Identification of new subtypes of breast cancer based on vasculogenic mimicry related genes and a new model for predicting the prognosis of breast cancer.

Breast cancer is a malignant tumor that poses a serious threat to women's health, and vasculogenic mimicry (VM) is strongly associated with bad prognosis in breast cancer. However, the relationship between VM and immune infiltration in breast cancer and the underlying mechanisms have not been fully studied. On the basis of the Cancer Genome Atlas (TCGA), Fudan University Shanghai Cancer Center (FUSCC) database, GSCALite database, and gene set enrichment analysis (GSEA) datasets, we investigated the potential involvement of VM-related genes in the development and progression of breast cancer. We analyzed the differential expression, mutation status, methylation status, drug sensitivity, tumor mutation burden (TMB), microsatellite instability (MSI), immune checkpoints, tumor microenvironment (TME), and immune cell infiltration levels associated with VM-related genes in breast cancer. We created two VM subclusters out of breast cancer patients using consensus clustering, and discovered that patients in Cluster 1 had better survival outcomes compared to those in Cluster 2. The infiltration levels of T cells CD4 memory resting and T cells CD8 were higher in Cluster 1, indicating an immune-active state in this cluster. Additionally, we selected three prognostic genes (LAMC2, PIK3CA, and TFPI2) using Lasso, univariate, and multivariate Cox regression and constructed a risk model, which was validated in an external dataset. The prognosis of patients is strongly correlated with aberrant expression of VM-related genes, which advances our knowledge of the tumor immune milieu and enables us to identify previously unidentified breast cancer subtypes. This could direct more potent immunotherapy approaches.

Tissue factor pathway inhibitor 2 as a serum biomarker for endometrial cancer: a single-center retrospective study.

BACKGROUND: Endometrial cancer is the most common gynecological malignancy; however, there is no useful blood diagnostic biomarker. This study aimed to determine the utility of tissue factor pathway inhibitor 2 (TFPI2), a biomarker of ovarian cancer, as a diagnostic marker for endometrial cancer. METHODS: We examined serum TFPI2 levels in patients with endometrial cancer (n = 328) compared to those in healthy controls (n = 65) and evaluated the performance of serum TFPI2 levels as a diagnostic marker. We investigated the clinicopathological characteristics of patients with TFPI2-negative and TFPI2-positive endometrial cancer. Using immunohistochemistry (IHC), we examined TFPI2 expression in tumor tissues of 105 patients with type II endometrial carcinoma and evaluated the correlation between serum and tissue TFPI2 positivity. RESULTS: Patients with endometrial cancer had significantly higher serum TFPI2 levels than controls (196.7 pg/mL vs. 83.3 pg/mL; p < 0.001). The sensitivity and specificity were 54.3% and 95.4%, respectively (cutoff value, 191 pg/mL). Serum TFPI2 levels were significantly elevated along with the stage progression (stage I, 189.6 pg/mL; stage III, 230.9 pg/mL; stage IV, 312.5 pg/mL; p < 0.001). Patients with high-risk histology showed significantly elevated serum TFPI2 levels than those with low-risk histology (220.8 pg/mL vs. 187.7 pg/mL; p < 0.001). The positivity rate for TFPI2 was the highest among tumor markers, including CA125, CA19-9, and CEA. Serum TFPI2 and CA125 levels were almost independent (r = 0.203, p < 0.001), and the combined sensitivity increased to 58.8%. The 5-year survival rate was significantly worse in TFPI2-positive patients (≥ 191 pg/mL, n = 178) than in TFPI2-negative patients (< 191 pg/mL, n = 150) (hazard ratio, 8.22; 95% confidence interval, 2.49-27.1; p < 0.001). TFPI2 immunostaining revealed that 37.1% (39/105) of the samples were positive for TFPI2, with an IHC score of > 0. There was no significant difference in the immunostaining score according to histological type. Serum TFPI2 levels and immunostaining score showed poor agreement (kappa coefficient, -0.039). CONCLUSIONS: The serum TFPI2 level is a promising marker for diagnosing and predicting the prognosis of endometrial cancer. No correlation exists between serum and tissue TFPI2 levels. Further multicenter clinical trials are needed to test the utility of TFPI2 as a diagnostic marker.

