miR-513c-5p Suppression Aggravates Pyroptosis of Endothelial Cell in Deep Venous Thrombosis by Promoting Caspase-1

Abstract

Deep vein thrombosis (DVT) is a common peripheral vascular disease. Secondary pulmonary embolism (PE) caused by DVT leads to substantial patient death. Inflammation has been suggested as a key factor in the pathophysiology of DVT, however, involvement of pyroptosis-related inflammatory factors in DVT formation remains unclear. Here, we proposed that post-transcriptional modification of caspase-1 might be a crucial trigger for enhanced pyroptosis in vascular endothelial cells (VECs), and consequently contributed to severer symptoms in DVT patients. In order to explore the involvement of pyroptosis in DVT, peripheral blood mononuclear cells were collected from 30 DVT patients, and compared with the healthy controls, we found caspase-1 was increased both in mRNA and protein levels. miRNA microarray analysis demonstrated that down-regulated miR-513c-5p was significantly negatively correlated with the expression of caspase-1. In vitro assays suggested that miR-513c-5p overexpression could ameliorate the expression of caspase-1, and thus decreased the production of cleaved gasdermin D (GSDMD) and interleukin (IL)-1β and IL-18 in VECs. The dual-luciferase reporter assay identified direct binding between miR-513c-5p and the 3' untranslated region of caspase-1 encoding gene. The administration of miR-513c-5p mimics through tail vein injection or caspase-1 inhibitor (vx-765) by intraperitoneal injection remarkably decreased the volume of blood clots in vivo, whereas miR-513c-5p inhibitor aggravated thrombosis formation and this effect was dramatically weakened when treated in combination with vx-765. Collectively, these results revealed that the pyroptosis of VECs induced by decreased miR-513c-5p was involved in DVT progression and indicated a potential therapeutic strategy of targeting the miR-513c-5p/caspase-1/GSDMD signal axis for DVT management.

Keywords: caspase-1; deep venous thrombosis; miR-513c-5p; pyroptosis; vascular endothelial cell.

FIGURE 1

Expressions of caspase-1, GSDMD, IL-1β, and IL-18 are up-regulated in DVT patients. (A) mRNA level of caspase-1 in the PBMCs from 30 DVT patients and 30 controls was determined by qRT-PCR. (B) Caspase-1 and GSDMD protein levels were measured in PBMCs of DVT subjects and controls by Western blot. (C,D) Protein levels of IL-1β and IL-18 in the serum from 12 DVT patients and 12 controls were determined by ELISA. ****p < 0.0001.

FIGURE 2

miR-513c-5p is down-regulated in DVT patients and negatively correlated with caspase-1. (A) The Venn diagram shows miR-513c-5p and miR-6850-5p predicted by TargetScan and Chip. (B) The expression level of miR-6850-5p in PBMCs from 30 DVT patients and 30 controls was measured by qRT-PCR. ns means no statistical difference. (C) The expression level of miR-513c-5p in PBMCs from 30 DVT patients and 30 controls was measured by qRT-PCR. (D) The correlation between miR-513c-5p and caspase-1 was analyzed using Pearson correction analysis (n = 30). (E) Diagnostic value of miR-513c-5p for DVT was evaluated by ROC curve. ****p < 0.0001.

FIGURE 3

miR-513c-5p negatively regulates caspase-1 expression in HUVECs. (A) The expression of miR-513c-5p after NC, miR-513c-5p mimics, INC and miR-513c-5p inhibitor transfection was evaluated by qRT-PCR. (B) The expression level of caspase-1 mRNA after NC, miR-513c-5p mimics, INC and miR-513c-5p inhibitor transfection was detected by qRT-PCR. (C,D) Expression of caspase-1 in HUVECs were detected after transfection NC, miR-513c-5p mimics, INC, miR-513c-5p inhibitor by immunofluorescence staining (magnification, ×40). Scale bar = 200 μm. (E,F) Protein expression levels of caspase-1 and GSDMD were examined by Western blot. (G) The expression of IL-1β and IL-18 was detected by ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001.

