Metabolic landscape in venous thrombosis: insights into molecular biology and therapeutic implications.

The findings of the last decade suggest a complex link between inflammatory cells, coagulation, and the activation of platelets and their synergistic interaction to promote venous thrombosis. Inflammation is present throughout the process of venous thrombosis, and various metabolic pathways of erythrocytes, endothelial cells, and immune cells involved in venous thrombosis, including glucose metabolism, lipid metabolism, homocysteine metabolism, and oxidative stress, are associated with inflammation. While the metabolic microenvironment has been identified as a marker of malignancy, recent studies have revealed that for cancer thrombosis, alterations in the metabolic microenvironment appear to also be a potential risk. In this review, we discuss how the synergy between metabolism and thrombosis drives thrombotic disease. We also explore the great potential of anti-inflammatory strategies targeting venous thrombosis and the complex link between anti-inflammation and metabolism. Furthermore, we suggest how we can use our existing knowledge to reduce the risk of venous thrombosis.

Regulation of macrophage fibrinolysis during venous thrombus resolution.

BACKGROUND: Venous thromboembolism (VTE), which includes pulmonary embolism (PE) and deep vein thrombosis (DVT), is a serious cardiovascular disease with significant mortality and morbidity. Clinically, patients with faster resolution of a venous thrombi have improved prognosis. Urokinase-plasminogen activator (uPA), produced by macrophages, is a key mediator of fibrinolysis required for resolving venous thrombi and restoring vascular integrity. The major macrophage protein, plasminogen activator inhibitor type-2 (PAI-2), was originally identified as an inhibitor of uPA and is implicated in the modulation of pathways affecting fibrinolytic uPA activity, however its direct role in blocking uPA-mediated clot lysis is not known. OBJECTIVE: To determine the contribution of macrophage PAI-2 in inhibiting uPA-mediated fibrinolysis during resolution of DVT. METHODS: Using a murine model of venous thrombosis and resolution, we determined histological changes and molecular features of fibrin degradation in venous thrombi from WT mice and mice genetically deficient in PAI-2 and PAI-1, and determined the fibrinolytic activities of macrophages from these genotypes ex vivo. RESULTS: Acceleration of venous thrombus resolution by PAI-2-/- mice increases fibrin degradation in venous thrombi showing a pattern similar to genetic deficiency of PAI-1, the major attenuator of fibrinolysis. PAI-2 deficiency was not associated with increased macrophage infiltration into thrombi or changes in macrophage PAI-1 expression. uPA-initiated fibrinolysis by macrophages in vitro could be accelerated by PAI-1 deficiency, but not PAI-2 deficiency. CONCLUSION: PAI-2 has an alternate anti-fibrinolytic activity that is macrophage uPA independent, where PAI-1 is the dominant uPA inhibitor during DVT resolution.

GSDMD-Dependent Neutrophil Extracellular Traps Mediate Portal Vein Thrombosis and Associated Fibrosis in Cirrhosis.

Portal vein thrombosis (PVT) is a challenging and controversial complication of cirrhosis. Experimental models that reproduce cirrhotic PVT and effective pharmacological therapies are limited. We aimed to investigate the nature course and mechanisms of PVT in cirrhosis. A novel PVT model was developed via two-step total portal vein ligation in healthy and thioacetamide (TAA)-cirrhotic rats. Circulating and liver-infiltrating neutrophils were isolated from individuals with cirrhosis to examine neutrophil extracellular traps (NETs) and explore their unique characteristics and implications in PVT-associated fibrosis in cirrhosis. We further validated macrophage-myofibroblast transition (MMT) via multiplex immunofluorescence and single-cell sequencing. In the experimental model, cirrhosis promoted PVT development and portal vein intimal thickening. Interestingly, cirrhosis promoted spontaneous resolution of PVT due to instability of thrombus structure, along with pulmonary and intrahepatic clots. NETs-MMT mediate cirrhotic PVT and PVT-associated fibrosis, including fibrotic thrombus remodeling and increased hepatic collagen deposition. Mechanistically, caspase-4-dependent activation of neutrophils and GSDMD mediated the formation of NETs. The extracellular DNA of NETs promoted TGF-ß1/Smad3-driven MMT. Inhibiting GSDMD with disulfiram suppressed cirrhotic PVT and prevented associated fibrosis. The cirrhotic PVT model reflected the following three main characteristics of cirrhotic PVT: spontaneous resolution, immunothrombosis, and intimal fibrosis. Targeting NETs with GSDMD inhibitors may serve as a new therapeutic concept to treat cirrhotic PVT.

