Downregulation of the endothelial histone demethylase JMJD3 is associated with neointimal hyperplasia of arteriovenous fistulas in kidney failure

Jumonji domain–containing protein-3 (JMJD3), a histone H3 lysine 27 (H3K27) demethylase, promotes endothelial regeneration, but its function in neointimal hyperplasia (NIH) of arteriovenous fistulas (AVFs) has not been explored. In this study, we examined the contribution of endothelial JMJD3 to NIH of AVFs and the mechanisms underlying JMJD3 expression during kidney failure. We found that endothelial JMJD3 expression was negatively associated with NIH of AVFs in patients with kidney failure. JMJD3 expression in endothelial cells (ECs) was also downregulated in the vasculature of chronic kidney disease (CKD) mice. In addition, specific knockout of endothelial JMJD3 delayed EC regeneration, enhanced endothelial mesenchymal transition, impaired endothelial barrier function as determined by increased Evans blue staining and inflammatory cell infiltration, and accelerated neointima formation in AVFs created by venous end to arterial side anastomosis in CKD mice. Mechanistically, JMJD3 expression was downregulated via binding of transforming growth factor beta 1–mediated Hes family transcription factor Hes1 to its gene promoter. Knockdown of JMJD3 enhanced H3K27 methylation, thereby inhibiting transcriptional activity at promoters of EC markers and reducing migration and proliferation of ECs. Furthermore, knockdown of endothelial JMJD3 decreased endothelial nitric oxide synthase expression and nitric oxide production, leading to the proliferation of vascular smooth muscle cells. In conclusion, we demonstrate that decreased expression of endothelial JMJD3 impairs EC regeneration and function and accelerates neointima formation in AVFs. We propose increasing the expression of endothelial JMJD3 could represent a new strategy for preventing endothelial dysfunction, attenuating NIH, and improving AVF patency in patients with kidney disease. Jumonji domain–containing protein-3 (JMJD3), a histone H3 lysine 27 (H3K27) demethylase, promotes endothelial regeneration, but its function in neointimal hyperplasia (NIH) of arteriovenous fistulas (AVFs) has not been explored. In this study, we examined the contribution of endothelial JMJD3 to NIH of AVFs and the mechanisms underlying JMJD3 expression during kidney failure. We found that endothelial JMJD3 expression was negatively associated with NIH of AVFs in patients with kidney failure. JMJD3 expression in endothelial cells (ECs) was also downregulated in the vasculature of chronic kidney disease (CKD) mice. In addition, specific knockout of endothelial JMJD3 delayed EC regeneration, enhanced endothelial mesenchymal transition, impaired endothelial barrier function as determined by increased Evans blue staining and inflammatory cell infiltration, and accelerated neointima formation in AVFs created by venous end to arterial side anastomosis in CKD mice. Mechanistically, JMJD3 expression was downregulated via binding of transforming growth factor beta 1–mediated Hes family transcription factor Hes1 to its gene promoter. Knockdown of JMJD3 enhanced H3K27 methylation, thereby inhibiting transcriptional activity at promoters of EC markers and reducing migration and proliferation of ECs. Furthermore, knockdown of endothelial JMJD3 decreased endothelial nitric oxide synthase expression and nitric oxide production, leading to the proliferation of vascular smooth muscle cells. In conclusion, we demonstrate that decreased expression of endothelial JMJD3 impairs EC regeneration and function and accelerates neointima formation in AVFs. We propose increasing the expression of endothelial JMJD3 could represent a new strategy for preventing endothelial dysfunction, attenuating NIH, and improving AVF patency in patients with kidney disease. 