MO037EPIGENETIC REGULATION OF KLOTHO IN PERIPHERAL BLOOD CIRCULATING CELLS IS ASSOCIATED CELLS IS ASSOCIATED WITH SOLUBLE PROTEIN SERUM IN CARDIOVASCULAR DISEASE
Authors: Pérez-Castro Atteneri , Martín-Núñez Ernesto , Donate-Correa Javier , Ferri Carla , Tagua Víctor G , González-Luis Ainhoa , Delgado-Molinos Alejandro , López-Castillo Angel , Rodríguez-López Sergio , Cerro-López Purificación , Mora-Fernández Carmen , Navarro-González Juan F 
Published: May 2021

Abstract

Background and Aims
The antiaging factor α-Klotho (KL) has been related to cardiovascular diseases (CVD), including coronary artery disease, heart failure and peripheral arterial disease. The soluble form of this protein (sKL), which is mainly produced by the kidneys, have demonstrated to display different protective effects on the cardiovascular system, e.g., prevention of vascular calcification, oxidative stress, or cardiac fibrosis. Likewise, different clinical studies have pointed out that CVD is a state of sKL deficiency, even when the renal function is still preserved. It has been demonstrated that peripheral blood circulating cells (PBCCs) also express KL, but little is known about its expression profile during CVD. The aim of this work is to determinate if the expression of KL gene and its promoter methylation in PBCCs are correlated with serum levels of the soluble protein, and if there exist any association with the underlying inflammatory process that occurs during CVD.

Method
Forty-four patients diagnosed with clinical atherosclerotic vascular disease (case group) and 15 subjects without CVD background (control group) were included in the study. Whole blood and serum samples were obtained from all participants. We performed gene expression analysis for KL expression in PBCCs by qPCR, as well as for DNMT1 and DNMT3A (members of the DNA-methyltransferases family), TNF, IL10 and NFKB1 (inflammatory parameters). We assessed the degree of methylation of the KL gene promoter in these cells by bisulfite sequencing. Furthermore, we determined circulating levels of sKL, TNFα and IL10 by ELISA immunoassay in serum samples of both groups.

Results
Results showed a lower expression of KL gene in PBCCs of patients with CVD compared to controls (45% reduction, P&lt;0.001), which was accompanied by a higher degree of methylation of its promoter (36.7±6.3% vs. 15.9±4.8%, P&lt;0.05). The case group also presented significantly higher levels of DNMT1 and DNMT3A transcripts in these cells (P&lt;0.0001 for both), as well as higher values of proinflammatory parameters (gene expression of TNF and NFKB1, and TNF/IL10 ratio; P&lt;0.001 for all of them). Serum levels of sKL were decreased in the CVD group compared to controls (a reduction of 57.5%, P&lt;0.0001). KL expression in PBCCs correlated directly with systemic levels of sKL (r=0.20, P&lt;0.05), but inversely with expression of DNMT1 (r=-0.33, P&lt;0.01) and inflammatory parameters (r=-0.37, P&lt;0.01 for NFKB1 expression; r=-0.38, P&lt;0.01 for TNF/IL10 expression ratio; and r=-0.39, P&lt;0.01 for serum TNFα/IL10 ratio). No significant associations were found between inflammatory markers and methylation degree of KL gene promoter.

Conclusion
These results indicate an association between epigenetic regulation of KL gene in PBCC and systemic levels of the soluble protein in CVD, which could be influenced by the proinflammatory state present in these disorders.