A high-calcium environment induced ectopic calcification of renal interstitial fibroblasts via TFPI-2-DCHS1-ALP/ENPP1 axis to participate in Randall's plaque formation.

Randall's plaques (RP) serve as anchoring sites for calcium oxalate (CaOx) stones, but the underlying mechanism remains unclear. Renal interstitium with a high-calcium environment is identified as pathogenesis of RP formation where the role of human renal interstitial fibroblasts (hRIFs) was highlighted. Our study aims to elucidate the potential mechanism by which a high-calcium environment drives ectopic calcification of hRIFs to participate in RP formation. Alizarin Red staining demonstrated calcium nodules in hRIFs treated with high-calcium medium. Utilizing transcriptome sequencing, tissue factor pathway inhibitor-2 (TFPI-2) was found to be upregulated in high-calcium-induced hRIFs and RP tissues, and TFPI-2 promoted high-calcium-induced calcification of hRIFs. Subsequently, the downstream regulator of TFPI2 was screened by transcriptome sequencing analysis of hRIFs with TFPI-2 knockdown or overexpressed. Dachsous Cadherin Related 1 (DCHS1) knockdown was identified to suppress the calcification of hRIFs enhanced by TFPI-2. Further investigation revealed that TFPI-2/DCHS1 axis promoted high-calcium-induced calcification of hRIFs via disturbing the balance of ENPP1/ALP activities, but without effect on the canonical osteogenic markers, such as osteopontin (OPN), osteogenic factors runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2). In summary, our study mimicked the high-calcium environment observed in CaOx stone patients with hypercalciuria, and discovered that the high-calcium drove ectopic calcification of hRIFs via a novel TFPI-2-DCHS1-ALP/ENPP1 pathway rather than adaption of osteogenic phenotypes to participate in RP formation.

Restoration of TFPI2 by LSD1 inhibition suppresses tumor progression and potentiates antitumor immunity in breast cancer.

Histone lysine-specific demethylase 1 (LSD1) is frequently overexpressed in triple negative breast cancer (TNBC), which is associated with worse clinical outcome in TNBC patients. However, the underlying mechanisms by which LSD1 promotes TNBC progression remain to be identified. We recently established a genetically engineered murine model by crossing mammary gland conditional LSD1 knockout mice with Brca1-deficient mice to explore the role of LSD1 in TNBC pathogenesis. Cre-mediated Brca1 loss led to higher incidence of tumor formation in mouse mammary glands, which was hindered by concurrent depletion of LSD1, indicating a critical role of LSD1 in promoting Brca1-deficient tumors. We also demonstrated that the silencing of a tumor suppressor gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), is functionally associated with LSD1-mediated TNBC progression. Mouse Brca1-deficient tumors exhibited elevated LSD1 expression and decreased TFPI2 level compared to normal mammary tissues. Analysis of TCGA database revealed that TFPI2 expression is significantly lower in aggressive ER-negative or basal-like BC. Restoration of TFPI2 through LSD1 inhibition increased H3K4me2 enrichment at the TFPI2 promoter, suppressed tumor progression, and enhanced antitumor efficacy of chemotherapeutic agent. Induction of TFPI2 by LSD1 ablation downregulates activity of matrix metalloproteinases (MMPs) that in turn increases the level of cytotoxic T lymphocyte attracting chemokines in tumor environment, leading to enhanced tumor infiltration of CD8+ T cells. Moreover, induction of TFPI2 potentiates antitumor effect of LSD1 inhibitor and immune checkpoint blockade in poorly immunogenic TNBC. Together, our study identifies previously unrecognized roles of TFPI2 in LSD1-mediated TNBC progression, therapeutic response, and immunogenic effects.

Unveiling heterogeneity and prognostic markers in ductal breast cancer through single-cell RNA-seq.