Monocyte/macrophage-mediated venous thrombus resolution.

Venous thromboembolism (VTE) poses a notable risk of morbidity and mortality. The natural resolution of the venous thrombus might be a potential alternative treatment strategy for VTE. Monocytes/macrophages merge as pivotal cell types in the gradual resolution of the thrombus. In this review, the vital role of macrophages in inducing inflammatory response, augmenting neovascularization, and facilitating the degradation of fibrin and collagen during thrombus resolution was described. The two phenotypes of macrophages involved in thrombus resolution and their dual functions were discussed. Macrophages expressing various factors, including cytokines and their receptors, adhesion molecules, chemokine receptors, vascular endothelial growth factor receptors, profibrinolytic- or antifibrinolytic-related enzymes, and other elements, are explored for their potential to promote or attenuate thrombus resolution. Furthermore, this review provides a comprehensive summary of new and promising therapeutic candidate drugs associated with monocytes/macrophages that have been demonstrated to promote or impair thrombus resolution. However, further clinical trials are essential to validate their efficacy in VTE therapy.

Expression of fibroblast activation protein-α in human deep vein thrombosis.

BACKGROUND: Fibroblast activation protein-α (FAP), a type-II transmembrane serine protease, is associated with wound healing, cancer-associated fibroblasts, and chronic fibrosing diseases. However, its expression in deep vein thrombosis (DVT) remains unclear. Therefore, this study investigated FAP expression and localization in DVT. METHODS: We performed pathological analyses of the aspirated thrombi of patients with DVT (n = 14), classifying thrombotic areas in terms of fresh, cellular lysis, and organizing reaction components. The organizing reaction included endothelialization and fibroblastic reaction. We immunohistochemically examined FAP-expressed areas and cells, and finally analyzed FAP expression in cultured dermal fibroblasts. RESULTS: All the aspirated thrombi showed a heterogeneous mixture of at least two of the three thrombotic areas. Specifically, 83 % of aspirated thrombi showed fresh and organizing reaction components. Immunohistochemical expression of FAP was restricted to the organizing area. Double immunofluorescence staining showed that FAP in the thrombi was mainly expressed in vimentin-positive or α-smooth muscle actin-positive fibroblasts. Some CD163-positive macrophages expressed FAP. FAP mRNA and protein levels were higher in fibroblasts with low-proliferative activity cultured under 0.1 % fetal bovine serum (FBS) than that under 10 % FBS. Fibroblasts cultured in 10 % FBS showed a significant decrease in FAP mRNA levels following supplementation with hemin, but not with thrombin. CONCLUSIONS: The heterogeneous composition of venous thrombi suggests a multistep thrombus formation process in human DVT. Further, fibroblasts or myofibroblasts may express FAP during the organizing process. FAP expression may be higher in fibroblasts with low proliferative activity.

Enhancement of endothelial function and attenuation of portal vein injury using mesenchymal stem cells carrying miRNA-25-3p.