FIGURE 4

miR-513c-5p targets caspase-1 mRNA 3′UTR directly. (A) Schematic representation of caspase-1 mRNA 3′UTR demonstrating putative miRNA target site, luciferase activities of WT and Mut constructs. (B,C) The luciferase activity was determined by co-transfecting the vectors (caspase-1 3′UTR-WT or Mut) combined with NC, miR-513c-5p mimics into 293T cells, respectively. ****p < 0.0001.

FIGURE 5

Caspase-1 inhibitor (vx-765) inhibits the formation of DVT in vivo. (A,B) H&E staining of serial cross sections of IVC from Ctrl, Sham, DVT, DVT+vehicle, DVT+vx-765, DVT+vehicle+miR-513c-5p inhibitor, DVT+vx-765+miR-513c-5p inhibitor at 48 h (magnification, ×40). Scale bars = 500 μm. (C,D) Thrombus length and weight at 48 h post-operation in the different treatment groups after the administration of caspase-1 inhibitor (vx-765) (n = 15 in each group). (E) Representative images of thrombi in each treatment group were detected by vascular ultrasound at 48 h post-operation. (F) Caspase-1 and GSDMD protein levels were determined by Western blot in vascular tissue of DVT animal group and vx-765-treated groups. (G) Expression of IL-1β and IL-18 in vascular tissue were detected by ELISA with vx-765 treated each group. **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6

Knockdown of miR-513c-5p promotes pyroptosis of VECs and DVT formation by increasing caspase-1. (A) The expression level of miR-513c-5p in vascular tissue was detected by qRT-PCR in DVT mice treated with INC, miR-513c-5p inhibitor, respectively. (B) Expression of miR-513c-5p in PBMCs was detected by qRT-PCR in DVT mice treated with INC, miR-513c-5p inhibitor, respectively. (C) Confocal microscopy images of miR-513c-5p expression in vascular tissues (miR-513c-5p, red; DAPI, blue) (magnification, ×200). Scale bars = 100 μm. (D) H&E staining of serial cross sections of inferior vena cava (IVC) from DVT mice treated with INC, miR-513c-5p inhibitor at 48 h (magnification, ×40). Scale bars = 500 μm. (E) Representative images of thrombi in each treatment group were detected by vascular ultrasound at 48 h post-operation. (F) Thrombus length and weight at 48 h post-operation in the different treatment groups (n = 15 in each group). (G) Caspase-1 mRNA level was determined by qRT-PCR in vascular tissue of each treatment group. (H) Caspase-1 and GSDMD protein levels were determined by Western blot in vascular tissue of DVT mice treated with INC, miR-513c-5p inhibitor. (I) IL-1β and IL-18 protein levels in vascular tissue were determined by ELISA in INC, miR-513c-5p inhibitor treated DVT mice models, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7

Overexpression of miR-513c-5p suppresses pyroptosis of VECs and DVT formation by inhibiting caspase-1. (A) Expression of miR-513c-5p in vascular tissue was detected by qRT-PCR in DVT mice treated with NC, miR-513c-5p mimics, respectively. (B) Relative expression of miR-513c-5p was measured by qRT-PCR in PBMCs of DVT each treatment group. (C) Confocal microscopy images of miR-513c-5p expression in vascular tissues (miR-513c-5p, red; DAPI, blue) (magnification, ×200). Scale bars = 100 μm. (D) H&E staining of serial cross sections of inferior vena cava (IVC) from DVT mice treated with NC, miR-513c-5p mimics at 48 h (magnification, ×40), respectively. Scale bars = 500 μm. (E) Representative images of thrombi in each treatment group were detected by vascular ultrasound at 48 h post-operation. (F) Thrombus length and weight at 48 h post-operation in DVT mice (n = 15) treated with NC, miR-513c-5p mimics. (G) Caspase-1 mRNA level was determined by qRT-PCR in vascular tissue of each treatment group. (H) Caspase-1 and GSDMD protein levels were determined by Western blot in vascular tissue of DVT each treatment group. (I) IL-1β and IL-18 protein levels in vascular tissue were determined by ELISA in DVT each treatment group. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 8

Schematic diagram of miR-513c-5p/caspase-1/GSDMD signal axis alteration in DVT pathogenesis. The down-regulation of miR-513c-5p in vascular endothelial cells relieved the inhibition of caspase-1 expression and resultant enhances of pyroptosis caused injury of endothelial cell, which lead to DVT formation.