BACKGROUND: Breast cancer (BC) is a heterogeneous disease, with the ductal subtype exhibiting significant cellular diversity that influences prognosis and response to treatment. Single-cell RNA sequencing data from the GEO database were utilized in this study to investigate the underlying mechanisms of cellular heterogeneity and to identify potential prognostic markers and therapeutic targets. METHODS: Bioinformatics analysis was conducted using R packages to analyze the single-cell sequencing data. The presence of highly variable genes and differences in malignant potency within the same BC samples were examined. Differential gene expression and biological function between Type 1 and Type 2 ductal epithelial cells were identified. Lasso regression and Cox proportional hazards regression analyses were employed to identify genes associated with patient prognosis. Experimental validation was performed in vitro and in vivo to confirm the functional relevance of the identified genes. RESULTS: The analysis revealed notable heterogeneity among BC cells, with the presence of highly variable genes and differences in malignant behavior within the same samples. Significant disparities in gene expression and biological function were identified between Type 1 and Type 2 ductal epithelial cells. Through regression analyses, CYP24A1 and TFPI2 were identified as pivotal genes associated with patient prognosis. Kaplan-Meier curves demonstrated their prognostic significance, and experimental validation confirmed their inhibitory effects on malignant behaviors of ductal BC cells. CONCLUSION: This study highlights the cellular heterogeneity in ductal subtype breast cancer and delineates the differential gene expressions and biological functions between Type 1 and Type 2 ductal epithelial cells. The genes CYP24A1 and TFPI2 emerged as promising prognostic markers and therapeutic targets, exhibiting inhibitory effects on BC cell malignancy in vitro and in vivo. These findings offer the potential for improved BC management and the development of targeted treatment strategies.

Deletion of tissue factor pathway inhibitor isoform beta or gamma, but not alpha, improves clotting in hemophilic mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) regulates tissue factor-triggered coagulation. Humans and mice express transcripts encoding for multidistributed (endothelial, platelet, and plasma) 3-Kunitz domain TFPIα and endothelial membrane-anchored 2-Kunitz TFPIß. Mice express a third transcript, γ, that encodes plasma lipoprotein-associated 2-Kunitz TFPI. In humans, proteolysis of α and/or ß produces plasma lipoprotein-associated 2-Kunitz TFPI at lower levels. In clinical trials, monoclonal antibodies that target all TFPI isoforms extend coagulation and correct bleeding in hemophilic patients but with some thrombosis risks. OBJECTIVES: To determine the impact of TFPI isoform-specific deletions on promoting clotting in hemophilic mice. METHODS: Engineered TFPI isoform-specific, hemophilic (factor VIII-null) mice were evaluated for clotting. RESULTS: Mice expressing any single TFPI isoform were healthy. Thrombin generation assays identified TFPIγ as the dominant anticoagulation isoform in mouse plasma. Hemostasis was assessed by serial bleeding times from a tail vein laceration. Repeatedly, after a clot forms, it was manually disrupted; the number of clots/disruptions occurring over a 15-minute period were reported. C57BL/6 and hemophilic mice clot on average 25.6 vs 5.4 times, respectively. On a hemophilia background, TFPIß or TFPIγ-specific deletion improved clotting to 14.6 and 15.2 times, respectively (P < .0001). TFPIα-specific deletion was without impact, clotting 5.1 times. Heterozygous deletion of TFPIß was effective, clotting 11.8 times (P < .0001). Heterozygous deletion of TFPIα or TFPIγ alone was ineffective, clotting 3.0 and 6.1 times, respectively, but heterozygous TFPIαγ deletion improved clotting to 11.2 times (P < .001). CONCLUSION: In hemophilic mice, endothelial TFPIß and plasma γ-derived 2-Kunitz TFPI individually contribute more to bleeding than total TFPIα.

Elevated TFPI is a prognostic factor in hepatocellular carcinoma: Putative role of miR-7-5p and miR-1236-3p.

