Hyperhomocysteinemia induces vascular calcification by activating the transcription factor RUNX2 via Krüppel-like factor 4 up-regulation in mice

Abstract

Edited by Qi-Qun Tang One of the main characteristics of atherosclerosis is vascular calcification, which is linked to adverse cardiovascular events. Increased homocysteine (Hcy), a feature of hyperhomocysteinemia, is correlated with advanced vascular calcification and phenotypic switching of vascular smooth muscle cells (VSMCs). Oxidative stress and high phosphate levels also induce VSMC calcification, suggesting that the Krüppel-like factor 4 (KLF4) signaling pathway may also contribute to vascular calcification. In this study, we investigated this possibility and the role and mechanisms of Hcy in vascular calcification. We found that in atherosclerotic apolipoprotein E-deficient (ApoE ؊/؊) mice, Hcy significantly increases vascular calcification in vivo, as well as VSMC calcification in vitro. Of note, the Hcy-induced VSMC calcification was correlated with elevated KLF4 levels. Hcy promoted KLF4 expression in calcified atherosclerotic lesions in vivo and in calcified VSMCs in vitro. shRNA-mediated KLF4 knockdown blocked the Hcy-induced up-regulation of runt-related transcription factor 2 (RUNX2) and VSMC calcification. RUNX2 inhibition abolished Hcy-induced VSMC calcification. Using ChIP analysis, we demonstrate that KLF4 interacts with RUNX2, an interaction promoted by Hcy stimulation. Our experiments also revealed that the KLF4 knockdown attenuates Hcy-induced RUNX2 transactivity, indicating that KLF4 is important in modulating RUNX2 transactivity. These findings support a role for Hcy in regulating vascular calcification through a KLF4-RUNX2 interaction and indicate that Hcy-induced, enhanced RUNX2 transactivity increases VSMC calcification. These insights reveal possible opportunities for developing interventions that prevent or manage vascular calcification. Atherosclerosis increases the occurrence of peripheral vascular disease, cerebral infarction, and coronary heart disease. Among its characteristics, arterial calcification is the most common feature (1-7). Abnormal deposition of hydroxyapatite mineral in the arteries leads to arterial wall thickening and

Vitamin D Treatment Prevents Uremia-Induced Reductions in Aortic microRNA-145 Attenuating Osteogenic Differentiation despite Hyperphosphatemia

“…These results are in agreement with previous studies demonstrating that miR-145 regulates the expression of contractile phenotype markers in VSMC, such as α-actin, among others [10,11]. Regarding VSMC osteogenic differentiation, miR145 reductions not only directly target osterix [36], but also the transcription factor KLF4, which directly controls Runx2 [17][18][19].…”

“…Hyperphosphatemia, a recognized risk factor for VC and cardiovascular mortality, even in individuals with normal renal function [13], also reduces the miR-145 content of normal aortas [14]. Similar to what has been described for miR-125b, the first microRNA directly associated with human artery calcification, miR-145 down-regulates the expression of osteogenic genes in osteoblasts [15,16] and directly targets the transcription factor krüppel-like factor 4 (KLF4), which also controls osteogenic gene expression [17][18][19]. Furthermore, miR-145 overexpression increases, and its silencing decreases the expression of α-actin and other markers of the integrity of the arterial elastic layer.…”

Six Shades of Vascular Smooth Muscle Cells Illuminated by KLF4 (Krüppel-Like Factor 4)

“… 117 KLF4 was also demonstrated to regulate RUNX2 transcription, with knockdown of KLF4 inhibiting upregulation of RUNX2 and vSMC calcification. 118 In addition, KLF4 enhanced chondrocyte differentiation as characterized by upregulation of SOX9 and downregulation of the chondrocyte dedifferentiation marker Col1α1. 119 In the context of scRNA-seq, loss of KLF4 in vSMCs coincided with reduction of an osteogenic phenotype.…”

The regulation of Ero1-alpha in homocysteine-induced macrophage apoptosis and vulnerable plaque formation in atherosclerosis

“…Total proteins were extracted from macrophages and aortic tissues using RIPA as previously described [27]. Equal amounts of protein samples were loaded on to SDS-PAGE and then transferred onto nitrocellulose membranes.…”