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The endothelial tumor suppressor p53 is essential for venous thrombus formation in aged mice.

ABSTRACT: Venous thromboembolism (VTE) is a leading cause of morbidity and mortality in elderly people. Increased expression of tumor suppressor protein 53 (p53) has been implicated in vascular senescence. Here, we examined the importance of endothelial p53 for venous thrombosis and whether endothelial senescence and p53 overexpression are involved in the exponential increase of VTE with age. Mice with conditional, endothelial-specific deletion of p53 (End.p53-KO) and their wild-type littermates (End.p53-WT) underwent subtotal inferior vena cava (IVC) ligation to induce venous thrombosis. IVC ligation in aged (12-month-old) End.p53-WT mice resulted in higher rates of thrombus formation and greater mean thrombus size vs adult (12-week-old) End.p53-WT mice, whereas aged End.p53-KO mice were protected from vein thrombosis. Analysis of primary endothelial cells from aged mice or human vein endothelial cells after induction of replicative senescence revealed significantly increased early growth response gene-1 (Egr1) and heparanase expression, and plasma factor Xa levels were elevated in aged End.p53-WT, but not in End.p53-KO mice. Increased endothelial Egr1 and heparanase expression also was observed after doxorubicin-induced p53 overexpression, whereas p53 inhibition using pifithrin-α reduced tissue factor (TF) expression. Importantly, inhibition of heparanase activity using TF pathway inhibitor-2 (TFPI2) peptides prevented the enhanced venous thrombus formation in aged mice and restored it to the thrombotic phenotype of adult mice. Our findings suggest that p53 accumulation and heparanase overexpression in senescent endothelial cells are critically involved in mediating the increased risk of venous thrombosis with age and that heparanase antagonization may be explored as strategy to ameliorate the prothrombotic endothelial phenotype with age.

SUBMITTER: Bochenek ML

PROVIDER: S-EPMC5998933 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RationaleChronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGF? (transforming growth factor-?) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGF? in the pathophysiology of CTEPH is unknown.ObjectiveTo determine whether defective TGF? signaling in endothelial cells contributes to thrombus nonresolution and fibrosis.Methods and resultsVenous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGF?1 in platelets (Plt.TGF?-KO) or TGF? type II receptors in endothelial cells (End.TGF?RII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGF?1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGF?RII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGF?1 levels, endothelial TGF?RI/ALK1 (activin receptor-like kinase), and TGF?RI/ALK5 expression were detected in End.TGF?RII-KO mice, and activated TGF? signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGF?RII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGF?1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGF?RII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGF?1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGF?RII-KO mice.ConclusionsEndothelial TGF?1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.

Project description:A comprehensive understanding of thrombus formation as a physicochemical process that has evolved to protect the integrity of the human vasculature is critical to our ability to predict and control pathological states caused by a malfunctioning blood coagulation system. Despite numerous investigations, the spatial and temporal details of thrombus growth as a multicomponent process are not fully understood. Here, we used computational modeling to investigate the temporal changes in the spatial distributions of the key enzymatic (i.e., thrombin) and structural (i.e., platelets and fibrin) components within a growing thrombus. Moreover, we investigated the interplay between clot structure and its mechanical properties, such as hydraulic resistance to flow. Our model relied on the coupling of computational fluid dynamics and biochemical kinetics, and was validated using flow-chamber data from a previous experimental study. The model allowed us to identify the distinct patterns characterizing the spatial distributions of thrombin, platelets, and fibrin accumulating within a thrombus. Our modeling results suggested that under the simulated conditions, thrombin kinetics was determined predominantly by prothrombinase. Furthermore, our simulations showed that thrombus resistance imparted by fibrin was ∼30-fold higher than that imparted by platelets. Yet, thrombus-mediated bloodflow occlusion was driven primarily by the platelet deposition process, because the height of the platelet accumulation domain was approximately twice that of the fibrin accumulation domain. Fibrinogen supplementation in normal blood resulted in a nonlinear increase in thrombus resistance, and for a supplemented fibrinogen level of 48%, the thrombus resistance increased by ∼2.7-fold. Finally, our model predicted that restoring the normal levels of clotting factors II, IX, and X while simultaneously restoring fibrinogen (to 88% of its normal level) in diluted blood can restore fibrin generation to ∼78% of its normal level and hence improve clot formation under dilution.

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The endothelial tumor suppressor p53 is essential for venous thrombus formation in aged mice.

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