The aims of this study were to determine whether human umbilical cord mesenchymal stem cells (hucMSCs) modified by miRNA-25-3p (miR-25-3p) overexpression could promote venous endothelial cell proliferation and attenuate portal endothelial cell injury. HucMSCs and human umbilical vein endothelial cells (HUVEC) were isolated and cultured from human umbilical cord and characterized. Lentiviral vectors expressing miRNA-25-3p were transfected into hucMSCs and confirmed by PCR. We verified the effect of miR-25-3p-modified hucMSCs on HUVEC by cell co-culture and cell supernatant experiments. Subsequently, exosomes of miR-25-3p-modified hucMSCs were isolated from cell culture supernatants and characterized by WB, NTA and TEM. We verified the effects of miR-25-3p-modified exosomes derived from hucMSCs on HUVEC proliferation, migration, and angiogenesis by in vitro cellular function experiments. Meanwhile, we further examined the downstream target genes and signaling pathways potentially affected by miR-25-3p-modified hucMSC-derived exosomes in HUVEC. Finally, we established a rat portal vein venous thrombosis model by injecting CM-DiR-labeled hucMSCs intravenously into rats and examining the homing of cells in the portal vein by fluorescence microscopy. Histological and immunohistochemical experiments were used to examine the effects of miRNA-25-3p-modified hucMSCs on the proliferation and damage of portal vein endothelial cells. Primary hucMSCs and HUVECs were successfully isolated, cultured and characterized. Primary hucMSCs were modified with a lentiviral vector carrying miR-25-3p at MOI 80. Co-culture and cell supernatant intervention experiments showed that overexpression of miRNA-25-3p in hucMSCs enhanced HUVEC proliferation, migration and tube formation in vitro. We successfully isolated and characterized exosomes of miR-25-3p-modified hucMSCs, and exosome intervention experiments demonstrated that miR-25-3p-modified exosomes derived from hucMSCs similarly enhanced the proliferation, migration, and angiogenesis of HUVECs. Subsequent PCR and WB analyses indicated PTEN/KLF4/AKT/ERK1/2 as potential pathways of action. Analysis in a rat portal vein thrombosis model showed that miR-25-3p-modified hucMSCs could homing to damaged portal veins. Subsequent histological and immunohistochemical examinations demonstrated that intervention with miR-25-3p overexpression-modified hucMSCs significantly reduced damage and attenuated thrombosis in rat portal veins. The above findings indicate suggest that hucMSCs based on miR-25-3p modification may be a promising therapeutic approach for use in venous thrombotic diseases.

Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice.

ABSTRACT: Neutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NET formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux from oxidative phosphorylation toward aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4-/-) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava-stenosis model at day 2 after surgery. Compared with wild-type (WT) mice, the venous thrombus obtained from PDK2/4-/- mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)-stimulated PDK2/4-/- neutrophils displayed reduced NETosis and secretion of cathepsin G and elastase compared with PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4-/- neutrophils were significantly reduced compared with PMA-stimulated WT neutrophils. Finally, PDK2/4-/- neutrophils exhibited reduced levels of intracellular Ca2+ concentration, extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and glycolytic proton efflux rate (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate, to our knowledge, for the first time, the fundamental role of PDK2/4 in regulating NETosis and acute DVT.

Preliminary clinical analysis and pathway study of S100A8 as a biomarker for the diagnosis of acute deep vein thrombosis.

Herein, we aimed to identify blood biomarkers that compensate for the poor specificity of D-dimer in the diagnosis of deep vein thrombosis (DVT). S100A8 was identified by conducting protein microarray analysis of blood samples from patients with and without DVT. We used ELISA to detect S100A8, VCAM-1, and ICAM-1 expression levels in human blood and evaluated their correlations. Additionally, we employed human recombinant protein S100A8 to induce human umbilical vein endothelial cells and examined the role of the TLR4/MAPK/VCAM-1 and ICAM-1 signaling axes in the pathogenic mechanism of S100A8. Simultaneously, we constructed a rat model of thrombosis induced by inferior vena cava stenosis and detected levels of S100A8, VCAM-1, and ICAM-1 in the blood of DVT rats using ELISA. The associations of thrombus tissue, neutrophils, and CD68-positive cells with S100A8 and p38MAPK, TLR4, and VCAM-1 expression levels in vein walls were explored. The results revealed that blood S100A8 was significantly upregulated during the acute phase of DVT and activated p38MAPK expression by combining with TLR4 to enhance the expression and secretion of VCAM-1 and ICAM-1, thereby affecting the occurrence and development of DVT. Therefore, S100A8 could be a potential biomarker for early diagnosis and screening of DVT.