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However, the role of JMJD3 in ECs during NIH development in AVFs and the mechanism of its expression regulation in CKD remain unknown.Herein, we investigate the association of endothelial JMJD3 with NIH of AVFs in patients with kidney failure and in mouse AVF models created by venous end to arterial side anastomosis configuration with chronic renal failure. We also explore the regulatory mechanism of endothelial JMJD3 expression in CKD.ResultsDecreased expression of endothelial JMJD3 is associated with NIH of AVFs from kidney failure patientsTo confirm the role of epigenetic regulation in NIH of AVFs, we examined histone modifications in NIH of AVFs from kidney failure patients. We found that JMJD3 was expressed in the nuclei of ECs (von Willebrand factor plus), and the expression of JMJD3 in the endothelium of venous anastomosis of AVF was reversely correlated with the size of neointima area of AVFs (p < 0.05, Fig. 1, A–D). The lesser expression level of JMJD3 in ECs, the larger area of NIH observed. Furthermore, lower expression of endothelial JMJD3 was accompanied by higher expression of mesenchymal marker α-smooth muscle actin (α-SMA) and endothelial injury component fibrinogen. Positive staining of α-SMA and fibrinogen can be found in the endothelium of venous anastomosis of AVF with varying degrees (Fig. 1, E and F), which indicate endothelial–mesenchymal transition (EndMT) and endothelium injury. Therefore, these results suggested that decreased JMJD3 expression in the endothelium could be associated with NIH in AVFs probably via regulating endothelial dysfunction.KO of JMJD3 in ECs promotes NIH of AVFs in CKD miceTo assess whether JMJD3 expression in ECs was regulated by uremia, we measured the expression of JMJD3 in blood vessel in normal control and CKD mice. The serum level of blood urea nitrogen (BUN) was remarkably increased from 6 weeks after subtotal nephrectomy in mice, which indicated that the CKD model was successfully established (Fig. 2A). JMJD3 expression was decreased significantly in vessels of CKD mice, especially in ECs (CD31+) of vein and aortas accompanied by increased H3K27me3 level (Fig. 2, B–E and S1). To examine the role of endothelial JMJD3 in vascular remodeling of AVF in CKD, JMJD3 was conditionally knocked out in ECs by breeding JMJD3f/f mice with vascular endothelial-cadherin (VE-cadherin)-CreERT2 mice (Fig. 3A). As expected, KO of JMJD3 in ECs led to a significant increase in H3K27me3 expression (Fig. 3, B and C). Subtotal nephrectomy and AVF were then created in ECJMJD3+/+ and ECJMJD3 KO mice (Fig. 3D). Though the serum BUN level was significantly higher in mice after subtotal nephrectomy, there was no statistically significant difference of BUN levels between ECJMJD3 KO mice and ECJMJD3+/+ mice observed either at 6 weeks or 10 weeks after CKD surgery, indicating that endothelial KO of JMJD3 did not aggravate renal dysfunction in mice (Fig. 3E). In AVFs, JMJD3 KO in ECs dramatically increased NIH and the ratio of neointima to lumen when compared with that in ECJMJD3+/+ mice (Fig. 3, F and G). There were more α-SMA+ and proliferating cell nuclear antigen-positive (PCNA+) cells in AVFs created in ECJMJD3 KO mice versus that in ECJMJD3+/+ mice (Fig. 3, H and I). Therefore, these findings indicated that JMJD3 KO in ECs could promote NIH and VSMC proliferation of AVF.Figure 2Expression of endothelial JMJD3 was decreased in vessels of CKD mice. A, serum BUN levels were detected in CKD mice before and after operation (n = 6). Data are presented as means ± SD. Statistical significance was measured using one-way ANOVA. B and C, Western blotting of JMJD3 and H3K27me3 expression in aortas from control (Ctl, n = 6) and CKD mice (n = 6). Quantification analysis of Western blotting shown as means ± SD. D and E, immunofluorescent staining of JMJD3 (green) or H3K27me3 (green) and CD31 (red) in the external jugular veins of control (Ctl, n = 6) and CKD mice (n = 6). Percentage of JMJD3 or H3K27me3-positive cells in total nuclei of endothelium from each sample was calculated (E). BUN, blood urea nitrogen; CKD, chronic kidney disease; H3K27, histone H3 lysine 27; JMJD3, Jumonji domain–containing protein-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3JMJD3 KO in ECs accelerates neointima formation. A, schematic for generating ECJMJD3 KO mice. B, ECJMJD3 KO and ECJMJD3+/+ were treated with tamoxifen. Immunofluorescent staining of JMJD3 (green) or H3K27me3 (green) and CD31 (red) in the external jugular veins of ECJMJD3+/+ and ECJMJD3 KO mice (n = 6). C, percentage of JMJD3 or H3K27me3-positive cells in total nuclei of endothelium from each sample was calculated. D, schematic for CKD-AVF mice model. CKD and AVFs were performed in the two groups of mice (ECJMJD3+/+ and ECJMJD3 KO). Sera were collected at the day before surgery of CKD, 6 weeks postsurgery of CKD and 4 weeks after creating AVF. AVFs were collected after 4 weeks of AVF surgery. E, serum BUN levels in ECJMJD3+/+ and ECJMJD3 KO CKD mice (n = 6) were measured. Data were presented as mean ± SD. Statistical significance was measured using two-way ANOVA. F and G, H&E staining of venous anastomosis of AVFs from ECJMJD3+/+ or ECJMJD3 KO CKD mice (n = 6). The black dotted line represents the boundary between neointima (N), lumen (L), media (M), and adventitia (A). Areas of neointima area of AVFs were calculated (G). H, immunostaining of α-SMA and PCNA in venous anastomosis of AVFs from ECJMJD3+/+ or ECJMJD3 KO (n = 6). Sections incubated with anti-rabbit secondary antibody but without primary antibody were used as negative control (NC). I, total numbers of α-SMA+ or PCNA+ cells in neointima area were calculated from five visual fields (40×) of each sample. All statistical analyses between groups were performed by Mann–Whitney test. Data were shown as means ± SD. α-SMA, α-smooth muscle actin; AVF, arteriovenous fistula; BUN, blood urea nitrogen; CKD, chronic kidney disease; EC, endothelial cell; H3K27, histone H3 lysine 27; JMJD3, Jumonji domain–containing protein-3; PCNA, proliferating cell nuclear antigen.View Large Image Figure ViewerDownload Hi-res image Download (PPT)JMJD3 deficiency in ECs associates with decreased EC regeneration, increased EndMT, inflammation, and fibrosis of venous anastomosisTo evaluate whether JMJD3 KO affects ECs' postsurgical reendothelialization, we examined Evans blue staining of which positive staining represents disruption of the endothelium. We found that the intensity and area of Evans blue in AVFs in ECJMJD3 KO CKD mice were increased approximately threefold as compared with the levels present in AVFs in ECJMJD3+/+ CKD mice (Fig. 4, A and B). In addition, compared with ECJMJD3+/+ mice, there was a dramatic decreased signal of CD31 (Fig. 4, C and D) and a large increased signal of mesenchymal marker, fibroblast-specific protein 1 (FSP-1), in endothelium of AVFs from ECJMJD3 KO mice (Fig. 4, E and F). These results indicate that endothelial JMJD3 KO delays endothelial regeneration and promotes EndMT in AVF.Figure 4JMJD3 deficiency in ECs is associated with increased barrier dysfunction, EndMT, inflammation, and fibrosis at venous anastomosis. A and B, at 4 weeks after placing AVF, Evans blue was administered intravenously followed by perfusion with PBS. The intensity of Evans blue leak was analyzed by ImageJ. C and D, immunofluorescent staining and intensity analysis of CD31 (red) in venous anastomosis of AVF from ECJMJD3+/+ CKD mice and ECJMJD3 KO CKD mice. E and F, IHC staining of FSP-1 in venous anastomosis from ECJMJD3+/+ and ECJMJD3 KO mice. Sections incubated with anti-rabbit secondary antibody but without primary antibody were used as negative control. Total numbers of FSP-1+ cell number in endothelium of venous anastomosis in AVFs were calculated. G and H, IHC staining of CD45 and Mac2 in venous anastomosis from ECJMJD3+/+ mice and ECJMJD3 KO mice. Sections incubated with antirat or anti-rabbit secondary antibody but without primary antibody were used as negative control (NC). Total numbers of CD45+ or Mac2+ cells in neointima area in AVFs were calculated. I, distribution of collagen was detected by Gomori's Trichrome staining (red color indicates muscle fibers, and green/blue color indicates collagen) and Sirius red staining (green color indicates type III collagen, and red–yellow indicates type I collagen). J, the ratio of area with red–yellow color to the whole area in Sirius red staining was calculated. There are six samples of each group. All statistical analyses between groups were performed by Mann–Whitney test. Data were represented as means ± SD. AVF, arteriovenous fistula; EC, endothelial cell; EndMT, endothelial–mesenchymal transition; FSP-1, fibroblast-specific protein 1; IHC, immunohistochemistry; JMJD3, Jumonji