Advances in microRNA regulation of deep vein thrombosis through venous vascular endothelial cells (Review).

Deep vein thrombosis (DVT) is a prevalent clinical venous thrombotic condition that often manifests independently or in conjunction with other ailments. Thrombi have the propensity to dislodge into the circulatory system, giving rise to complications such as pulmonary embolism, thereby posing a significant risk to the patient. Virchow proposed that blood stagnation, alterations in the vessel wall and hypercoagulation are primary factors contributing to the development of venous thrombosis. Vascular endothelial cells (VECs) constitute the initial barrier to the vascular wall and are a focal point of ongoing research. These cells exert diverse stimulatory effects on the bloodstream and secrete various regulatory factors that uphold the dynamic equilibrium between the coagulation and anticoagulation processes. MicroRNAs (miRNAs) represent a class of non­coding RNAs present in eukaryotes, characterized by significant genetic and evolutionary conservation and displaying high spatiotemporal expression specificity. Typically ranging from 20 to 25 bases in length, miRNAs can influence downstream gene transcription through RNA interference or by binding to specific mRNA sites. Consequently, advancements in understanding the molecular mechanisms of miRNAs, including their functionalities, involve modulation of vascular­associated processes such as cell proliferation, differentiation, secretion of inflammatory factors, migration, apoptosis and vascular remodeling regeneration. miRNAs play a substantial role in DVT formation via venous VECs. In the present review, the distinct functions of various miRNAs in endothelial cells are outlined and recent progress in comprehending their role in the pathogenesis and clinical application of DVT is elucidated.

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.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

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.

Protective Role of Endothelial SIRT1 in Deep Vein Thrombosis and Hypoxia-induced Endothelial Dysfunction Mediated by NF-κB Deacetylation.

Venous hypoxia is considered as the major pathogenetic mechanism linking blood flow stagnancy with deep vein thrombosis (DVT). Our previous study showed that activating SIRT1 may attenuate inferior vena cava (IVC) stenosis-induced DVT in rats. This study was aimed to investigate the role of endothelial SIRT1 in DVT and hypoxia-induced endothelial dysfunction as well as the underlying mechanism. Protein profiling of IVCs and blood plasma of DVT rats induced by IVC stenosis was analysed by 4D Label free proteomics analysis. To verify the independent role of SIRT1 in DVT and oxygen-glucose deprivation (OGD)-induced endothelial dysfunction, SIRT1 specific activator SRT1720 and SIRT1 knockdown in both local IVCs and endothelial cells were employed. Moreover, the role of the NF-κB were investigated using NF-κB inhibitor caffeic acid phenethyl ester (CAPE). SRT1720 significantly inhibited thrombus burden, leukocytes infiltration, protein expressions of cell adhesion molecules and chemokines, as well as acetylation level of NF-κB/p65 in wild DVT rats, while these protective effects of SRT1720 were abolished in rats with SIRT1 knockdown in local IVCs. In vitro, SRT1720 protected endothelial cells against OGD-induced dysfunction characterized with enhanced adhesion of monocytes as well as the protein expressions of cell adhesion molecules and chemokines, whereas these protective effects of SRT1720 were vanished by SIRT1 stable knockdown. Furthermore, CAPE attenuated endothelial cell dysfunction and abolished these effects of SIRT1 knockdown. Collectively, these data suggested that endothelial SIRT1 plays an independent role in ameliorating hypoxia-induced endothelial dysfunction and thrombotic inflammation in DVT, and this effect is mediated by NF-κB deacetylation.

Sleep like a bear.

Reduced expression of a platelet protein protects against thrombosis during chronic immobilization.

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.

Pathophysiology of deep vein thrombosis.

Deep venous thrombosis is a frequent, multifactorial disease and a leading cause of morbidity and mortality. Most of the time deep venous thrombosis is triggered by the interaction between acquired risk factors, such as hip fracture, pregnancy, and immobility, and hereditary risk factors such as thrombophilias. The mechanisms underlying deep venous thrombosis are not fully elucidated; however, in recent years, important advances have shed light on the role of venous flow, endothelium, platelets, leukocytes, and the interaction between inflammation and hemostasis. It has been described that the alteration of venous blood flow produces endothelial activation, favoring the adhesion of platelets and leukocytes, which, through tissue factor expression and neutrophil extracellular traps formation, contribute to the activation of coagulation, trapping more cells, such as red blood cells. Thus, the concerted interaction of these phenomena allows the formation and growth of the thrombus. In this work, the main mechanisms involved in the pathophysiology of deep vein thrombosis will be described.

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.

Molecular Detection of Venous Thrombosis in Mouse Models Using SPECT/CT.