PDIA2 has a dual function in promoting androgen deprivation therapy induced venous thrombosis events and castrate resistant prostate cancer progression.

Androgen deprivation therapy (ADT) is the first line of treatment for metastatic prostate cancer (PCa) that effectively delays the tumor progression. However, it also increases the risk of venous thrombosis event (VTE) in patients, a leading cause of mortality. How a pro-thrombotic cascade is induced by ADT remains poorly understood. Here, we report that protein disulfide isomerase A2 (PDIA2) is upregulated in PCa cells to promote VTE formation and enhance PCa cells resistant to ADT. Using various in vitro and in vivo models, we demonstrated a dual function of PDIA2 that enhances tumor-mediated pro-coagulation activity via tumor-derived extracellular vehicles (EVs). It also stimulates PCa cell proliferation, colony formation, and xenograft growth androgen-independently. Mechanistically, PDIA2 activates the tissue factor (TF) on EVs through its isomerase activity, which subsequently triggers a pro-thrombotic cascade in the blood. Additionally, TF-containing EVs can activate the Src kinase inside PCa cells to enhance the AR signaling ligand independently. Androgen deprivation does not alter PDIA2 expression in PCa cells but enhances PDIA2 translocation to the cell membrane and EVs via suppressing the clathrin-dependent endocytic process. Co-recruitment of AR and FOXA1 to the PDIA2 promoter is required for PDIA2 transcription under androgen-deprived conditions. Importantly, blocking PDIA2 isomerase activity suppresses the pro-coagulation activity of patient plasma, PCa cell, and xenograft samples as well as castrate-resistant PCa xenograft growth. These results demonstrate that PDIA2 promotes VTE and tumor progression via activating TF from tumor-derived EVs. They rationalize pharmacological inhibition of PDIA2 to suppress ADT-induced VTE and castrate-resistant tumor progression.

Mucin 1 and venous thrombosis in tumor-bearing mice and patients with cancer.

INTRODUCTION: Mucins released from epithelial tumors have been proposed to play a role in cancer-associated thrombosis. Mucin1 (MUC1) is a transmembrane mucin that is overexpressed in a variety of human malignancies, including breast and pancreatic cancer. We analyzed the association of MUC1 and venous thrombosis in a mouse tumor model and in patients with cancer. MATERIALS AND METHODS: We used a human pancreatic cancer cell line HPAF-II that expresses a high level of MUC1. We grew HPAF-II tumors in the pancreas of Crl:NU-Foxn1nu male mice. MUC1 in plasma and extracellular vesicles (EVs) isolated from plasma was measured using an enzyme-linked immunosorbent assay. MUC1 in EVs and venous thrombi from tumor-bearing mice was assessed by western blotting. We measured MUC1 in plasma from healthy controls and patients with stomach, colorectal or pancreatic cancer with or without venous thromboembolism. RESULTS AND DISCUSSION: MUC1 was detected in the plasma of mice bearing HPAF-II tumors and was associated with EVs. MUC1 was present in venous thrombi from mice bearing HFAP-II tumors. Recombinant MUC1 did not induce platelet aggregation. Levels of MUC1 were higher in patients with pancreatic cancer compared with healthy controls. In contrast to the mouse model, MUC1 was present in EV-free plasma in samples from healthy controls and patients with cancer. There was no significant difference in the levels of MUC1 in cancer patients with or without VTE. Our data did not find any evidence that MUC1 contributed to VTE in patients with cancer.

Astragaloside IV induces endothelial progenitor cell angiogenesis in deep venous thrombosis through inactivation of PI3K/AKT signaling.

