The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease

J A F Demandt,C G Schalkwijk,K Van Kuijk,M Zaťovičová,S W Van Der Laan,S Pastoreková,C P M Reutelingsperger,L J Dubois,H Jin,C D A Stehouwer,K B J M Cleutjens,B M E Mees,L Jelenska,J C Sluimer,M M J Van Greevenbroek,S Parkkila,C J H Van Der Kallen,E A L Biessen,G Pasterkamp

doi:10.1038/s41598-020-79978-5

Abstract

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

Highlights

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD)
carbonic anhydrase IX (CAIX) mRNA expression in unstable human plaques correlated with pro-inflammatory macrophages, lipid core size, and CD105+ new angiogenic microvasculature (Fig. 1F)
Since CAIX mRNA correlated with pro-atherogenic plaque traits, and CAIX knock-out (CAIXko) led to metabolic changes in BMDMs, we further investigated its role in macrophage functions relevant for atherosclerosis

Summary

Introduction

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Traits associated to rupture[2], plasma biomarkers of plaque hypoxia may offer a cost-effective alternative to identify patients with rupture-prone atherosclerotic plaques To this end, carbonic anhydrase IX (CAIX) could be a suitable biomarker, as it is a hypoxia-induced transmembrane protein of which the extracellular 4 kDa component can be shed into body fluids by a disintegrin and metalloprotease (ADAM)[174]. Inhibiting CAIX function was shown to potentiate radiation and chemotherapy in multiple types of cancer, underlining the importance of CAIX in tumor cell s urvival[9] How do these findings of the cancer field translate to macrophage function in the atherosclerotic plaque? Several pro-atherogenic lipid modifications are induced by intraplaque acidification, potentially promoting plaque progression Deficiency of another pH regulator, Na+ H+ exchanger 1, led to reduced plaque formation[21]. In this study, we aimed to explore the expression of CAIX in human atherosclerosis, a role for CAIX in atherogenic macrophage functions and its potential as a biomarker for atherosclerotic disease

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Conclusion