MO031KLOTHO GENE PROMOTER METHYLATION IN THE VASCULATURE IS RELATED TO INFLAMMATION IN HUMAN ATHEROSCLEROSIS
Authors: Martín-Núñez Ernesto , Donate-Correa Javier , Ferri Carla , Tagua Víctor G , González-Luis Ainhoa , Rodríguez-López Sergio , Cerro-López Purificación , López-Castillo Angel , Delgado-Molinos Alejandro , Ontoria-Aguilera Eduardo , Mora-Fernández Carmen , Navarro-González Juan F 
Published: May 2021

Abstract

Background and Aims
Atherosclerosis, a vascular pathological process with an important inflammatory component, underlies most of the cardiovascular diseases (CVD). Deficiencies in the antiaging factor α-Klotho (KL) have been related to the appearance and progression of atherosclerotic damage associated with CVD. In addition, some studies have demonstrated that KL gene is expressed in human arteries and that low vascular KL expression levels are related with clinical atherosclerotic disease. Among the epigenetic mechanisms that regulate the expression of the KL gene, methylation of the CpG islands in the promoter region is one of the most studied. In this pilot study, we propose that this mechanism participates in the regulation of vascular KL expression and contributes to the deficiency in vascular KL levels observed in CVD. Moreover, we determined the influence of different inflammatory components in the methylation status of KL promoter.

Method
We developed a case-control study that included 25 patients with diagnosis of clinical atherosclerotic vascular disease undergoing elective vascular surgery and 15 cadaveric organ donors with no medical history of CVD, respectively. Vascular fragments were retrieved from all subjects, and the degree of methylation of the KL gene promoter was assessed by bisulfite sequencing. Additionally, vascular gene expression in both groups was assessed by qPCR for KL, two DNA methyltransferases (DNMT1 and DNMT3A), and three inflammatory-related loci (TNF, IL10 and NFKB1). Serum concentrations of TNFα and IL10 were measured by ELISA immunoassay, and complementary inflammatory parameters (CRP and neutrophil/lymphocyte ratio) by standardized routine methods.

Results
The vascular tissue of patients in the case group presented a higher percent level of methylated positions in the KL promoter region (71.4±8.3% vs. 39.4±12.3%, P&lt;0.05), as well as a significant decrease in the expression of KL gene (a 40% reduction as compared to controls, P&lt;0.05). Considering the whole study population, the methylation degree was inversely related to the endogenous transcript levels of KL gene (r=-0.66, P&lt;0.0001). Moreover, the expression of DNMT1 was significantly increased in the case group (P&lt;0.05), as well as those of the proinflammatory genes TNF and NFKB1 (P&lt;0.0001 and P&lt;0.05, respectively), being all of them directly associated with the vascular methylation levels of KL gene (r=0.51, P&lt;0.0001 for DNMT1; r=0.45, P&lt;0.001 for TNF; r= 0.37, P&lt;0.05 for NFKB1). Furthermore, vascular expression of DNMT1 and DNMT3A directly correlated with both inflammatory markers in this tissue (P&lt;0.0001, for all). Systemic levels of IL10 presented a direct association with vascular KL gene expression (r=0.28, P&lt;0.05), while inverse associations were observed with the methylation degree of KL gene (r=-0.38, P&lt;0.05) and DNMT1 expression (r=-0,29, P&lt;0.05).

Conclusion
These results suggest that the methylation of KL gene promoter in the vasculature is a mechanism associated with the inflammatory response that would partly explain its reduction during atherosclerotic injury.