BACKGROUND: Deep vein thrombosis (DVT), referred to as venous thromboembolism, is the third most frequent cardiovascular disease. Endothelial progenitor cells (EPCs) contribute to the recanalization of DVT. Astragaloside IV (AS-IV) has been suggested to have angiogenesis-enhancing effects. Here, we investigate the roles and mechanisms of AS-IV in EPCs and DVT. METHODS: The experimental DVT model was established by inferior vena cava stenosis in rats. EPCs were collected from patients with DVT. Transwell assays were performed to detect cell migration. Tube formation was determined using Matrigel basement membrane matrix and ImageJ software. The thrombus weight and length were measured. Pathological changes were examined by hematoxylin-eosin staining. The production of proinflammatory cytokines was estimated by ELISA. The level of PI3K/AKT-related proteins was measured by western blotting. RESULTS: AS-IV administration facilitated the migrative and angiogenic functions of human EPCs in vitro. Additionally, AS-IV inhibited thrombosis and repressed the infiltration of leukocytes into the thrombus and the production of proinflammatory cytokines in rats. Mechanistically, AS-IV inactivated PI3K/AKT signaling in rats. CONCLUSION: AS-IV prevents thrombus in an experimental DVT model by facilitating EPC angiogenesis and decreasing inflammation through inactivation of PI3K/AKT signaling.

Platelets and neutrophils cooperate to induce increased neutrophil extracellular trap formation in JAK2V617F myeloproliferative neoplasms.

BACKGROUND: Neutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. OBJECTIVES: This study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. METHODS: NETosis was studied in PF4iCre;Jak2V617F/WT mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2V617F/WT mice in which JAK2V617F is expressed only in leukocytes. RESULTS: In PF4iCre;Jak2V617F/WT mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2V617F/WT mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2V617F/WT mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. CONCLUSION: Our study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified.

Platelet-Derived TGF-ß1 Promotes Deep Vein Thrombosis.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) modulates multiple cellular functions during development and tissue homeostasis. A large amount of TGF-ß1 is stored in platelet α-granules and released upon platelet activation. Whether platelet-derived TGF-ß1 plays a role in venous thrombosis remains unclear. This study intends to assess the role of platelet-derived TGF-ß1 in the development of venous thrombosis in mice. MATERIAL AND METHODS: TGF-ß1flox/flox and platelet-specific TGF-ß1-/- mice were utilized to assess platelet function in vitro, arterial thrombosis induced by FeCl3, tail bleeding time, prothrombin time (PT), activated partial thromboplastin time (APTT), and deep vein thrombosis induced through ligation of the inferior vena cava (IVC). The IVC sample was collected to measure accumulation of neutrophils, monocytes, and the formation of neutrophil extracellular traps (NETs) by immunofluorescence staining. RESULTS: TGF-ß1 deficiency in platelets did not affect the number of circulating platelets, platelet aggregation, adenosine triphosphate release, and integrin αIIbß3 activation. Meanwhile, TGF-ß1 deficiency did not alter the arterial thrombus formation, hemostasis, and coagulation time (PT and APTT), but significantly impaired venous thrombus formation, inhibited the recruitment and accumulation of neutrophils and monocytes in thrombi, as well as reduced formation of NETs and platelet-neutrophil complex. In addition, adoptive transfer of TGF-ß1flox/flox platelets to TGF-ß1-/- mice rescued the impaired venous thrombus formation, recruitment of leukocytes and monocytes, as well as the NETs formation. CONCLUSION: In conclusion, platelet-derived TGF-ß1 positively modulates venous thrombus formation in mice, indicating that targeting TGF-ß1 might be a novel approach for treating venous thrombosis without increasing the risk of bleeding.

ADAMTS13 inhibits H2O2-induced human venous endothelial cell injury to attenuate deep-vein thrombosis by blocking the p38/ERK signaling pathway.

