DUSP1 and TNFSF9.

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.

Antithrombin Together with NETs Inhibitor Protected Against Postoperative Adhesion Formation in Mice.

BACKGROUND/AIMS: Postoperative adhesions may induce adverse outcomes in patients. Adhesion formation is initiated by fibrin accumulation at the surgical site which is followed by local neutrophilia and the establishment of neutrophil extracellular traps (NET). Previous reports have suggested that the preventive efficacy of reagents designed to reduce postoperative adhesion is inversely correlated with neutrophilia and NET production. Antithrombin (AT) is a natural inhibitor of thrombin, a key factor in coagulation. Here, we evaluate whether treatment with AT and/or NET inhibitors prevent or reduce postoperative adhesion formation in mice. METHODS: Mice were treated with AT and/or NET inhibitors before and/or after cecum cauterization and their adhesion scores were evaluated on day 7 post-operation. Immunochemistry/ immunofluorescence analyses were also performed and we used GSK484, an inhibitor of peptidyl arginine deiminase 4 (PAD4), as the NET inhibitor. RESULTS: AT or GSK484 partially rescued postoperative adhesion formation in mice. AT prevented thrombin-induced plasminogen activator inhibitor 1 and interleukin-6 expression in mesothelial cells in vitro. However, AT could not prevent neutrophilia or NETs formation around the injured serosa. Finally, we investigated a combination of AT and a PAD4 inhibitor and found that this could inhibit almost all adhesion formation in these animals. Since AT-inactivating proteases are liberated following NET release, they might dampen the biological action of the AT treatment. This suggests that NET inhibitors might allow AT to exert its full action in the surgically injured serosa. CONCLUSION: Combined treatment with AT and GSK484 may effectively attenuate postoperative adhesion production in mice.

Bioinformatics analysis reveals a stem cell-expressed circ-Serpine2-mediated miRNA-mRNA regulatory subnetwork in the malignant progression of glioma.

BACKGROUND: High-grade glioma has a poor prognosis, and GSCs can have pivotal roles in glioma pathology. This study investigated GSC exosome-containing circRNA mechanisms affecting the malignant progression of glioma. METHODS: In this study, we identified differentially expressed circRNAs and constructed a circRNA-miRNA-mRNA regulatory network through circRNA sequencing/bioinformatics analysis. Then, we identified circRNAs that were upregulated in GSC23 cells and employed them as downstream targets in subsequent investigations. Such investigations included downstream target knockout to assess any influence on A172 cell proliferation, invasion, migration and apoptosis. In addition, in vivo investigations using tumor-bearing animals evaluated the in vivo influences of the selected targets. RESULTS: This study identified circ-Serpine2/miR-124-3p/KIF20A as a regulatory pathway in glioma. Our in vitro analysis confirmed that circ-Serpine2 could upregulate KIF20A by sponging miR-124-3p, consequently promoting A172 cell proliferation, migration and invasion. Such a signaling channel could also inhibit glioma cell apoptosis. Additionally, our research indicated that circ-Serpine2 inhibited glioma apoptosis and promoted in vivo tumor progression. CONCLUSION: Circ-Serpine2 exacerbated the malignant progression of glioma mediated by the miR-124-3p/KIF20A nexus, thus providing novel predictive/prognostic biomarkers and drug targets against glioma.

Targeting protease nexin-1, a natural anticoagulant serpin, to control bleeding and improve hemostasis in hemophilia.

Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.

Targeting of plasminogen activator inhibitor-1 activity promotes elimination of chronic myeloid leukemia stem cells.

Therapeutic strategies that target leukemic stem cells (LSCs) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here, we show that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSCs to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSCs and leads to sustained remission of the disease. We demonstrated for the first time that TGF-ß-PAI-1 axis was selectively augmented in CML-LSCs in the bone marrow (BM), whereby protecting CML-LSCs from TKI treatment. Furthermore, the combined administration of TKI plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSCs. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSCs, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI-1 activity, for CML patients.

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.

A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis.

Cancer metastasis requires that primary tumour cells evolve the capacity to intravasate into the lymphatic system or vasculature, and extravasate into and colonize secondary sites. Others have demonstrated that individual cells within complex populations show heterogeneity in their capacity to form secondary lesions. Here we develop a polyclonal mouse model of breast tumour heterogeneity, and show that distinct clones within a mixed population display specialization, for example, dominating the primary tumour, contributing to metastatic populations, or showing tropism for entering the lymphatic or vasculature systems. We correlate these stable properties to distinct gene expression profiles. Those clones that efficiently enter the vasculature express two secreted proteins, Serpine2 and Slpi, which were necessary and sufficient to program these cells for vascular mimicry. Our data indicate that these proteins not only drive the formation of extravascular networks but also ensure their perfusion by acting as anticoagulants. We propose that vascular mimicry drives the ability of some breast tumour cells to contribute to distant metastases while simultaneously satisfying a critical need of the primary tumour to be fed by the vasculature. Enforced expression of SERPINE2 and SLPI in human breast cancer cell lines also programmed them for vascular mimicry, and SERPINE2 and SLPI were overexpressed preferentially in human patients that had lung-metastatic relapse. Thus, these two secreted proteins, and the phenotype they promote, may be broadly relevant as drivers of metastatic progression in human cancer.

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.