The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links α2-AP to fibrin to protect it from early lysis. This was exploited to develop an α2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using 111In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and 111In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl3) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with 111In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16 imaging probe to fibrin was obtained. In vivo IVC thrombosis models yielded stable platelet-rich thrombi with FeCl3 and fibrin and red cell-rich thrombi with stenosis. In the stenosis model, clot formation in the vena cava corresponded with a SPECT hotspot using an A16 imaging probe as a molecular tracer. The fibrin-targeting A16 probe showed specific binding to mouse thrombi in in vitro assays and the in vivo DVT model. The use of specific and covalent fibrin-binding probes might enable the clinical non-invasive imaging of early and active thrombosis.

Fibrinogen and Factor XIII in Venous Thrombosis and Thrombus Stability.

As the third most common vascular disease, venous thromboembolism is associated with significant mortality and morbidity. Pathogenesis underlying venous thrombosis is still not fully understood. Accumulating data suggest fibrin network structure and factor XIII-mediated crosslinking are major determinants of venous thrombus mass, composition, and stability. Understanding the cellular and molecular mechanisms mediating fibrin(ogen) and factor XIII production and function and their ability to influence venous thrombosis and resolution may inspire new anticoagulant strategies that target these proteins to reduce or prevent venous thrombosis in certain at-risk patients. This article summarizes fibrinogen and factor XIII biology and current knowledge of their function during venous thromboembolism.

Impact of Neutrophil Extracellular Traps on Thrombosis Formation: New Findings and Future Perspective.

Thrombotic diseases seriously endanger human health, neutrophils and neutrophil extracellular traps (NETs) play an important role in abnormal thrombus formation. NETs are extracellular structures released by neutrophils upon stimulation by pathogens. NETs include neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G and other active substances. The network structure provided by NETs can prevent the spread of pathogens and effectively kill and eliminate pathogens. However, the components of NETs can also abnormally activate the coagulation pathway and participate in the formation of pathological thrombi. This review aims to summarize the mechanisms of NETs formation in detail; the research progress of NETs in venous thrombosis, arterial thrombosis, acquired disease-associated thrombosis, sepsis coagulation disorder; as well as the strategies to target NETs in thrombosis prevention and treatment.

MicroRNA-136-5p from Endothelial Progenitor Cells-released Extracellular Vesicles Mediates TXNIP to Promote the Dissolution of Deep Venous Thrombosis.

OBJECTIVE: Endothelial progenitor cells-released extracellular vesicles (EPCs-EVs) have previously been reported to promote the dissolution of deep venous thrombosis (DVT) through delivery of microRNA (miR). Given that, this research was projected to search the relative action of EPCs-EVs transferring of miR-136-5p in DVT. METHODS: From EPCs transfected with miR-136-5p agomir or antagomir, EVs were extracted and then injected into DVT mice. Meanwhile, based on the treatment with EPCs-EVs loading miR-136-5p antagomir, silenced thioredoxin-interacting protein (TXNIP) lentivirus was injected into DVT mice to perform the rescue experiments. Afterwards, the length and weight of venous thrombosis, EPC apoptosis and inflammatory factors, plasmin, fibrinogen, and thrombin-antithrombin were measured. miR-136-5p and TXNIP expression in DVT mice, and their targeting relationship were evaluated. RESULTS: miR-136-5p expression was suppressed and TXNIP expression was elevated in DVT mice. EPCs-EV reduced the length and weight of venous thrombosis, suppressed cell apoptosis and inflammatory reaction, as well as elevated level of plasmin, and reduced levels of fibrinogen and thrombin-antithrombin in DVT mice. Restored miR-136-5p loaded by EPCs-EV further attenuated DVT but EPCs-EV transfer of depleted miR-136-5p resulted in the opposite consequences. miR-136-5p targeted TXNIP and silenced TXNIP rescued the effect of EPCs-EV transfer of depleted miR-136-5p on DVT. CONCLUSION: miR-136-5p from EPCs-EV suppresses TXNIP expression to reduce the thrombus size in DVT, offering a promising treatment target for DVT.

Upregulated miR-206 Aggravates Deep Vein Thrombosis by Regulating GJA1-Mediated Autophagy of Endothelial Progenitor Cells.