Deep vein thrombosis (DVT) is a common complication in hematologic malignancies and immunologic disorders. Endothelial cell injury and dysfunction comprise the critical contributor for the development of DVT. A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13), a plasma metalloprotease that cleaves von Willebrand factor, acts as a critical regulator in normal hemostasis. This study was aimed to explore the role of ADAMTS13 in endothelial cell injury during DVT and the possible mechanism. First, human umbilical vein endothelial cells (HUVECs) were exposed to hydrogen peroxide (H2O2). Then, the mRNA and protein expressions of ADAMTS13 were evaluated with the reverse transcription-quantitative polymerase chain reaction and western blot. After treatment with recombinant ADAMTS13 (rADAMTS13; rA13), the viability and apoptosis of H2O2-induced HUVECs were assessed by cell counting kit-8 assay and terminal-deoxynucleoitidyl transferase-mediated nick end labeling staining. In addition, the levels of prostaglandin F1-alpha, endothelin-1, and reactive oxygen species were detected using the enzyme-linked immunosorbent assay and dichloro-dihydro-fluorescein diacetate assay. The expressions of proteins related to p38/extracellular signal-regulated kinase (ERK) signaling pathway were estimated with the western blot. Then, p79350 (p38 agonist) was used to pretreat cells to analyze the regulatory effects of rA13 on p38/ERK signaling in H2O2-induced HUVEC injury. The results revealed that ADAMTS13 expression was significantly downregulated in H2O2-induced HUVECs. The reduced viability and increased apoptosis of HUVECs induced by H2O2 were revived by ADAMTS13. ADAMTS13 also suppressed the oxidative stress in HUVECs after H2O2 treatment. Besides, ADAMTS13 was found to block p38/ERK signaling pathway, and p79350 reversed the impacts of ADAMTS13 on the damage of HUVECs induced by H2O2. To sum up, ADAMTS13 could alleviate H2O2-induced HUVEC injury through the inhibition of p38/ERK signaling pathway.

Crocin improves lower extremity deep venous thrombosis by regulating the PIM1/FOXO3a axis.

Lower extremity deep venous thrombosis (LEDVT) has a high incidence and mortality. Crocin has the potential to ameliorate thrombosis. The study aimed to clarify whether crocin affects LEDVT. Human umbilical vein endothelial cells (HUVECs) were exposed to thrombin and crocin (0, 5, 10, 20, 40, and 80 µM). Cell viability was assessed by MTT assay. Cellular behaviors were assessed using flow cytometry, TUNEL assay, and tube formation assay. The binding relationship between crocin and PIM1 was analyzed by molecular docking. The underlying mechanism of PIM1 was determined by reverse transcription-quantitative PCR, dual-luciferase reporter assay, and RIP. We found that crocin (5, 10, 20, and 40 µM) promoted thrombin-treated HUVEC viability in a dose-dependent manner. Crocin inhibited apoptosis and promoted the angiogenesis of HUVECs induced by thrombin. PIM1 was a target of crocin and was upregulated in patients with LEDVT and thrombin-treated cells. Foxo3a could interact with PIM1 and positively related to PIM1 expression. Moreover, the knockdown of PIM1 suppressed apoptosis and promoted angiogenesis in thrombin-HUVECs treated with crocin, while overexpression of Foxo3a reversed the effects. In conclusion, crocin inhibited apoptosis and promoted the angiogenesis of HUVECs induced by thrombin via the PIM1/Foxo3a axis, suggesting that crocin may be effective for LEDVT therapy.

Cancer-associated fibroblasts promote venous thrombosis through podoplanin/CLEC-2 interaction in podoplanin-negative lung cancer mouse model.

