High glucose decreased cell viability and increased inflammation and ROS production in HUVECs
HUVECs exposed to different concentrations of high glucose (25 and 30 mM) exhibited lower cell survival, with 53.77 and 51.69% of cells surviving relative to normal conditions for 24 h and 49.29 and 52.1% for 48 h, respectively, showing the significantly cytotoxic effect of high glucose (p < 0.05, n = 3) (Fig. 1a and Additional file 1: Figure S1). Furthermore, to assess the inflammatory status of HUVECs after high-glucose- and hypoxia-induced injury, we first sought to determine the effects of the two different glucose concentrations on protein secretion of IL-6, IL-8, ICAM-1, and MCP-1. As shown in Fig. 1b, treatment with various concentrations of high glucose for 48 h increased IL-6, IL-8, and MCP-1 protein secretion (by 1.12-, 1.41-, and 2.20-fold at 25 mM glucose and 1.06-, 1.80-, and 2.36-fold at 30 mM glucose) compared with controls. Similarly, various concentrations of high glucose also affected ROS production in HUVECs. Compared with untreated normal cultures, 48 h stimulation with high glucose (25 and 30 mM) resulted in a 1.24- and 1.47-fold promotion of ROS production (p < 0.05, n = 3) (Fig. 1c). In addition, high-glucose treatment at 25 and 30 mM produced similar increases. Therefore, cells were treated under high-glucose conditions for 25 mM in subsequent experiments.
Hypoxia enhanced high-glucose-induced inflammation and oxidative production in HUVECs
To analyze the pathogenic role of hypoxia in diabetic vascular injuries, a HUVEC high-glucose and hypoxia model was established. We assayed the inflammation of HUVECs induced by high glucose (25 mM) for 6, 12, 24, and 48 h. Our results showed that high glucose affected IL-6 (by 19.2-, 9.39-, 6.83-, 3.75-fold), IL-8 (by 6.75-, 5.37-, 5.49-, 5.10-fold), ICAM-1 (by 5.43-, 6.35-, 5.21-, 6.29-fold), and MCP-1 (by 1.66-, 1.17-, 1.45-, 0.49-fold) mRNA expression in HUVECs cultured for 6, 12, 24 and 48 h compared with controls. Next, we addressed the effects of the combination of the two stimuli on the mRNA expression of inflammatory factors. Compared with the combined stimulus, 6 and 12 h stimulation with high glucose significantly increased the protein secretion of IL-6 (to 2.79- and 1.95-fold) and IL-8 (to 1.67- and 1.52-fold), respectively. However, a large, time-dependent increase in IL-6, IL-8, ICAM-1, and MCP-1 mRNA expression compared with the control or glucose was observed at 48 h. Specifically, the combined stimulus increased mRNA expression of IL-6 (by 2.60- and 3.75- fold), IL-8 (by 1.34- and 6.82-fold), ICAM-1 (by 2.24- and 14.08-fold), and MCP-1 (by 5.84- and 2.84-fold) significantly, compared with the control or high glucose for 48 h, respectively (Fig. 2a–d). Meanwhile, we addressed the effects of the single and combination stimuli for 48 h on the protein secretion of inflammatory factors. A single stimulus with glucose induced IL-8 and MCP-1 protein secretion by 1.40- and 2.40-fold relative to the control, although IL-6 and ICAM-1 protein secretions were not significantly affected. Importantly, the combined stimulus increased the secretion of IL-6, IL-8, ICAM-1, and MCP-1 by 3.48-, 2.90-, 5.52-, and 2.65-fold, respectively, compared with controls, and induced a 2.75-, 2.06-, and 3.42-fold increase in IL-6, IL-8, and ICAM-1 protein secretion compared with glucose alone (Fig. 2e). These findings indicated that a regulatory network involving inflammatory factors might participate in HUVEC damage induced by these two factors. All cells were treated under hypoxic conditions for 48 h in subsequent experiments.
Conversely, our study showed that simultaneous incubation with high glucose (25 mM) and hypoxia for 48 h increased ROS production and MDA levels (by 3.82- and 2.14-fold) compared with controls, and decreased SOD activity by 0.40-fold, respectively (Fig. 2f–j). Overall, these results suggested that high-glucose and hypoxic conditions could increase inflammation and the expression of oxidative stress factors in HUVECs, particularly during simultaneous exposure to both stimuli.
