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.

中文翻译：

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单细胞RNA-seq和bulk-seq鉴定RAB17是糖尿病足溃疡中人真皮微血管内皮细胞血管生成的潜在调节剂

背景 血管生成对于糖尿病伤口愈合至关重要，并且在糖尿病足溃疡 (DFU) 中常常受到损害。人真皮微血管内皮细胞（HDMEC）是真皮血管生成的重要组成部分；然而，它们在 DFU 患者中的功能和转录组特征尚不清楚。本研究旨在全面分析 DFU 患者和健康对照的 HDMEC，寻找 DFU 血管生成的潜在调节因子。方法通过磁激活细胞分选从 DFU 患者和健康对照的皮肤标本中分离 HDMEC。然后比较实验组之间细胞的增殖、迁移和管形成能力。批量 RNA 测序 (bulk-seq) 和单细胞 RNA 测序 (scRNA-seq) 均用于鉴定 RAB17 作为血管生成的潜在标志物，并通过加权基因共表达网络分析 (WGCNA) 和最小化分析进一步证实了这一点。绝对收缩和选择算子（LASSO）回归。通过体外和体内实验检查了 RAB17 在血管生成中的作用。结果分离的HDMECs显示出典型的内皮细胞标志物。与健康对照相比，从 DFU 患者中分离的 HDMEC 表现出管形成明显受损，而不是增殖或迁移。Bulk-seq 和 scRNA-seq 的基因集富集分析 (GSEA)、fGSEA 和基因集变异分析 (GSVA) 表明 DFU-HDMEC 中血管生成下调。LASSO回归确定了两个基因，RAB17和CD200，作为DFU-HDMEC的特征；此外，与健康对照的 HDMEC 相比，DFU-HDMEC 中 RAB17 的表达显着降低。研究发现，RAB17 的过表达可增强血管生成、缺氧诱导因子 1α 和血管内皮生长因子 A 的表达以及糖尿病伤口愈合，部分是通过丝裂原激活蛋白激酶/细胞外信号调节激酶信号通路实现的。结论 我们的研究结果表明，DFU 中血管生成能力受损可能与 HDMEC 中 RAB17 的表达失调有关。RAB17 作为潜在分子靶点的鉴定为治疗 DFU 中血管生成受损提供了潜在途径。