HIF-1α/JMJD1A signaling regulates inflammation and oxidative stress following hyperglycemia and hypoxia-induced vascular cell injury

Abstract

Background: Endothelial cell (EC) injury accelerates the progression of diabetic macrovascular complications. Hypoxia is an important cause of EC injury. Hypoxia-inducible factor-1 alpha (HIF-1α) is an important hypoxia regulatory protein. Our previous studies showed that high-glucose and hypoxic conditions could upregulate HIF-1α expression and enhance EC inflammatory injury, independently of the nuclear factor kappa-B (NF-κB) pathway. However, it is not clear whether HIF-1α plays a role in vascular disease through epigenetic-related mechanisms.

Methods: We conducted gene expression analysis and molecular mechanistic studies in human umbilical vein endothelial cells (HUVECs) induced by hyperglycemia and hypoxia using RNA sequencing (RNA-seq) and small interfering HIF-1α (si-HIF-1α). We determined HIF-1α and Jumonji domain-containing protein 1 A (JMJD1A) expression by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot, analyzed inflammatory protein secretion in the cell supernatant by enzymelinked immunosorbent assay (ELISA), and assessed protein interaction between HIF-1α and JMJD1A by chromatin immunoprecipitation (Ch-IP). We used the Cell Counting Kit8 (CCK-8) assay to analyze cell viability, and assessed oxidative stress indicators by using a detection kit and flow cytometry.

Results: High glucose and hypoxia up-regulated HIF-1α expression, and down-regulated HIF-1α decreased the level of inflammation and oxidative stress in HUVECs. To determine the downstream pathways, we observed histone demethylases genes and related pathway by RNA-sEq. Among these, JMJD1A was the most upregulated gene in histone demethylases. Moreover, we observed that HIF-1α bound to the promoter of JMJD1A, and the ameliorative effects of si-HIF-1α on oxidative stress and inflammatory cytokines in high-glucose and hypoxia-induced HUVECs were reversed by JMJD1A overexpression. Furthermore, knockdown of JMJD1A decreased inflammatory and oxidative stress injury. To determine the JMJD1A-related factors, we conducted gene expression analysis on JMJD1A-knockdown HUVECs. We observed that downregulation of inflammation and the oxidative stress pathway were enriched and FOS and FOSB might be important protective transcription factors.

Conclusions: These findings provide novel evidence that the HIF-1α/JMJD1A signaling pathway is involved in inflammation and oxidative stress in HUVECs induced by high glucose and hypoxia. Also, this pathway might act as a novel regulator of oxidative stress and inflammatory-related events in response to diabetic vascular injury and thus contribute to the pathological progression of diabetes and vascular disease.

Keywords: Diabetes; Epigenetics; Hypoxia-inducible factor-1 alpha; Vascular disease.

Fig. 1

Stimulation with high glucose decreases cell survival and increases ROS levels in HUVECs. a Cells treated with various concentrations of glucose (25 or 30 mM) for 6, 12, 24, or 48 h reduced cell viability. High glucose (25 or 30 mM) decreased (b) cell viability in a dose- and time-dependent manner, c increased IL-6, IL-8, ICAM-1, and MCP-1 protein secretion by ELISA, and c increased ROS production compared with control. n = 3; *p < 0.05 and **p < 0.01 vs. control; NG, control

Fig. 2

Combined stimulation with high glucose and hypoxia increases inflammation and oxidative stress in HUVECs. Cells were treated with high glucose (25 mM) or with combined exposure to high glucose and hypoxia. Glucose alone or combined stimulation with both factors significantly increased mRNA expression of IL-6 (a), IL-8 (b), ICAM-1 (c), MCP-1 (d) after 6, 12, 24, 48 h, and similarly increased protein secretion at 48 h (e), increased cell ROS production (f–h), increased MDA level (i), and reduced SOD activity (j) for 48 h compared with controls. n = 3; p < 0.05 and **p < 0.01 vs. control. #p < 0.05 vs. glucose. HG, high glucose; HG + Hypoxia, combined stimulus with high-glucose and hypoxia; NG, control

Fig. 3

Effects of inhibition of HIF-1α on expression of inflammation after hyperglycemic and hypoxia. a Cells were treated with glucose (25 mM) or with combined exposure to high glucose and hypoxia for 6, 12, 24, and 48 h. Exposure to glucose alone or combined stimulation with hypoxia significantly increased gene expression of HIF-1α. b Cells were treated with KC7F2 (10 µM) or si-HIF-1α for 48 h following combined stimulation, and IL-6, IL-8, ICAM-1, MCP-1, and c HIF-1α levels were analyzed. The relative density of IL-6, IL-8, ICAM-1, and MCP-1 (d) was normalized according to GAPDH expression. Importantly, the downregulation of HIF-1α decreased the secretion of IL-6, IL-8, ICAM-1 and MCP-1 based on ELISA and qRT-PCR assays (e–f). n = 3; *p < 0.05 and **p < 0.01 vs. DMSO. DMSO-treated cells, DMSO; HIF-1α inhibitors KC7F2 (10 µM)-treated cells, KC7F2 (10 µM); si-HIF-1α, small interfering RNA HIF-1α; HG, high glucose; HG + Hypoxia, combined stimulus with high glucose and hypoxia; NG, control