Inhibition of HIF-1α in hyperglycemia-hypoxia decreased inflammation and oxidative stress in HUVECs
HIF-1α is a hypoxia regulatory protein that plays an essential role in regulating the process of inflammatory and oxidative stress in hypoxia. To assess the role of HIF-1α during hypoxic progression in HUVECs after high-glucose- and hypoxia-induced injury, we first tried to verify the effects of the two stimuli individually or in combination with HIF-1α expression. In our study, HUVECs exposed to 6, 12, 24, and 48 h of glucose (25 mM) exhibited an increase in HIF-1α mRNA levels of 8.15-, 5.93-, 7.63-, and 8.78-fold compared with controls, respectively. The simultaneous incubation with high glucose (25 mM) followed by exposure to hypoxia for 24 and 48 h increased HIF-1α levels (by 1.51-, 11.55-, 2.00-, and 17.62-fold at the mRNA level) compared with high glucose or controls, respectively. However, when cells were treated for 6 and 12 h, the combined stimuli increased HIF-1α expression (7.37- and 8.70-fold at the mRNA level) compared with controls, but there was no statistically significant difference compared with high glucose (Fig. 3a). These results suggested that high glucose and hypoxia could induce HIF-1α expression in cultured HUVECs, particularly during simultaneous exposure to both stimuli.
It was previously reported that HIF-1α was closely associated with cell inflammatory and oxidative stress proteins. In order to clarify the potential regulatory network related to HIF-1α after exposure to high glucose and hypoxia, a cell high-glucose and hypoxia model was established. Cells were then treated with a specific inhibitor of HIF-1α KC7F2 and siRNA HIF-1α. The relative protein and gene expression levels of inflammatory factors were analyzed by qRT-PCR, western blotting, and ELISA tests under hypoxic and high-glucose environments (Fig. 3a–f). The results revealed that the protein secretion of IL-6, IL-8, ICAM-1, and MCP-1 in the KC7F2 and si-HIF-1α groups decreased significantly in comparison to that of the DMSO and empty vector-treated groups after the combined stimulus (Fig. 3a–f). In addition, the downregulation of HIF-1α reduced ROS production and MDA release (by 0.64- and 0.66-fold) respectively, and increased SOD activity by 5.28-fold compared with the DMSO-treated group (Fig. 4a–f). These results suggested that downregulation of HIF-1α inhibited inflammation and oxidative stress in HUVECs exposed to combined stimulus-mediated injury. Therefore, these data supported the hypothesis that HIF-1α might accelerate cell inflammation and oxidative stress injury under hyperglycemic and hypoxic conditions.
RNA sequencing revealed that JMJD1A could be a potential epigenetic regulator in endothelial injury induced by hyperglycemia and hypoxia
To uncover the underlying mechanisms by which HIF-1α promoted cellular oxidative stress and inflammatory progression, we analyzed the gene expression profiles of high glucose and hypoxia by using high-throughput RNA sequencing. Three samples of each group were sequenced. A total of 269,878,950 reads in the control group (on average 89,959,650 reads per sample, ranging from 41,113,776 to 123,552,542 reads) and 125,009,922 reads in the high-glucose and hypoxia group (average 41,669,974 reads per sample, range from 39,797,748 to 43,600,994 reads) passed the quality control. A total of 96.33% of total reads were mapped to the Homo sapiens genome Ensembl GRCh38. 24,366 genes with counts greater than 10 in six samples were identified for further analysis.
DEGs were analyzed using DEseq2. When the threshold was adjusted to a p-value < 0.05, 1353 DEGs were selected and of these 554 genes were up-regulated, while 799 were down-regulated. DEGs are listed in Additional file 3: Table S2. Importantly, IL-8, ICAM-1, and IL-6 were significantly up-regulated, induced by high glucose and hypoxia (Fig. 5a).