BACKGROUND: Cancer-associated thrombosis (CAT) is the leading cause of morbidity and mortality. Cancer-associated fibroblasts (CAFs) are a prominent component of the tumor microenvironment that contributes to cancer progression through direct cell-cell interactions and the release of extracellular vesicles (EVs). However, the role of CAFs in CAT remains unclear. OBJECTIVE: This study aims to investigate whether CAFs aggravate CAT and the underlying molecular mechanism using a preclinical mouse lung cancer model. METHODS: We designed a Lewis lung carcinoma (LLC) tumor-bearing mouse model. CAFs were characterized using fluorescence immunohistostaining. The presence of podoplanin, a platelet-activating membrane protein through C-type lectin-like receptor 2 (CLEC-2), in EVs isolated from primary CAFs or LLC tumor tissues was assessed by immunoblotting. The platelet activation and aggregation abilities of the EVs were quantified using flow cytometry. Podoplanin plasma levels were measured by enzyme-linked immunosorbent assay. Venous thrombosis was induced in the femoral vein using 2.5% ferric chloride. The anti-CLEC-2 monoclonal antibody 2A2B10 was used to deplete CLEC-2 on the surface of the platelets. RESULTS: CAFs expressing CD90, PDGFRß, HSP47, CD34, and vimentin, co-expressed podoplanin and induced platelet activation and aggregation in a CLEC-2-dependent manner. Tumor-bearing mice showed elevated podoplanin plasma levels. CAF-EV injection and tumor-bearing mice showed shorter occlusion time in the venous thrombosis model. Although tumor growth was not altered, antibody-induced CLEC-2 depletion suppressed venous thrombosis in the tumor-bearing state but not in the healthy condition. CONCLUSION: CAFs and CAF-derived EVs induce CLEC-2-dependent platelet aggregation and aggravate venous thrombosis.

Downregulation of miR-125a-5p Promotes Endothelial Progenitor Cell Migration and Angiogenesis and Alleviates Deep Vein Thrombosis in Mice Via Upregulation of MCL-1.

Endothelial progenitor cells (EPCs) contribute to recanalization of deep vein thrombosis (DVT). MicroRNAs (miRNAs) play regulatory roles in functions of EPCs, which is becoming a promising therapeutic choice for thrombus resolution. The main purpose of this study was to explore the effect of miR-125a-5p on EPC functions in deep vein thrombosis (DVT). EPCs were isolated from the peripheral blood of patients with DVT. In DVT mouse models, DVT was induced by stenosis of the inferior vena cava (IVC). The levels of miR-125a-5p and myeloid cell leukemia sequence 1 (MCL-1) in EPCs and thrombi of DVT mice were detected by RT-qPCR. EPC migration, angiogenesis, and apoptosis were estimated by Transwell assay, tube formation assay, and flow cytometry analysis. Luciferase reporter assay was utilized for detecting the binding of miR-125a-5p and MCL-1. The phosphorylation of PI3K and AKT was estimated by western blot. DVT formation in vivo was observed through hematoxylin-eosin (H&E) staining. The expression of thrombus resolution marker, CD34 molecule (CD34), in the thrombi was measured by immunofluorescence staining. MiR-125a-5p upregulation repressed EPC migration and angiogenesis and facilitated apoptosis. MiR-125a-5p downregulation showed the opposite effect. MCL-1 was targeted and negatively regulated by miR-125a-5p. Additionally, miR-125a-5p inhibited the PI3K/AKT pathway in EPCs. Inhibition of MCL-1 or PI3K/AKT pathway reversed the effect of miR-125a-5p knockdown on EPC functions. The in vivo experiments revealed that miR-125a-5p downregulation repressed thrombus formation and promoted the homing capability of EPCs to the thrombosis site, thereby alleviating DVT mice. Downregulation of miR-125a-5p promotes EPC migration and angiogenesis by upregulating MCL-1, thereby enhancing EPC homing to thrombi and facilitating thrombus resolution.

Impact of repeated intravenous infusions of umbilical cord-derived versus bone marrow-derived mesenchymal stem cells on angiogenesis in a pregnant experimentally induced deep venous thrombosis rat model.