BACKGROUND: Primary liver cancer is the third leading cause of cancer related deaths worldwide, and the disease is associated with high incidence rate of thrombosis. Studies indicate that Tissue Factor Pathway Inhibitor (TFPI) plays a role in cancer development. We aimed to study its expression, clinical role and regulation by micro RNAs (miRNAs) in hepatocellular carcinoma (HCC). METHODS: Publically available datasets were used for clinical analysis of TFPI and miRNAs expression by web analysis tools. miRNA mimics targeting TFPIα 3'untranslated region (UTR) were selected from target prediction programs and verified by luciferase reporter assay. In vitro effects of miRNAs overexpression in HCC cell lines on TFPI expression and cell proliferation and apoptosis were analysed. RESULTS: TFPI expression was significantly increased in HCC tumours compared to normal tissue. Low TFPI tumour expression was associated with better survival probability. Four candidate miRNAs were selected from the target prediction programs. miR-7-5p and miR-1236-3p were validated in HepG2 and Huh7 cells to reduce TFPI mRNA and protein levels following overexpression. Furthermore, miR-7-5p and miR-1236-3p reduced TFPIα-3'UTR-controlled luciferase activity. The two validated miRNAs inhibited proliferation of HepG2 cells, and had clinical significance in HCC. CONCLUSIONS: TFPI was increased in HCC tumours compared to normal tissue and high TFPI expression was associated with an unfavorable outcome in HCC patients. miR-7-5p and miR-1236-3p were identified as novel regulators of TFPI in vitro.

Relationship between plasma tissue Factor Pathway Inhibitor (TFPI) levels, thrombin generation and clinical risk of bleeding in patients with severe haemophilia A or B.

INTRODUCTION: Bleeding severity in severe haemophilic patients, with low thrombin generation (TG) capacity, can vary widely between patients, possibly reflecting differences in tissue factor pathway inhibitor (TFPI) level. AIM: To compare free TFPI (fTFPI) levels in patients with severe haemophilia A (sHA) and severe haemophilia B (sHB) and to investigate in these patients as a whole the relationships between bleeding and TG potential, between TG potential and fTFPI level and between fTFPI level and bleeding tendency. METHODS: Data on bleeding episodes retrospectively recorded during follow-up visits over 5-10 years were collected and used to calculate the annualised joint bleeding rate (AJBR). fTFPI levels and basal TG parameters were determined in platelet-poor plasma (PPP) and platelet-rich plasma (PRP) using calibrated automated tomography (CAT). RESULTS: Mean fTFPI levels did not differ significantly between sHA (n = 34) and sHB (n = 19) patients. Mean values of endogenous thrombin potential (ETP) and thrombin peak (peak) in PPP and PRP were two-fold higher when fTFPI levels < 9.4 versus > 14.3 ng/mL. In patients treated on demand, ETP and peak in PRP were doubled when AJBR was ≤ 4.9 $ \le 4.9$ , AJBR being halved in patients with a low fTFPI level (9.4 ng/mL). In patients on factor prophylaxis, no association was found between TG parameters and either fTFPI level or AJBR. CONCLUSION: In patients treated on demand, bleeding tendency was influenced by fTFPI levels, which in turn affected basal TG potential. In patients on prophylaxis, bleeding tendency is probably determined primarily by the intensity of this treatment.

Effect of tissue factor pathway inhibitor on the pyroptosis of vascular smooth muscle cells induced by angiotensin II.