To examine processes and pathways that may be altered upon exposure to high glucose and hypoxia, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. Dozens of biological processes (BPs) were identified, including the inflammatory response, regulation of immune response, response to hypoxia, response to oxidative stress, regulation of angiogenesis, and histone demethylation (Fig. 5b and Additional file 4: Table S3). Regulation of transcription, DNA-template, multicellular organism development, cell differentiation, positive regulation of transcription by RNA polymerase II, negative regulation of transcription by RNA polymerase II, cell adhesion, ion transport, transmembrane transport, angiogenesis, and response to hypoxia were the top 10 enriched BP terms. These results showed that the BPs that involved cell regulation of transcription were unbalanced, especially in BPs including angiogenesis and response to hypoxia, and cell adhesion. The top 30 GO terms with the highest enrichment factor are shown in Fig. 5b. We performed the KEGG pathway enrichment analysis to gain further insights into the function of genes and their interaction for high glucose and hypoxia in HUVECs, and found 322 pathway terms including 55 pathway terms with p < 0.05. The top 10 pathways with the greatest enrichment were the MAPK signaling pathway, transcriptional mis-regulation in cancer, human T-cell leukemia virus 1 infection, cell adhesion molecules, cytokine-cytokine receptor interaction, Hippo signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, advanced glycation end products, and the receptor for advanced glycation end products (AGE-RAGE) signaling pathway in diabetic complications and cellular senescence. The top 20 enriched pathways are presented in Fig. 5c. Further, the specific genes associated with signaling pathways or biological processes including the associated inflammatory response, regulation of the immune response, response to hypoxia, response to oxidative stress, regulation of angiogenesis, and histone demethylation in high-glucose and hypoxic conditions are shown in Fig. 6a–g; they were consistent with previous studies.
Inhibition of HIF-1α decreased inflammation and oxidative stress in HUVECs via JMJD1A induction by exposure to high glucose and hypoxia
We screened the expression of H3K9 demethylating enzymes of the JMJD families by RNA-sEq. Of these, three demethylating enzymes, KDM3A, KDM4B, and KDM7A, were differentially expressed between the two groups. JMJD1A (KDM3A) showed more than a 2-fold increase under high-glucose and hypoxic conditions in HUVEC (Fig. 7a). The differential expression for JMJD1A was further confirmed by qRT-PCR (Fig. 7b) and western blotting (Fig. 7c). These data suggested that JMJD1A was up-regulated in the high-glucose and hypoxic conditions and might be involved in the specific regulation of HUVEC injury. Next, to analyze the pathogenic role of JMJD1A in vascular injuries induced by HIF-1α under high-glucose and hypoxic conditions, inhibition of HIF-1α using KC7F2 and a si-HIF-1α assay was performed. Our study showed that high-glucose and hypoxia stimulated the expression of HIF-1α and JMJD1A, which was attenuated by pre-treatment with KC7F2 (Fig. 7d). Under the same conditions, a CHIP-qPCR assay was also performed in which chromatin was immunoprecipitated with an anti-HIF-1α antibody and the JMJD1A promoter region (from − 432 bp to − 371 bp) was amplified by PCR. As expected, the conventional CHIP-qPCR assay in HUVECs confirmed that JMJD1A promoter fragments containing potential hypoxia responsive element (HRE) sites could be more significantly immunoprecipitated by a specific HIF-1α antibody than by the anti-lgG antibody (Fig. 7e–f). The ameliorative effects of si-HIF-1α and KC7F2 on ROS and inflammation cytokines, specifically IL-6 and ICAM-1 in high-glucose and hypoxia-induced HUVECs, respectively, were reversed by JMJD1A overexpression (Fig. 7g–h). These data suggested that HIF-1α could reduce the inflammatory and oxidative stress levels in a JMJD1A-dependent manner in the presence of high glucose and hypoxia.
Knockdown of JMJD1A inhibited expression of inflammation and oxidative stress in HUVECs after hyperglycemia and hypoxia stimulus
Although it is known how inflammatory and oxidative stress genes function in the high-glucose and hypoxic environment of diabetic vascular disease, it is not clear whether the histone H3K9 demethylation enzyme JMJD1A regulates this process. Therefore, we aimed to analyze the effects of JMJD1A on inflammatory proteins including IL-6, IL-8, ICAM-1, and MCP-1 under hyperglycemia and hypoxic stimulation of HUVECs. We transduced a green fluorescent protein (GFP)-scramble control and a GFP-shJMJD1A into cells, using a lentiviral vector (Fig. 8a). Changes in expression of JMJD1A were also examined under high-glucose and hypoxic conditions for 6, 12, 24, and 48 h to identify the reduction compared with GFP-scrambled cells (Fig. 8b–c). We found that although the downregulation of JMJD1A in cells exposed to the combined stimulus did not affect ICAM-1 secretion, significant decreases were observed in IL-6, IL-8, and MCP-1 secretion (0.46-, 0.33-, and 0.56-fold at the mRNA level and 0.13-, 0.18-, and 0.11-fold at the protein level, respectively), compared with control shRNA transfection (Fig. 8d–e). We therefore inferred that JMJD1A played an important role in co-stimulation-induced injury through the effect on the secretion or function of inflammatory proteins.
