#3986 THE ROLE AND MECHANISM OF EZH2 IN VASCULAR CALCIFICATION ASSOCIATED WITH CHRONIC KIDNEY DISEASE
Authors: Pinglan Lin , Ye Chaoyang , Wu Ming 
Published: June 2023

Abstract

Background and Aims
Vascular calcification (VC) is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. EZH2 (Enhancer of zeste 2 homolog-2) is reported as a key epigenetic regulator involved in various kidney diseases such as acute kidney injury, renal fibrosis, diabetic nephropathy, and lupus nephritis. In this study, we aimed to investigated the role of EZH2 in chronic kidney disease associated vascular calcification.

Method
1. Patients and Radial Artery Analysis
Radial arteries with or without calcification from patients with end-stage renal disease that underwent arterial venous fistular surgery were collected for Von Kossa staining and immunohistochemical (IHC) staining.
2. Animal model of Chronic renal failure-Vascular Calcification (CRF-VC) (in vivo)
An adenine and phosphate (1.2%) diet-induced CRF mouse model was designed following an 8-week program. The thoracoabdominal arteries of mice and rats were dissected to assay Ca deposition and histological analysis. Plasma levels of Alkaline phosphate (ALP), and phosphate (Pi) were measured. The transcription level of EZH2 and Runt-related transcription factor 2 (Runx2) were measured by real-time PCR and IHC staining.
3. Aortic ring calcification (ex vivo)
Thoracic aortas were dissected from SD rat. To induce calcification, Pi (NaH2PO4/Na2HPO4 [pH = 7.4]) was added to HG-DMEM at a final concentration of 2.6 mmol/l. After the indicated incubation periods (3, 5, 7 days), samples were taken for Calcium deposition assay, Western blotting and histological analysis.
4. Cell culture and treatment (in vitro)
Calcification of rat smooth muscle embryonic thoracic aorta cell line A7r5 (VSMC) was induced by incubation in calcifying media. 3-deazaneplanocin A (3-DZNeP), a carbocyclic analog of adenosine was added right after Pi to block EZH2 activation. After the indicated incubation periods, samples were collected for Calcium deposition assay and Western blotting analysis.

Results
1. In vivo, EZH2 protein level was increased in calcified radial arteries from patients with end-stage renal disease that underwent arterial venous fistular operation. In CRF-VC mice model, the aortic calcium deposition markedly aggravated in the group induced by adenine diet. Severe vascular calcification was also induced as shown by increased intensity of von Kossa. Moreover, IHC and q-PCR results showed that the protein and transcriptional levels of EZH2 expression were upregulated in calcified aortas of CRF mice, in paralleled with the increased expression Runx2 in protein and transcriptional levels.
2. Ex vivo, histological analysis revealed significant medial vascular calcification in the aortic ring culture with this calcifying medium for 7 days. Similarly, WB results showed that the expression of EZH2 and its downstream, methylation of Histone H3 at lysine 27 (H3K27me3), were significantly enhanced after high Pi medium stimulation while the osteogenic markers Runx2, Osteopontin (OPN) and Msh homeobox 2 (Msx2) protein levels were upregulated and the expression of VSMC differentiation markers α-Smooth muscle actin (α-SMA), Calponin (CNN) and Smooth muscle protein 22 (SM22) were downregulated.
3. In vitro, the osteogenic markers Runx2, OPN and Msx2 were significantly upregulated in high Pi treated VSMCs compared to control VSMCs. Moreover, the epigenetic markers EZH2 and H3K27me3a were markedly repressed in calcifying VSMCs by time.
4. 3-DZNep treatment reduced the calcium deposition in a concentration dependent manner. Western blotting showed that 3-DZNep treatment blocked the increased expression of osteogenic markers Runx2, Bone morphogenetic protein 2 (BMP2) as well as epigenetic markers EZH2 and H3K27me3a by high Pi treatment, indicating that inhibition of EZH2 attenuates VSMCs Calcification.

Conclusion
Our study revealed that EZH2 is highly expressed in calcified vascular tissues of CRF patients and CRF mice, rat aorta culture and VSMCs calcification models, and EZH2 inhibitor 3-DZNep attenuated calcification of VSMCs. EZH2 could be a promising target for treatment of vascular calcification in CRF patients.