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. 2023 Aug 18:11:tkad020.
doi: 10.1093/burnst/tkad020. eCollection 2023.

Single-cell RNA-seq and bulk-seq identify RAB17 as a potential regulator of angiogenesis by human dermal microvascular endothelial cells in diabetic foot ulcers

Hengyu Du  1 , Shenghong Li  1 , Jinqiang Lu  1 , Lingzhi Tang  1 , Xiao Jiang  1 , Xi He  1 , Jiaji Liang  1 , Xuan Liao  1 , Taixing Cui  2 , Yuesheng Huang  3 , Hongwei Liu  1
Affiliations

Single-cell RNA-seq and bulk-seq identify RAB17 as a potential regulator of angiogenesis by human dermal microvascular endothelial cells in diabetic foot ulcers

Hengyu Du et al. Burns Trauma. .

Abstract

Background: Angiogenesis is crucial in diabetic wound healing and is often impaired in diabetic foot ulcers (DFUs). Human dermal microvascular endothelial cells (HDMECs) are vital components in dermal angiogenesis; however, their functional and transcriptomic characteristics in DFU patients are not well understood. This study aimed to comprehensively analyse HDMECs from DFU patients and healthy controls and find the potential regulator of angiogenesis in DFUs.

Methods: HDMECs were isolated from skin specimens of DFU patients and healthy controls via magnetic-activated cell sorting. The proliferation, migration and tube-formation abilities of the cells were then compared between the experimental groups. Both bulk RNA sequencing (bulk-seq) and single-cell RNA-seq (scRNA-seq) were used to identify RAB17 as a potential marker of angiogenesis, which was further confirmed via weighted gene co-expression network analysis (WGCNA) and least absolute shrink and selection operator (LASSO) regression. The role of RAB17 in angiogenesis was examined through in vitro and in vivo experiments.

Results: The isolated HDMECs displayed typical markers of endothelial cells. HDMECs isolated from DFU patients showed considerably impaired tube formation, rather than proliferation or migration, compared to those from healthy controls. Gene set enrichment analysis (GSEA), fGSEA, and gene set variation analysis (GSVA) of bulk-seq and scRNA-seq indicated that angiogenesis was downregulated in DFU-HDMECs. LASSO regression identified two genes, RAB17 and CD200, as characteristic of DFU-HDMECs; additionally, the expression of RAB17 was found to be significantly reduced in DFU-HDMECs compared to that in the HDMECs of healthy controls. Overexpression of RAB17 was found to enhance angiogenesis, the expression of hypoxia inducible factor-1α and vascular endothelial growth factor A, and diabetic wound healing, partially through the mitogen-activated protein kinase/extracellular signal-regulated kinase signalling pathway.

Conclusions: Our findings suggest that the impaired angiogenic capacity in DFUs may be related to the dysregulated expression of RAB17 in HDMECs. The identification of RAB17 as a potential molecular target provides a potential avenue for the treatment of impaired angiogenesis in DFUs.