Background: In recent years, a mass of studies have shown that pyroptosis plays an important role in the proliferation of vascular smooth muscle cells (VSMCs). We investigated whether angiotensin II (Ang II) induces the pyroptosis of rat aortic VSMCs and the role of NOD-like receptor family pyrin domain containing 3 (NLRP3) in this process. Additionally, we explored the effect and related mechanism of recombinant tissue factor pathway inhibitor (rTFPI) in Ang II-induced VSMC pyroptosis. Methods: Cultured VSMCs were divided into five groups: control group, Ang II group (1×10-5 mol/L), MCC950 group (NLRP3 inhibitor, 15 nmol/L), Ang II + MCC950 group and Ang II + rTFPI (50 µg/L) group. Cell viability was measured by cell counting kit-8 (CCK8) assays and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays. Propidium iodide (PI) staining and immunofluorescence were performed to determine the pyroptosis of VSMCs. Changes in VSMC ultrastructure were evaluated through transmission electron microscopy. The expression levels of NLRP3, pro-caspase-1, gasdermin D-N (GSDMD-N), and interleukin-1ß (IL-1ß) were determined by western blot analysis. Results: The cell viability, the positive rate of PI staining, and the expression level of GSDMD detected by immunofluorescence in the Ang II group were higher than that in the control group, whereas they all decreased in Ang II + MCC950 group and Ang II + rTFPI group compared with Ang II group (P<0.05). Electron microscopy analysis revealed less extracellular matrix, increased myofilaments, and decreased endoplasmic reticulum, Golgi complex, and mitochondria in Ang II + rTFPI-treated VSMCs than in Ang II-treated VSMCs. The protein expression levels of the pyroptosis-related molecules NLRP3, pro-caspase-1, GSDMD-N, and IL-1ß in Ang II group showed an increasing trend compared with those in control group (P<0.05); however, these expression levels in Ang II + MCC950 and Ang II + rTFPI groups were significantly lower than those in Ang II group (P<0.05). Conclusions: Ang II may induce pyroptosis in VSMCs by activating NLRP3. rTFPI can inhibit Ang II-induced VSMC pyroptosis. Furthermore, rTFPI might exert this effect by inhibiting the NLRP3 pathway and therefore play an important role in the treatment of vascular remodeling induced by hypertension.

Site-directed mutagenesis of tissue factor pathway inhibitor-binding exosite D60A on factor VII results in a new factor VII variant with lower coagulant activity.

Background: Recombinant factor (F)VIIa (rFVIIa) has been approved by the US Food and Drug Administration for the treatment of hemophilia A and B with inhibitors and congenital FVII deficiency. Moreover, the investigational uses of rFVIIa are becoming of interest since it can be used to treat various clinical bleeding conditions. However, there is evidence showing that rFVIIa is a potent procoagulant agent that potentially leads to an increased risk of thrombotic complications. Objectives: To design a new rFVII with lower coagulant activity that could potentially be used as an alternative hemostatic agent aiming to minimize the risk of thrombogenicity. Methods: D60A was introduced into the F7 sequence by polymerase chain reaction-based mutagenesis. Wild type (WT) and D60A were generated in human embryonic kidney 293T cells by stable transfection. FVII coagulant activities were determined by amidolytic cleavage of the FVIIa-specific substrate, 2-step FXa generation, thrombin generation (TG), and clot-based assays. Results: WT and D60A demonstrated similar FVIIa amidolytic activity. However, D60A showed approximately 50% activity on FX activation and significantly longer lag time in the TG assay than that shown by WT. The clotting time produced by D60A spiked in FVII-deficient plasma was significantly prolonged than that of WT. Additionally, the ex vivo plasma half-lives of WT and D60A were comparable. Conclusion: D60A demonstrated lower coagulant activities, most likely due to the weakening of FX binding, leading to impaired FX activation and delayed TG and fibrin formation. Considering that a plasma FVII level of 15% to 25% is adequate for normal hemostasis, D60A is a molecule of interest for future development of an rFVII with a lesser extent of thrombogenicity.

FTO overexpression expedites wound healing and alleviates depression in burn rats through facilitating keratinocyte migration and angiogenesis via mediating TFPI-2 demethylation.

Burn injury is a serious traumatic injury that leads to severe physical and psychosocial impairment. Wound healing after burn injury is a substantial challenge in medical community. This study investigated the biological effects of the demethylase fat mass and obesity-associated protein (FTO) on burn injury. FTO protein level in burn skin tissues of patients was measured with Western blot assay. Keratinocytes (HaCaT cells) were given heat stimulation to induce an in vitro burn injury model, and then transfected with overexpression plasmids of FTO (pcDNA-FTO) or small interfering RNA against FTO (si-FTO). Cell proliferation, migration, and angiogenesis in keratinocytes were evaluated with CCK-8, Transwell, and tube formation assays, respectively. Tissue factor pathway inhibitor-2 (TFPI-2) m6A methylation level was detected with MeRIP­qPCR assay. Then rescue experiments were conducted to explore the effects of FTO/TFPI-2 axis on keratinocyte functions. Lentivirus carrying FTO overexpression plasmids was injected into a burn rat model to detect its effects on wound healing and depressive-like behaviors in burn rats. FTO was downregulated in burn skin and heat-stimulated keratinocytes. FTO prominently augmented proliferation, migration and angiogenesis in heat-stimulated keratinocytes, while FTO knockdown showed the opposite results. FTO inhibited TFPI-2 expression by FTO-mediated m6A methylation modification. TFPI-2 overexpression abrogated FTO mediated enhancement of proliferation, migration and angiogenesis in keratinocytes. Additionally, FTO overexpression accelerated wound healing and improved depressive-like behaviors in burn rat model. FTO prominently augmented proliferation, migration and angiogenesis in heat-stimulated keratinocytes though inhibiting TFPI-2, and then improved wound healing and depressive-like behaviors.