Deep venous thrombosis (DVT) therapy during pregnancy warrants special consideration for the woman and the fetus. This study aimed to evaluate the impact of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) in terms of pro-angiogenic capacity and amelioration of pregnancy outcomes. The pregnant DVT rat model was successfully established by the "stenosis" method. Three consecutive injections of both UC-MSCs and BM-MSCs improved angiogenesis and ameliorated the embryo absorption rate in pregnant SD rats with DVT, in which UC-MSCs promoted angiogenesis more significantly. Furthermore, the levels of serum vascular endothelial growth factor-A (VEGF-A) and epidermal growth factor (EGF) were significantly higher in the UC-MSC group compared to those of the BM-MSC group. Thereafter, differentially expressed genes (DEGs) in thrombosed inferior vena cava tissues in the UC-MSC and BM-MSC groups were identified using transcriptome sequencing and further assessed by RT-qPCR and western blotting. The bioinformatics analysis indicated that the enriched DEG terms occurred in the cytokine activity, and the DEG pathways were significantly enriched in the cytokine-cytokine receptor interaction. In addition, both the mRNA and protein levels of angiogenic genes and their receptors, including VEGF-A, VEGF receptor-1, EGF, and EGF receptor, were significantly higher in the UC-MSC group. In conclusion, the BM-MSCs and UC-MSCs both significantly stimulate angiogenesis and ameliorate the embryo absorption rate in pregnant SD rats with DVT, but the difference in cytokine secretion causes UC-MSCs to have more potent angiogenic effects than BM-MSCs.

MicroRNA-342-3p loaded by human umbilical cord mesenchymal stem cells-derived exosomes attenuates deep vein thrombosis by downregulating EDNRA.

Exosomes (exos) exert biological functions to maintain the dynamic balance of cells and tissues by transferring biological cargo to recipient cells. Thus, this study explored human umbilical cord mesenchymal stem cells (hucMSCs)-derived exo transfer of microRNA (miR)-342-3p in deep vein thrombosis (DVT). DVT rat models were established via inferior vena cava (IVC) ligation. HucMSCs-exos were extracted and injected into rats with DVT to observe whether they could influences thrombus formation in vivo. HucMSCs-exos were co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro to observe angiogenesis. miR-342-3p and endothelin A receptor (EDNRA) expression in rats with DVT, as well as their interaction was analyzed. miR-342-3p was downregulated and EDNRA was upregulated in rats with DVT. HucMSCs-exos inhibited the formation of thrombus in rats with DVT, as well as promoted angiogenesis of HUVECs. Upregulated miR-342-3p delivery by hucMSCs-exos alleviated DVT in rats and improved angiogenesis of HUVECs. miR-342-3p targeted EDNRA, and the effect of hucMSCs-exos transfer of upregulated miR-342-3p was rescued by overexpressing EDNRA. Briefly, miR-342-3p loaded by hucMSCs-exos attenuates DVT by downregulating EDNRA, offering a novel direction to treat DVT.

TNFα activation and TGFß blockage act synergistically for smooth muscle cell calcification in patients with venous thrombosis via TGFß/ERK pathway.

Venous calcification has been observed in post-thrombotic syndrome (PTS) patients; yet, the cell types and possible mechanisms regulating this process are still unclear. We evaluated the calcium deposition within the venous wall, the cell type involved in the calcified remodelling of the venous wall after thrombosis and explored possible mechanisms in vitro. Calcium deposition was found in human specimens of superficial thrombotic veins and was co-localized with VSMCs markers αSMA and TAGLN (also known as SM22α). Besides, the expression of osteogenesis-related genes was dramatically changed in superficial thrombotic veins. Moreover, the inhibition of the TGFß signalling pathway after TNFα treatment effectively induced the expression of osteogenic phenotype markers, the calcium salt deposits and the obvious phosphorylation of ERK1/2 and JNK2 in the VSMCs calcification model. Supplementing TGFß2 or blocking the activation of the ERK/MAPK signalling pathway prevented the transformation of VSMCs into osteoblast-like cells in vitro. Taken together, VSMCs have an important role in venous calcification after thrombosis. Supplementing TGFß2 or inhibiting the ERK/MAPK signalling pathway can reduce the appearance of VSMCs osteogenic phenotype. Our findings may present a novel therapeutic approach to prevent of vascular calcification after venous thrombosis.