Keywords: Angiogenesis; Diabetic foot ulcers; Diabetic wound healing; Human dermal microvascular endothelial cells; RAB17; Single-cell RNA-seq.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Patient-derived HDMECs showed typical characteristics of endothelial cells. (a) Bright-field microscopy showed typical cobblestone-like morphological features of patient-derived HDMECs (scale bar: 50 μm). (b) Flow cytometric analysis detecting the positive rates of EC-specific markers CD31 and CDH5 in MACS+ cells. (c) Western blotting showing EC markers and SMC markers in MACS+ cells and NC cells. (d) mRNA levels of EC markers and SMC markers in MACS+ cells and NC cells measured by q-PCR. (e) Immunofluorescent staining showing the expression of EC markers and SMC markers in MACS+ cells and NC cells (Scale bar: 50 µm). The data represent the mean ± SD; *p < 0.05, ***p < 0.001. HDMECs human dermal microvascular endothelial cells, MACS magnetic-activated cell sorting, EC endothelial cell, NC negative control, SMC smooth muscle cells, CDH5, cadherin 5, α-SMA actin alpha 2 smooth muscle, vWF Von Willebrand Factor, SD standard deviation
Figure 2
Figure 2
DFU-HDMECs showed impaired angiogenesis rather than proliferation and migration. (a) Cell counting assay for determining the proliferation of Nor-HDMECs and DFU-HDMECs. (b) EdU (red) proliferation assay and (c) quantitative analysis showing the positive rates of proliferating cells in Nor-HDMECs and DFU-HDMECs. Nuclei were stained blue by DAPI (scale bar: 200 μm). (d) Wound scratch assay images and (f) quantitative analysis for evaluation of the migration in monolayer of Nor-HDMECs and DFU-HDMECs (scale bar: 200 μm). (e) Transwell assay images and (g) quantitative analysis for evaluation of the migration through the membrane of Nor-HDMECs and DFU-HDMECs (scale bar: 200 μm). (h) Matrigel tube-formation assay showing the capillary-like structures of Nor-HDMECs and DFU-HDMECs (scale bar: 200 μm). (i) Quantitative analysis of tube formation using number of tubes and nodes. HIF-1α and VEGF-A expression levels in HDMECs between normal and DFU group were measured by (j) q-PCR and (k) western blotting. Results are represented as the mean ± SD; *p < 0.05, **p < 0.01; ns, not significant. DFU diabetic foot ulcer, Nor normal, HDMECs human dermal microvascular endothelial cells, EdU 5-ethynyl-2′-deoxyuridine, HIF-1α hypoxia inducible factor 1 subunit alpha, VEGF-A vascular endothelial growth factor A, SD standard deviation
Figure 3
Figure 3
RNA sequencing and functional enrichment analysis showed impaired angiogenesis in DFU-HDMECs. (a) Volcano plot of genes differentially expressed between Nor-HDMECs and DFU-HDMECs. Up- or down-regulated genes are highlighted in red and blue respectively, whereas grey represents non-significant genes. (b) Heatmap of expressions of DEGs between Nor-HDMECs and DFU-HDMECs. (c) GSEA enrichment plots of the negatively enriched BP terms of angiogenesis and endothelium development in DFU-HDMECs compared to Nor-HDMECs. (d) The GSVA results showing the significantly up- or down-regulated hallmark gene sets in DFU-HDMECs compared to Nor-HDMECs. (e) The fGSEA plots of the significantly up- or down-regulated hallmark gene sets in DFU-HDMECs. A p-value <0.05 was considered statistically significant. DFU diabetic foot ulcer, Nor normal, HDMECs human dermal microvascular endothelial cells, DEGs differentially expressed genes, GSEA gene set enrichment analysis, GSVA gene set variation analysis, NES normalized enrichment score
Figure 4
Figure 4
Single-cell RNA-seq reveals heterogeneity and impaired angiogenesis in HDMECs of DFU and normal skins. (a) t-SNE dimensionality reduction plot of the dataset GSE165816 with 63,190 cells passed quality control. The cells are coloured by manually annotated cell types. (b) Dot plot of the expression of the well-accepted cell-specific marker genes used for annotations of cell types. The colour indicates averaged expression levels of the scaled genes, and the size represents percentage of the marker genes expressed. (c) Stacked bar plots of the proportions of different types of cells in normal and DFU skins. (d, e) Split t-SNE plots of the HDMECs split from the whole dataset, shown by groups, depicting eight sub-clusters of HDMECs. (f) Stacked bar plots of the proportions of sub-types of HDMECs in normal and DFU skins. (g) GSEA plots showing the selected biological processes that are significantly affected (p-adj < 0.01) in DFU-HDMECs of the single-cell dataset. (h) Violin plot of GSVA showing the statistically significant variations (p-adj < 0.05) in single-cell data of DFU-HDMECs. (i) fGSEA plots of the significantly up- or down-regulated (p-adj < 0.05) hallmark gene sets in DFU-HDMECs extracted from the scRNA-seq data. DFU diabetic foot ulcer, Nor normal, HDMECs human dermal microvascular endothelial cells, tSNE t-distributed stochastic neighbour embedding, GSEA gene set enrichment analysis, GSVA gene set variation analysis, scRNA-seq single-cell RNA sequencing, NES normalized enrichment score
Figure 5
Figure 5
RAB17 level was decreased under hyperglycemic conditions. RAB17 expression level in HDMECs between normal and DFU group was measured by (a) q-PCR and (b) western blotting. (c, d) Western blotting assessing the expression of RAB17 in Nor-HDMEC exposed to high-glucose medium (30 mM D-glucose) for 2 and 7 days; 30 mM mannitol was used as an osmotic control. (e) Immunohistochemical analysis of RAB17 in the skin of normal patients and DFU subjects. Scale bar: 200 μm (upper panel) and 40 μm (lower panel) respectively. (f) Immunohistochemical analysis of RAB17 in the skin of normal mice and diabetic mice. Scale bar: 200 μm (upper panel) and 40 μm (lower panel) respectively. Results are represented as the mean ± SD; *p < 0.05, **p < 0.01. DFU diabetic foot ulcer, Nor normal, HDMECs human dermal microvascular endothelial cells, HG high glucose, SD standard deviation
Figure 6
Figure 6
RAB17 affected angiogenic capacity of HDMECs in vitro. (a, b) Western blotting assessing the expression of RAB17 in DFU-HDMECs after RAB17-overexpressing lentivirus transfection, and the expressions of HIF-1α, VEGF-A, ERK and p-ERK in DFU-HDMECs after overexpression of RAB17. (c) Matrigel tube-formation assay of DFU-HDMECs following RAB17 overexpression and the quantitative analysis of tube formation using length of tubes and number of nodes. (d, e) Western blotting assessing the expressions of RAB17, HIF-1α, VEGF-A, ERK and p-ERK after knockdown by siRNA targeting RAB17. (f) Matrigel tube-formation assay and quantitative analysis of Nor-HDMECs following knock-down of RAB17. (g, h) Western blotting revealing the expression levels of HIF-1α, VEGF-A, ERK and p-ERK after overexpression of RAB17 with or without PD98059. (i) Matrigel tube-formation assay and quantitative analysis of DFU-HDMECs following RAB17 overexpression with or without PD98059. Results are represented as the mean ± SD; *p < 0.05, **p < 0.01. Lv lentivirus, Ctrl control, DFU diabetic foot ulcer, Nor normal, HDMECs human dermal microvascular endothelial cells, HIF-1α hypoxia inducible factor 1 subunit alpha, VEGF-A vascular endothelial growth factor A, si-RAB17 RAB17 small interfering RNA, si-NC negative control small interfering RNA, SD standard deviation
Figure 7
Figure 7
RAB17 overexpression promoted diabetic angiogenesis and wound healing in vivo. (a) Immunohistochemical analysis of RAB17 between groups after injection of rAAV-RAB17 or rAAV-vector. Scale bar: 40 μm. (b, c) Western blotting assessing the expression of RAB17 in rAAV-RAB17 or rAAV-vector transfected mice skin. (d) Laser speckle contrast imager recording the wound perfusion images and (e) relative wound perfusion ratio in rAAV-RAB17 group and the control vector group; high- or low blood flow are represented in red and blue respectively. (f) Wound area monitored at the indicated days post-wounding and (g) quantitative analysis in rAAV-RAB17 group and the control vector group. Results are represented as the mean ± SD; *p < 0.05. rAAV recombinant adeno-associated viral, SD standard deviation
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References