Curcumin inhibits the invasion and migration of pancreatic cancer cells by upregulating TFPI-2 to regulate ERK- and JNK-mediated epithelial-mesenchymal transition.

PURPOSE: Pancreatic cancer (PC) is one of the most deadly human malignancies. Curcumin is a natural polyphenolic compound with wide-ranging pharmacological effects. Growing evidence suggests that curcumin has anticancer activity against PC, but the mechanism remains incompletely elucidated. This study aimed to investigate the effects and mechanisms of curcumin on the invasion and migration of PC cells. METHODS: Effect of curcumin on tissue factor pathway inhibitor (TFPI)-2 mRNA expression in PC cells was initially identified using qRT-PCR. Cytotoxicity of curcumin was assessed with MTT assays and IC50 was calculated. Involvement of ERK and JNK pathways, as well as protein expression of TFPI-2 and epithelial-mesenchymal transition (EMT)-related markers, were detected using immunoblotting. Invasion and migration of PC cells were examined using Transwell assays. TFPI-2 expression was manipulated by transfection with siRNA and shRNA. Rescue assays were used to validate the effect of curcumin on cell invasion and migration via TFPI-2. RESULTS: Curcumin increased the expression of TFPI-2 mRNA and protein in PC cells and attenuated cell invasion and migration. Curcumin also inhibited ERK and JNK pathways and EMT in PC cells. Knockdown of TFPI-2 partially reversed the inhibition of ERK and JNK pathways and EMT by curcumin. Mechanistically, curcumin upregulated TFPI-2, thereby inhibiting the ERK and JNK pathways, leading to the inhibition of EMT in PC cells. CONCLUSION: Collectively, curcumin inhibits ERK- and JNK-mediated EMT through upregulating TFPI-2, which in turn suppresses the migration and invasion of PC cells. These findings provide new insights into the antitumor mechanism of curcumin.

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation.

INTRODUCTION: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. MATERIALS AND METHODS: We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. RESULTS: In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. CONCLUSIONS: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.

SARS-CoV-2 Spike Proteins and Cell-Cell Communication Induce P-Selectin and Markers of Endothelial Injury, NETosis, and Inflammation in Human Lung Microvascular Endothelial Cells and Neutrophils: Implications for the Pathogenesis of COVID-19 Coagulopathy.

COVID-19 progression often involves severe lung injury, inflammation, coagulopathy, and leukocyte infiltration into pulmonary tissues. The pathogenesis of these complications is unknown. Because vascular endothelium and neutrophils express angiotensin-converting enzyme-2 and spike (S)-proteins, which are present in bodily fluids and tissues of SARS-CoV-2-infected patients, we investigated the effect of S-proteins and cell-cell communication on human lung microvascular endothelial cells and neutrophils expression of P-selectin, markers of coagulopathy, NETosis, and inflammation. Exposure of endothelial cells or neutrophils to S-proteins and endothelial-neutrophils co-culture induced P-selectin transcription and expression, significantly increased expression/secretion of IL-6, von Willebrand factor (vWF, pro-coagulant), and citrullinated histone H3 (cit-H3, NETosis marker). Compared to the SARS-CoV-2 Wuhan variant, Delta variant S-proteins induced 1.4-15-fold higher P-selectin and higher IL-6 and vWF. Recombinant tissue factor pathway inhibitor (rTFPI), 5,5'-dithio-bis-(2-nitrobenzoic acid) (thiol blocker), and thrombomodulin (anticoagulant) blocked S-protein-induced vWF, IL-6, and cit-H3. This suggests that following SARS-CoV-2 contact with the pulmonary endothelium or neutrophils and endothelial-neutrophil interactions, S-proteins increase adhesion molecules, induce endothelial injury, inflammation, NETosis and coagulopathy via the tissue factor pathway, mechanisms involving functional thiol groups, and/or the fibrinolysis system. Using rTFPI, effectors of the fibrinolysis system and/or thiol-based drugs could be viable therapeutic strategies against SARS-CoV-2-induced endothelial injury, inflammation, NETosis, and coagulopathy.

