HOXD9 regulated mitophagy to promote endothelial progenitor cells angiogenesis and deep vein thrombosis recanalization and resolution

Abstract

Background: Deep vein thrombosis (DVT) is a common vascular surgical disease caused by the coagulation of blood in the deep veins, and predominantly occur in the lower limbs. Endothelial progenitor cells (EPCs) are multi-functional stem cells, which are precursors of vascular endothelial cells. EPCs have gradually evolved into a promising treatment strategy for promoting deep vein thrombus dissolution and recanalization through the stimulation of various physical and chemical factors.

Methods: In this study, we utilized a mouse DVT model and performed several experiments including qRT-PCR, Western blot, tube formation, wound healing, Transwell assay, immunofluorescence, flow cytometry analysis, and immunoprecipitation to investigate the role of HOXD9 in the function of EPCs cells. The therapeutic effect of EPCs overexpressing HOXD9 on the DVT model and its mechanism were also explored.

Results: Overexpression of HOXD9 significantly enhanced the angiogenesis and migration abilities of EPCs, while inhibiting cell apoptosis. Additionally, results indicated that HOXD9 specifically targeted the HRD1 promoter region and regulated the downstream PINK1-mediated mitophagy. Interestingly, intravenous injection of EPCs overexpressing HOXD9 into mice promoted thrombus dissolution and recanalization, significantly decreasing venous thrombosis.

Conclusions: The findings of this study reveal that HOXD9 plays a pivotal role in stimulating vascular formation in endothelial progenitor cells, indicating its potential as a therapeutic target for DVT management.

Keywords: Angiogenesis; Deep vein thrombosis; Endothelial progenitor cells; HOXD9; Mitophagy.

Fig. 1

Characterization of EPCs. A. Spindle-shaped EPCs were observed on day 3 of BM-MNCs plating. B. The image illustrating the EPCs uptake DiI-Ac-LDL (red). The nuclei were stained with DAPI (blue). Scale Bar = 50 μm. C. EPC phenotypic analysis by flow cytometry. D. Senescent cells were stained with senescence β-galactosidase. Data are presented as the mean ± SD, n = 3

Fig. 2

HOXD9 enhanced EPCs cell function. A. Volcano plots of differentially expressed genes in GEO datasets (GSE17078). B. The mRNA expression of HOXD9 in venous tissue from mice. C. The mRNA expression of HOXD3 in EPCs transfected with si-HOXD9, si-NC, ad-HOXD9, or ad-NC for 24 h. D. The protein expression of HOXD3 in EPCs transfected with si-HOXD9, si-NC, ad-HOXD9, or ad-NC for 24 h. E. Effects of HOXD3 on tube development in EPCs. Scale Bar = 200 μm. F. Effects of HOXD9 on cell migration in EPCs. G. Wound healing assay of Effects of HOXD9 on cell migration in EPCs. H. Effects of HOXD9 on F-actin expression (red) in EPCs. DAPI (blue) was used to stain the nucleus. Scale Bar = 20 μm. I. Effects of HOXD3 on apoptosis in EPCs by flow cytometry. Data are presented as the mean ± SD, n = 3

Fig. 3

HOXD9 transcriptionally regulates HRD1 in EPCs. A. JASPAR database predicts a putative HOXD9 binding site located on the promoter region of mouse HRD1. B. ChIP assay of the relative enrichment of HOXD9 on the promoter region of mouse HRD1. C. The mRNA expression of HRD1in EPCs transfected with si-HOXD9, si-NC, ad-HOXD9, or ad-NC for 24 h. D. The protein expression of HRD1 in EPCs transfected with si-HOXD9, si-NC, ad-HOXD9, or ad-NC for 24 h. E. Effects of HOXD9 and HRD1 on tube formation in EPCs. Scale Bar = 200 μm. F. Effects of HOXD9 and HRD1 on cell migration in EPCs as determined by Transwell assay. G. Effects of HOXD9 and HRD1 on cell migration in EPCs as evaluated by the wound healing assay. Data are presented as the mean ± SD, n = 3

Fig. 4

HOXD9/HRD1 regulated mitochondrial autophagy via PINK1 ubiquitination in EPCs. A. CHX tracking assay illustrating the effect of HRD1 in EPCs on the change in the half-life of PINK1. B. The effect of HRD1 on the ubiquitination of PINK1 in EPCs as determined by Immunoprecipitation assay. C. Western blot results showing the protein expression of autophagy-related markers (LC3 and p62), mitochondria-related markers (COX IV and TOMM20), and PINK1 in EPCs. D. Effects of HOXD9 and HRD1 on Mitotracker (green) and LC3B (red) in EPCs. The nuclei were stained with DAPI (blue). Scale Bar = 50 μm. E. After stably transfected with tandem-labeled mRFP-GFP-LC3, representative images of mRFP-GFP-LC3 vector were shown by immunofluorescent detection. Scale Bar = 20 μm. Data are presented as the mean ± SD, n = 3

Fig. 5

EPC-HOXD9 enhanced thrombus organization and recanalization in vivo. A. Representative HE staining images of the thrombus sections. B. Representative TF (red) and DAPI (blue) staining images of the thrombus sections. C. Representative images of CD31 (green) and DAPI (blue) staining for the thrombus sections. n = 3

Fig. 6

EPC-HOXD9 inhibited mitophagy in vivo. A. The mRNA expression of HOXD9 in venous tissue from mice. B. Representative images showing HOXD9 (red) and DAPI (blue) staining in the thrombus sections. C. The protein expression of autophagy-related markers (LC3 and p62), mitochondria-related markers (COX IV and TOMM20), and PINK1 in mice venous tissue. D. Representative images of LC3B (green) and DAPI (blue) staining of the thrombus sections. Data are presented as the mean ± SD, n = 3