    1. Chen H, Cheng Y, Tian J, Yang P, Zhang X, Chen Y, et al. Dissolved oxygen from microalgae-gel patch promotes chronic wound healing in diabetes. Sci Adv. 2020;6:eaba4311. 10.1126/sciadv.aba4311. - DOI - PMC - PubMed
    1. Fesseha BK, Abularrage CJ, Hines KF, Sherman R, Frost P, Langan S, et al. Association of Hemoglobin a(1c) and wound healing in diabetic foot ulcers. Diabetes Care. 2018;41:1478–85. 10.2337/dc17-1683. - DOI - PMC - PubMed
    1. Okonkwo UA, DiPietro LA. Diabetes and wound angiogenesis. Int J Mol Sci. 2017;18. 10.3390/ijms18071419. - DOI - PMC - PubMed
    1. Wu X, He W, Mu X, Liu Y, Deng J, Liu Y, et al. Macrophage polarization in diabetic wound healing. Burns. Dent Traumatol. 2022;10:tkac051. 10.1093/burnst/tkac051. - DOI - PMC - PubMed
    1. Ishihara J, Ishihara A, Starke RD, Peghaire CR, Smith KE, McKinnon TAJ, et al. The heparin binding domain of von Willebrand factor binds to growth factors and promotes angiogenesis in wound healing. Blood. 2019;133:2559–69. 10.1182/blood.2019000510. - DOI - PMC - PubMed
-