Downregulated DUXAP8 lncRNA impedes trophoblast cell proliferation and migration by epigenetically upregulating TFPI2 expression.

BACKGROUND: Preeclampsia (PE), a pregnancy complication characterized by new-onset hypertension and proteinuria during the second trimester, is the leading cause of neonatal and maternal morbidity and mortality. In the etiology of PE, failure of uterine spiral artery remodeling may be related to functioning abnormally of trophoblast cells, leading to the occurrence and progression of PE. Recently, long noncoding RNAs (lncRNAs) have been reported to play critical roles in PE nowadays. This study aimed to investigate the expression and functions of the TFPI2 pathway-related lncRNA DUXAP8. METHODS: DUXAP8 expression in the placenta from pregnancies was examined using qPCR. Then, the in vitro functions of DUXAP8 were investigated through MTT, EdU, colony, transwell, and flow cytometry experiments. The downstream gene expression profiles were assessed using RNA transcriptome sequencing analysis and verified using qPCR and western blot. Furthermore, Immunoprecipitation (RIP), chromatin immunoprecipitation (CHIP) and fluorescence in situ hybridization (FISH) were used to detect the interaction between lncDUXAP8/EZH2/TFPI2. RESULTS: The expression of lncRNA DUXAP8 in placenta of patients with eclampsia was significantly decreased. After knockout of DUXAP8, the proliferation and migration of trophoblasts were significantly decreased, and the percentage of apoptosis was increased. Flow cytometry showed that low expression of DUXAP8 increased the accumulation of cells in G2/M phase, while overexpression of DUXAP8 had the opposite effect. We also proved that DUXAP8 epigenetically inhibited TFPI2 expression by recruiting EZH2 and mediating H3K27me3 modification. CONCLUSION: Together, these resulting data clarify that aberrant expression of DUXAP8 is involved in the potential PE development and progress. Unraveling the role of DUXAP8 will provide novel insights into the pathogenesis of PE.

Single-cell RNA sequencing reveals a mechanism underlying the susceptibility of the left atrial appendage to intracardiac thrombogenesis during atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is associated with an increased risk of thrombosis of the left atrial appendage (LAA). However, the molecular mechanisms underlying this site-specificity remain poorly understood. Here, we present a comparative single-cell transcriptional profile of paired atrial appendages from patients with AF and illustrate the chamber-specific properties of the main cell types. METHODS: Single-cell RNA sequencing analysis of matched atrial appendage samples from three patients with persistent AF was evaluated by 10× genomics. The AF mice model was created using Tbx5 knockout mice. Validation experiments were performed by glutathione S-transferase pull-down assays, coimmunoprecipitation (Co-IP), cleavage assays and shear stress experiments in vitro. RESULTS: In LAA, phenotype switching from endothelial cells to fibroblasts and inflammation associated with proinflammatory macrophage infiltration were observed. Importantly, the coagulation cascade is highly enriched in LAA endocardial endothelial cells (EECs), accompanying the up-regulation of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) and the down-regulation of the tissue factor pathway inhibitor (TFPI) and TFPI2. Similar alterations were verified in an AF mouse model (Tbx5+/- ) and EECs treated with simulated AF shear stress in vitro. Furthermore, we revealed that the cleavage of both TFPI and TFPI2 based on their interaction with ADAMTS1 would lead to loss of anticoagulant activities of EECs. CONCLUSIONS: This study highlights the decrease in the anticoagulant status of EECs in LAA as a potential mechanism underlying the propensity for thrombosis, which may aid the development of anticoagulation therapeutic approaches targeting functionally distinct cell subsets or molecules during AF.