Background: Deep vein thrombosis (DVT) is the third most prevalent vascular disease worldwide. MicroRNAs (miRNAs) play regulatory roles in functions of endothelial progenitor cells (EPCs), which is becoming a promising therapeutic choice for thrombus resolution. Nevertheless, the role of miR-206 in EPCs is unclear. Methods: 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-206 and gap junction protein alpha 1 (GJA1) in EPCs and vascular tissues of DVT mice were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The proliferation, migration, apoptosis, and angiogenesis were tested by cell counting kit-8 (CCK-8) assay, Transwell assay, flow cytometry analysis, and in vitro tube formation assay. The levels of autophagy-related proteins as well as the level of GJA1 in EPCs and vascular tissues were evaluated by western blotting. DVT formation in vivo was observed through hematoxylin-eosin (HE) staining. The expression of thrombus resolution markers, CD34 molecule (CD34) and matrix metallopeptidase 2 (MMP2), in the thrombi was measured by immunofluorescence staining. Results: miR-206 overexpression inhibited proliferation, migration, and angiogenesis and promoted apoptosis of EPCs, while miR-206 knockdown exerted an opposite effect on EPC phenotypes. Downregulation of GJA1, the target of miR-206, abolished the influence of miR-206 on EPC phenotypes. Furthermore, silencing of miR-206 suppressed the autophagy of EPCs via upregulating GJA1. miR-206 knockdown repressed thrombus formation, enhanced the homing ability of EPCs to the thrombosis site, and facilitated thrombus resolution in DVT mouse models. Additionally, miR-206 was upregulated while GJA1 was downregulated in vascular tissues of DVT mice. miR-206 knockdown elevated GJA1 expression in vascular tissues of DVT mice. The expression of miR-206 was negatively correlated with that of GJA1 in DVT mice. Conclusion: miR-206 knockdown upregulates GJA1 to inhibit autophagy of EPCs and then promote EPC proliferation, migration, and angiogenesis, thereby enhancing EPC homing to thrombi and facilitating thrombus resolution.

Both G protein-coupled and immunoreceptor tyrosine-based activation motif receptors mediate venous thrombosis in mice.

Platelets are critical in hemostasis and a major contributor to arterial thrombosis (AT). (Pre)clinical studies suggest platelets also contribute to venous thrombosis (VT), but the mechanisms are largely unknown. We hypothesized that in VT, platelets use signaling machinery distinct from AT. Here we aimed to characterize the contributions of platelet G protein-coupled (GPCR) and immunoreceptor tyrosine-based activation motif (ITAM) receptor signaling to VT. Wild-type (WT) and transgenic mice were treated with inhibitors to selectively inhibit platelet-signaling pathways: ITAM-CLEC2 (Clec2mKO), glycoprotein VI (JAQ1 antibody), and Bruton's tyrosine kinase (ibrutinib); GPCR-cyclooxygenase 1 (aspirin); and P2Y12 (clopidogrel). VT was induced by inferior vena cava stenosis. Thrombin generation in platelet-rich plasma and whole-blood clot formation were studied ex vivo. Intravital microscopy was used to study platelet-leukocyte interactions after flow restriction. Thrombus weights were reduced in WT mice treated with high-dose aspirin + clopidogrel (dual antiplatelet therapy [DAPT]) but not in mice treated with either inhibitor alone or low-dose DAPT. Similarly, thrombus weights were reduced in mice with impaired ITAM signaling (Clec2mKO + JAQ1; WT + ibrutinib) but not in Clec2mKO or WT + JAQ1 mice. Both aspirin and clopidogrel, but not ibrutinib, protected mice from FeCl3-induced AT. Thrombin generation and clot formation were normal in blood from high-dose DAPT- or ibrutinib-treated mice; however, platelet adhesion and platelet-neutrophil aggregate formation at the vein wall were reduced in mice treated with high-dose DAPT or ibrutinib. In summary, VT initiation requires platelet activation via GPCRs and ITAM receptors. Strong inhibition of either signaling pathway reduces VT in mice.

Neutrophils, Cancer and Thrombosis: The New Bermuda Triangle in Cancer Research.

Spontaneous venous thrombosis is often the first clinical sign of cancer, and it is linked to a worsened survival rate. Traditionally, tumor-cell induced platelet activation has been the main actor studied in cancer-associated-thrombosis. However, platelet involvement alone does not seem to be sufficient to explain this heightened pro-thrombotic state. Neutrophils are emerging as key players in both thrombus generation and cancer progression. Neutrophils can impact thrombosis through the release of pro-inflammatory cytokines and expression of molecules like P-selectin and Tissue Factor (TF) on their membrane and on neutrophil-derived microvesicles. Their role in cancer progression is evidenced by the fact that patients with high blood-neutrophil counts have a worsened prognosis. Tumors can attract neutrophils to the cancer site via pro-inflammatory cytokine secretions and induce a switch to pro-tumoral (or N2) neutrophils, which support metastatic spread and have an immunosuppressive role. They can also expel their nuclear contents to entrap pathogens forming Neutrophil Extracellular Traps (NETs) and can also capture coagulation factors, enhancing the thrombus formation. These NETs are also known to have pro-tumoral effects by supporting the metastatic process. Here, we strived to do a comprehensive literature review of the role of neutrophils as drivers of both cancer-associated thrombosis (CAT) and cancer progression.