domain–containing protein-3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Furthermore, JMJD3 KO in ECs also stimulated more robust inflammatory responses evidenced by the infiltration of macrophages (Mac-2) and monocytes (CD45) in AVF versus that in ECJMJD3+/+ mice (Fig. 4, G and H). Trichrome and Sirius red staining showed that JMJD3 KO also increased the deposition of collagens in the media and neointima (Fig. 4, I and J).Decreased expression of JMJD3 attenuates EC functionTo further determine the function of JMJD3 in ECs, we analyzed its effects on the proliferation and migration of ECs. Compared with control, the expression of JMJD3 in ECs was successfully knocked down with single-guide RNA (sgRNA), whereas the methylation level of H3K27 was increased (Fig. 5A). Expression level of ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX) was not changed by knockdown of JMJD3 (Fig. 5A). Furthermore, expression of the PCNA was decreased by knockdown of JMJD3 (Fig. 5, A and B), whereas PCNA level was increased by overexpression of JMJD3 (Fig. 5, C and D). The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) results showed that knockdown of JMJD3 dramatically inhibited EC proliferation (Fig. 5E). To examine the effect of JMJD3 knockdown on endothelial migration, wound-healing assay was performed to examine the EC migration rate. Knockdown of JMJD3 resulted in a significant decrease in wound closure when compared with control (Fig. 5F). Moreover, inhibition of JMJD3 activity by GSK-J4 also significantly decreased EC proliferation and migration as well as the PCNA expression (Fig. S2).Figure 5Effects of downregulated JMJD3 on EC survival and migration. A, ECs infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl) for 2 days at 90% confluence in a 12-well plate. Western blot detected the expressions of JMJD3, UTX, PCNA and methylation of H3K27. Pictures are representative of three independent experiments. B, quantification of Western blot analyses of the expressions of JMJD3 and PCNA. Statistical significance was analyzed by one-way ANOVA. C, ECs infected with lentivirus-mediated overexpression of JMJD3 (JMJD3OE) for 2 days at 90% confluence in a 12-well plate. ECs infected with lentivirus packaging with empty vector were used as negative control (Ctl). Western blot was used to detect the expressions of PCNA and methylation of H3K27. Photos are representative of three independent experiments. D, quantification of Western blot analyses in B. Statistical significance was analyzed by one-way ANOVA. E, ECs were cultured in 96-well plate and infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl). EC proliferation was determined by MTS during different time points. Data are presented as mean ± SD from three replicates. Statistical significance between groups was analyzed by two-way ANOVA. F and G, ECs were cultured in 12-well plate and infected with lentivirus-mediated sgRNA targeting JMJD3 (sgJMJD3) or nonspecific sgRNA (Ctl). Wound-healing assay was used to detect the EC migration (F). The rate of migration was measured by quantifying the total distance that the cells moved from the edge of the scratch toward the center of the scratch at 2 days or 4 days when compared with 0 days (G). EC, endothelial cell; JMJD3, Jumonji domain–containing protein-3; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PCNA, proliferating cell nuclear antigen; sgRNA, single-guide RNA; UTX, ubiquitously transcribed tetratricopeptide repeat, X chromosome.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since the level of transforming growth factor beta 1 (TGFβ1) is increased in the plasma/serum of patients with kidney failure and CKD mice (34Fujisawa M. Haramaki R. Miyazaki H. Imaizumi T. Okuda S. Role of lipoprotein (a) and TGF-beta 1 in atherosclerosis of hemodialysis patients.J. Am. Soc. Nephrol. 2000; 11: 1889-1895Crossref PubMed Google Scholar, 35Junker U. Haufe C.C. Nuske K. Rebstock K. Steiner T. Wunderlich H. Junker K. Reinhold D. Elevated plasma TGF-beta1 in renal diseases: Cause or consequence?.Cytokine. 2000; 12: 1084-1091Crossref PubMed Scopus (33) Google Scholar, 36Loeffler I. Wolf G. 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