Endothelial-dependent S-Sulfhydration of tissue factor pathway inhibitor regulates blood coagulation.

Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. Here we investigated whether endothelial cell-driven oxidative post-translational modifications could have an impact on TFPI activity. We focused on S-sulfhydration, which is a hydrogen sulfide-dependent post-translational modification that, in endothelial cells, is regulated by the enzyme cystathionine γ-lyase (CSE). The study made use of human primary endothelial cells and blood from healthy individuals or subjects with atherosclerosis as well as from mice lacking endothelial CSE. TFPI was S-sulfhydrated in endothelial cells from healthy individuals and mice, while the loss of endothelial CSE expression/activity reduced its modification. Non-S-sulfhydrated TFPI was no longer able to interact with factor Xa, which facilitated the activation of tissue factor. Similarly, non-S-sulfhydratable TFPI mutants bound less protein S, while supplementation with hydrogen sulfide donors, preserved TFPI activity. Phenotypically, loss of TFPI S-sulfhydration increased clot retraction, suggesting that this post-translational modification is a new endothelial cell-dependent mechanism that contributes to the regulation of blood coagulation.

Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Natural anticoagulant discovery, the gift that keeps on giving: finding FV-Short.

The complex reactions of blood coagulation are balanced by several natural anticoagulants resulting in tuned hemostasis. During several decades, the knowledge base of the natural anticoagulants has greatly increased and we have also learned about antiinflammatory and cytoprotective activities expressed by antithrombin and activated protein C (APC). Some coagulation proteins have also been found to function as anticoagulants; e.g., thrombin when bound to thrombomodulin activates protein C. Another example is factor V (FV), which in addition to being a procofactor to FVa has emerged as an anticoagulant. The discovery of APC resistance, caused by FVLeiden, as a thrombosis risk factor resulted in the identification of FV as an APC cofactor working in synergy with protein S in the regulation of FVIIIa in the Xase complex. More recently, a natural anticoagulant FV splice isoform (FV-Short) was discovered when investigating the East Texas bleeding disorder. In FV-Short, the truncated B domain exposes a high-affinity binding site for tissue factor pathway inhibitor alpha (TFPIα), and together with protein S a high-affinity trimolecular complex is generated. The FXa-inhibitory activity of TFPIα is synergistically stimulated by FV-Short and protein S. The circulating FV-Short/protein S/TFPIα complex concentration is normally low (≈0.2 nM) but provides an anticoagulant threshold. In the East Texas bleeding, the concentration of the complex, and thus the threshold, is increased 10-fold, which results in bleeding manifestations. The anticoagulant properties of FV were discovered during investigations of individual patients and follow the great tradition of bed-to-bench and bench-to-bed research in the coagulation field.

Integrative analyses of RNA-seq and ChIP-seq Reveal MITF as a Target Gene of TFPI-2 in MDA231 Cells.

Breast cancer is the most prevalent cancer in female patients worldwide. Tissue factor pathway inhibitor 2 (TFPI-2) is identified as an important tumor suppressor in various cancers. Recent studies have shown that TFPI-2 translocates into the nucleus, where it modulates the transcription of the matrix metalloproteinase-2 (MMP-2) gene. However, its biological role and molecular mechanisms in the progression of breast cancer remain unclear. In this study, we identified 5125 differentially expressed genes (DEGs) from RNA sequencing (RNA-seq) in TFPI-2-overexpressing MDA231 cells compared with control cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) analysis shown that cell cycle, cell differentiation, proteoglycans in cancer, and pathways associated with cancer were highly enriched in downregulated DEGs. Integration of the RNA-seq and ChIP-sequencing (ChIP-seq) data identified 73 genes directly controlled by TFPI-2 in MDA231 cells. Among them, melanocyte inducing transcription factor (MITF) gene expression was repressed by TFPI-2, which was further verified by a luciferase reporter assay and ChIP-quantitative PCR. Our study provides evidence of a novel role of TFPI-2 in human breast cancer involving targeting of the MITF.