Fig. 4

Effects of inhibition of HIF-1α expression on oxidative stress under hyperglycemia and hypoxia. Assays showing ROS (a–d), MDA (e) and SOD activity (f) compared with control. Downregulation of HIF-1α decreased expression of ROS and MDA, and increased SOD activity significantly compared with the control group following combined stimulation. n = 3; *p < 0.05 and **p < 0.01 vs. DMSO. DMSO-treated cells, DMSO; HIF-1α inhibitors KC7F2 (10 µM)-treated cells, KC7F2 (10 µM); si-HIF-1α, small interfering RNA HIF-1α; HG, high glucose; HG + Hypoxia, combined stimulus with high glucose and hypoxia; NG, control

Fig. 5

Gene expression of HUVECs induced by high glucose and hypoxia exposure by RNA-sEq. a Volcano plot was used to show the difference in gene expression values between the control group and the high glucose hypoxia group. Grey dotted line indicates the threshold for p < 0.05 and log FC < 2. Blue and red points represent down-regulated and up-regulated DEGs respectively. GO (b) and KEGG enrichment analysis (c) of differentially expressed genes (correct p < 0.05). Biological processes are indicated in red, and red bubbles show KEGG pathways

Fig. 6

Heatmaps of HUVECs induced by high glucose and hypoxia exposure by RNA-sEq. a Heatmaps of differentially expressed genes related to the inflammatory response (b), regulation of immune response (c), response to oxidative stress (d), histone demethylation (e), response to angiogenesis (f), response to hypoxia (f), and regulation of angiogenesis (g)

Fig. 7

Inhibition of HIF-1α decreases inflammation and oxidative stress in HUVECs via JMJD1A induced by high glucose and hypoxia. a Quantitative gene expression changes of JMJD families under high glucose and hypoxia. Cells were treated with high glucose and hypoxia for 6, 12, 24, and 48 h, qRT-PCR (b) and western blotting (c) showing the expression of JMJD1A. d Cells were treated with or without KC7F2 (10 µM) before exposure to high glucose and hypoxia for 48 h. The total proteins of HIF-1α and JMJD1A were collected and analyzed using western blotting as described under Materials and Methods. The relative densities of HIF-1α and JMJD1A were calculated according to GAPDH. e Cells were treated with high glucose and hypoxia for 24 h. ChIP assay showing HIF-1α bound to the JMJD1A promoter in vitro. The promoter regions of JMJD1A (–2.5 kb to –500 bp) were amplified using the input and immunoprecipitated DNA as templates. g Cells were treated with si-HIF-1α/KC7F2 and JMJD1A overexpression induced by high glucose and hypoxia for 48 h. ELISA and flow cytometry showing the secretion of (g) IL-6, ICAM-1, and h ROS. n = 3; *p < 0.05 and **p < 0.01 vs. DMSO. DMSO-treated cells, DMSO; HIF-1α inhibitors KC7F2 (10 µM)-treated cells, KC7F2 (10 µM); HG, high glucose; HG + Hypoxia, combined stimulus with high glucose and hypoxia; NG, control

Fig. 8

Knockdown of JMJD1A in HUVECs decreases inflammation and oxidative stress injury induced by high glucose and hypoxia. a Knockdown of JMJD1A under high-glucose and hypoxic conditions in HUVECs. Western blotting analysis of JMJD1A levels (b, c) in HUVECs stably transduced with shJMJD1A and control scrambled shRNA (shCtrl), and cultured in normal or high-glucose and hypoxic conditions for 6, 12, 24, and 48 h. Cells were transfected with control shRNA or shJMJD1A for 48 h following combined stimulation, and qPCR (d) and ELISA for IL-6, IL-8, ICAM-1, and MCP-1 (e) were performed. Downregulation of JMJD1A decreased the secretion of IL-6, IL-8, and MCP-1, but did not affect ICAM-1 secretion. Assays for ROS (f and g), MDA (h), and SOD activity (i) are shown compared with controls. JMJD1A shRNA decreased the expression of ROS and MDA level, and increased SOD activity markedly compared with control shRNA-transfected cells. n=3; *p<0.05 and **p<0.01 vs. control shRNA-transfected cells. HG, high glucose; HG+Hypoxia, combined stimulus with high glucose and hypoxia; NG, control; shCtrl, control shRNA

Fig. 9

Gene expression of shJMJD1A HUVECs induced by high glucose and hypoxia exposure by RNA-sEq. a Volcano plot was used to show the difference in gene expression values between the shCtrl and shJMJD1A induced by high glucose and hypoxia. Grey dotted line indicates the threshold for p < 0.05 and log FC < 2. Blue points represent down-regulated and red points represent up-regulated differentially expressed genes. GO (b) and KEGG enrichment analysis (c) of differentially expressed genes (correct_P ≤ 0.05). Biological processes are indicated in red, and red bubbles show KEGG pathway

Fig. 10

RNA-seq revealed that JMJD1A knockdown ameliorates injury by FOS/FOSB under high glucose and hypoxia. a–b Heatmaps of differentially expressed genes related to response to oxidative stress (a) and inflammatory response, regulation of immune response (b). c–d Venn diagram shows genes regulated by JMJD1A only under high glucose and hypoxia in HUVECs. e PPI network of the high-glucose- and hypoxia-induced HUVECs by silencing JMJD1A. The size of the nodes was positively related to the degree of nodes; red triangles indicate transcription factors, while blue nodes indicate DEGs. f–g Results of RNA-seq (f) and qRT-PCR (g) for core transcription factors. PPI, protein–protein interaction; HG, high glucose; HG + Hypoxia, combined stimulus with high glucose and hypoxia; NG, control; shCtrl, control shRNA