Next, we tested whether JMJD1A modulated cell oxidative stress under this condition. The downregulation of JMJD1A reduced ROS and MDA production by 0.75- and 0.58-fold compared with control shRNA-transfected cells, and increased SOD activity by 2.24-fold (Fig. 8f–i). These results suggested that JMJD1A inhibited oxidative stress in the cells exposed to combined stimulus-mediated injury. Overall, these data supported the hypothesis that JMJD1A might accelerate cell inflammation and oxidative stress under the combined high-glucose and hypoxic-induced injury in HUVECs.
RNA-seq revealed that JMJD1A knockdown may ameliorate the high-glucose and hypoxia-induced endothelial injury via FOS/FOSB
Although JMJD1A expression was very weak in normal oxygen, it markedly increased under high-glucose and hypoxic conditions. This led us to investigate the transcription factors involved in JMJD1A induction under high glucose and hypoxia. In analysis by RNA-seq, using a threshold adjusted to p < 0.05, 777 DEGs were selected, of which 302 genes were up-regulated, while 475 were down-regulated in shJMJD1A-treated HUVECs compared with controls transfected with shRNA under high-glucose and hypoxic conditions (Fig. 9a). The DEGs are listed in Additional file 5: Table S4. Further, we performed a comprehensive expression analysis of genes that reversed the high-glucose and hypoxia-induced endothelial injury in the shJMJD1A-transduced HUVECs.
To further understand the association between JMJD1A and high-glucose and hypoxia-induced EC injury in HUVECs, we screened genes whose expression increased and decreased more than 2-fold in the sample and performed a GO analysis. Our findings showed that the inflammatory response, oxidation-reduction processes, cell adhesion, negative regulation of cell proliferation, angiogenesis, mitogen-activated protein kinase (MAPK) cascade, positive regulation of cell migration, positive regulation of gene expression, axon guidance, and cellular responses to fibroblast growth factor stimulus were the Top 10 enriched BP terms (Additional file 6: Table S5). These results showed that the cell response was unbalanced, especially in the inflammatory response, in oxidation-reduction processes, and in cell adhesion. The top 30 GO terms with the highest enrichment factor are shown in Figs. 9b and 10a–b. We performed KEGG pathway enrichment analysis to provide further insights into the function of genes and their interaction for cells harboring knockdown of JMJD1A, and found 301 pathway terms including 57 pathway terms with a p < 0.05. The top 10 pathways showing the greatest enrichment were cell adhesion molecules, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, axon guidance, human cytomegalovirus infection, human T-cell leukemia virus 1 infection, fluid shear stress, atherosclerosis, pertussis, hypertrophic cardiomyopathy, transcriptional mis-regulation in cancer, and focal adhesion pathways. The top 20 enriched pathways were presented in Fig. 9c.
Next, we identified 398 genes that showed increased expression under high-glucose and hypoxic conditions, which were downregulated by shJMJD1A, or genes with decreased expression in high glucose and hypoxia that were upregulated by shJMJD1A (Fig. 10c–d and Additional file 7: Table S6). Next, the 398 significant JMJD1A-related genes were used to construct a protein–protein interaction (PPI) network. As shown in Fig. 10e, the PPI network consisted of 50 nodes and 56 interactions. Many genes were highly connected, including: FOS, EGR1, FOSB, ATF3, NR4A1, E2F2, RELB, HOXA7, and BCL6. The relative gene expression levels of the core transcription factors were analyzed by qRT-PCR and RNA-seq under a hypoxic and high-glucose environment. The results indicated that the expression of FOS, EGR1, FOSB, ATF3, NR4A1, RELB, and BCL6 in the hypoxia and high-glucose treated group increased significantly in comparison to that of the control group, and decreased in the shJMJD1A group (Fig. 10f–g). Details for the top 10 JMJD1A-related genes and transcription factors are listed in Additional file 8: Table S7.