Hypercholesterolemia-induced impairment of VEGFR1 receptor signalling in monocytes is reversed by the attenuation of oxidative stress

Purpose: Immune cell dysfunction contributes to several cardiovascular diseases in patients with hypercholesterolemia (HC). Vascular endothelial growth factor receptor-1 (VEGFR1) signalling is central to monocyte function and is dysregulated in HC. Impaired monocyte function results in an impaired ability to respond to arteriogenic stimuli - including VEGFR-1 agonists VEGF/PlGF - and failure to promote arteriogenesis. The underlying mechanisms are unclear, and this is the aim of this study.

Methods: Primary monocytes were isolated from HC patients and age-matched controls by magnet-assisted cell sorting. In addition to primary human monocytes, the monocytic cell line THP-1 was also used in the experiments. To mimic HC conditions, monocytes were exposed to 50 µg/ml oxLDL for 24-48 hours. VEGFR1 receptor levels were analysed by FACS. WB and qPCR were used to analyse the altered expression of signalling molecules.  ERK inhibitor (U0126) and p38 inhibitor (SB239063) were used to inhibit signalling molecules. CellROX was used to analyse ROS accumulation. Thioredoxin mimetic peptide (TMP) and N-acetyl cysteine (NAC) were used as antioxidants. To analyse monocyte chemokinesis and chemotaxis, Boyden chamber migration assays were performed using VEGF and PlGF as VEGR1 agonists.

Results: We demonstrated a highly significant impairment of VEGFR1 responses in monocytes from HC patients. This was also confirmed in primary monocytes and THP-1 cells treated with ox-LDL. This impairment was not due to reduced surface expression of VEGFR1. HC monocytes also showed increased random migration (chemokinesis). Activation of p38 and ERK were responsible for the random motility of monocytes (chemokinesis), as pharmacological inhibition of p38 and ERK completely blunted chemokinesis of HC monocytes. Similar results were obtained when primary monocytes and THP-1 cells were exposed to oxLDL in vitro. oxLDL treatment promoted the accumulation of ROS in monocytes. The causal link between oxidative stress and blunted VEGFR1 responses was revealed as monocytes exposed to exogenous H2O2 showed refractoriness to VEGF and PlGF stimulation. Co-treatment of oxLDL-treated monocytes with the antioxidants NAC and TMP attenuated ROS accumulation, blocked chemokinesis and promoted VEGFR1 responses, but this was more robust for TMP. Most importantly, NAC and, to a greater extent, TMP were able to blunt the VEGF/PlGF refractoriness of HC monocytes and restore the chemotactic potential of these cells.

Conclusions: In this study, we provide evidence that oxidative stress is a primary driver of monocyte dysfunction in hyperlipidemic conditions and that attenuation of ROS accumulation by two different antioxidants improved monocyte function in HC. These findings identify TMP as a potential therapeutic target to attenuate random monocyte activation and improve VEGFR1 responses in hyperlipidemic conditions. Improving monocyte function in HC may promote arteriogenesis and represents an attractive therapeutic strategy in atherosclerotic disease.