Focus on vascular biology: new therapeutic targets in atherosclerosis, aortic dissection, and peripheral artery disease

For the podcast associated with this article, please visit https://academic.oup.com/eurheartj/pages/Podcasts. This Focus Issue on vascular biology and medicine contains the Fast Track Clinical Research article entitled ‘Critical limb-threatening ischaemia and microvascular transformation: clinical implications’, Santeri Tarvainen from the Kuopio University Hospital in Finland, and colleagues note that the role of microvascular dysfunction in cardiovascular disease is relevant, but it is still largely underestimated and, more importantly, not utilized for treatment.1–7 The aim of this study was to characterize the role of the microvasculature in critical limb-threatening ischaemia (CLTI) pathology.8 Clinical high-resolution imaging of CLTI patients (n = 50) and muscle samples from amputated CLTI limbs (n = 40) were used to describe microvascular pathology of CLTI at the level of resting muscle blood flow and microvascular structure, respectively. Furthermore, a chronic, low arterial driving pressure-simulating ischaemia model in rabbits (n = 24) was used together with adenoviral vascular endothelial growth factor A gene transfers to study the effect of microvascular alterations on muscle outcome. Resting microvascular blood flow was not depleted but displayed decreased capillary transit time (P < .01) in CLTI muscles. CLTI muscle microvasculature also exhibited capillary enlargement (P < .001) and further arterialization along with worsening of myofibre atrophy and detachment of capillaries from myofibres. Furthermore, CLTI-like capillary transformation was shown to worsen calf muscle force production (P < .05) and tissue outcome (P < .01) under chronic ischaemia in rabbits and in healthy, normal rabbit muscle (Figure 1). Hypoxic capillary enlargement is a major problem in chronic limb-threatening ischaemia (CLTI).8 Tarvainen et al. conclude that their findings depict a progressive, hypoxia-driven transformation of the microvasculature in CLTI muscles, which pathologically alters blood flow dynamics and aggravates tissue damage under low arterial driving pressure. Hypoxia-driven capillary enlargement can be highly important for CLTI outcomes and should therefore be considered in future diagnostic and treatment developments in the setting of CLTI. The contribution is accompanied by an Editorial by Vijay Ganta and Brian Annex from the Medical College of Georgia at Augusta University, Augusta, GA and W. Schuyler Jones from Duke University Health System and Duke Clinical Research Institute in Durham, NC, USA.9 The authors note that the findings of Tarvainen et al. highlight that the ultimate prize in CLTI will be achieved by focusing on the ischaemic muscle/tissue niche and determining if potential therapeutics should target solely the endothelium or simultaneously other cell types that can regulate endothelial function. Other cells can include proinflammatory macrophages, synthetic (non-quiescent) vascular smooth muscle cells, and myocytes. Furthermore, distinct from targeting cellular responses to ischaemia, biophysical approaches to determine whether normalizing the arterial pressure gradient in ischaemic muscle is sufficient to revert capillary shunting and endothelial phenotypic transitions are needed. They conclude that the combination of human and animal data in this study provides an important step forward in understanding the relationship between blood flow, perfusion, and tissue survival. Atherosclerosis is a complex chronic inflammatory disease.10–14 Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. The P2Y6 receptor (P2Y6R) when highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. In a Translational Research article entitled ‘Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cβ/store-operated calcium entry/calreticulin/scavenger receptor A pathways’, Yehong Li from the China Pharmaceutical University in Nanjing, China, and colleagues explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways.15 Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. The high-throughput Glide docking pipeline of the repurposing drug library was used to find potent P2Y6R antagonists. The P2Y6R deficiency alleviated atherogenesis characterized by reduced plaque formation and lipid deposition in the aorta. Deletion of macrophage P2Y6R significantly inhibited uptake of oxidized LDL through decreasing scavenger receptor A expression mediated by phospholipase Cβ/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to calreticulin, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found to be a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity (Figure 2). The pharmacodynamics and the effects on atherosclerosis severity were also investigated. Macrophage P2Y6 receptor deficiency inhibits foam cell formation and atherosclerosis via the phospholipase Cβ/store-operated calcium entry/calreticulin/scavenger receptor A axes. P2Y6 receptor regulates the formation of foam cells by coupling G protein-coupled receptors Gαq/11 to target the activation of phospholipase Cβ, and then phospholipase Cβ isoforms hydrolyse phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol. STIM1 is localized in the endoplasmic reticulum membrane and forms a complex with calreticulin. Depletion of Ca2+ stores triggers Ca2+ release from the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and consequent Ca2+ dissociation from the STIM–calreticulin complex. Consequently, STIM oligomerizes and translocates next to the plasma membrane, and STIM1 opens Orai1 channels, a process known as store-operated calcium entry, promoting extracellular Ca2+ entry. Moreover, calreticulin and scavenger receptor A form a complex. Thereby, increasing expression of scavenger receptor A facilitates the uptake of oxidized LDL and accumulation of cholesterol within macrophages, and further results in the promotion of atherosclerosis. Thiamine pyrophosphate is an antagonist of the P2Y6 receptor, which inhibits the P2Y6 receptor and its downstream response, thereby improving atherosclerosis. CALR, calreticulin; IP3R, inositol 1,4,5-trisphosphate receptor; ox-LDL, oxidized LDL; P2Y6R, P2Y6 receptor; PLCβ, phospholipase Cβ; SOCE, store-operated calcium entry; SR-A, scavenger receptor A; TPP, thiamine pyrophosphate.15 The authors conclude that macrophage P2Y6R regulates the phospholipase Cβ/store-operated calcium entry/calreticulin signalling pathway to increase the scavenger receptor A protein level, thereby promoting foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target using P2Y6R antagonists such as thiamine pyrophosphate. The contribution is accompanied by an Editorial by Katey Rayner from the University of Ottawa Heart Institute in Canada.16 Rayner note that overall, the study by Li et al. is an illustration of the power of combining observational studies in humans, mechanistic studies in mice, and a drug discovery pipeline to identify novel therapeutics for atherosclerosis. Aortic dissection (AD) is a degenerative aortic remodelling disease marked by an exceedingly high mortality without effective pharmacological therapies.17,18 In a Translational Research article entitled ‘The activator protein-1 complex governs a vascular degenerative transcriptional programme in smooth muscle cells to trigger aortic dissection and rupture’, Yongting Luo from the Beijing Advanced Innovation Center for Food Nutrition and Human Health in China, and colleagues note that smooth muscle cells (SMCs) lining the tunica media adopt a range of states, and their transformation from contractile to synthetic phenotypes fundamentally triggers AD.19 However, the underlying pathomechanisms governing this shift remain elusive. Ascending aortas from nine patients undergoing ascending aorta replacement and five individuals undergoing heart transplantation were subjected to single-cell RNA sequencing. The pathogenic targets governing the phenotypic switch of SMCs were identified by trajectory inference, functional scoring, single-cell regulatory network inference and clustering, regulon, and interactome analyses, and confirmed using human ascending aortas, primary SMCs, and a β-aminopropionitrile monofumarate-induced AD model. The transcriptional profiles of 93 397 cells revealed a dynamic temporal-specific phenotypic transition and marked elevation of the activator protein-1 (AP-1) complex, actively enabling synthetic SMC expansion. Mechanistically, tumour necrosis factor signalling enhanced AP-1 transcriptional activity by dampening mitochondrial oxidative phosphorylation (OXPHOS). Targeting this axis with the OXPHOS enhancer coenzyme Q10 or the AP-1-specific inhibitor T-5224 prevented phenotypic transition and aortic degeneration, and improved in the β-aminopropionitrile monofumarate-induced AD model. The authors conclude that this cross-sectional compendium of the cellular atlas of human ascending aortas during AD progression provides previously unappreciated insights into a transcriptional programme permitting aortic degeneration, highlighting a translational proof of concept for an antiremodelling intervention as an attractive strategy through modulation of the tumour necrosis factor–OXPHOS–AP-1 axis. This manuscript is accompanied by an Editorial by Kapka Miteva from the University of Geneva in Switzerland.20 Miteva concludes by pointing out that considering the significant advance achieved in revealing the mechanistic regulation of SMC synthetic transition in AD, as well as the identification of small selective molecule inhibiting the AP-1-associated signalling pathways, further clinical translational efforts remain an urgent need as a viable therapeutic strategy for human AD. In a Rapid Communications article entitled ‘Overnight exposure to high altitude in pulmonary hypertension: adverse events and effect of oxygen therapy’, Simon Schneider from the University Hospital Zurich in Switzerland, and colleagues remind us that pulmonary hypertension (PH) guidelines recommend that symptomatic pulmonary vascular disease (PVD) patients should not travel >1500 m or fly without supplemental oxygen therapy (SOT), but evidence is scarce.21 PVD patients were investigated at 470 m (Zurich, low altitude, LA) and 2500 m (high altitude, HA) during a 30 h overnight stay at Mount Säntis according to a randomized-sequence, cross-over design. The main outcomes were adverse events at high altitude (AEHA), pre-defined as: (i) severe hypoxaemia [oxygen saturation by pulse oximetry (SpO2) < 80% > 30 min]; (ii) acute mountain sickness (AMS) by Lake Louise score ≥4 with headache or AMSc score ≥0.7; or (iii) any new illness including symptomatic cardiac arrhythmia, severe rise in systolic/diastolic blood pressure (>200/100 mmHg), or angina. Of 65 patients assessed, 27 were included. Pre-defined AEHA occurred in 14/27: 10/27 had severe hypoxaemia, thereafter treated with SOT, and 7/27 scored positive for AMS, of which 3/7 were combined with severe hypoxaemia. The authors conclude that, stable, low-risk PVD patients tolerate a weekend getaway to 2500 m for up to 30 h generally well. In the case of severe hypoxaemia, SOT restores resting but not exercise low-altitude physiology. The results of this field study help to counsel PVD patients for HA sojourns and call for future longer term studies. The issue is also complemented by two Discussion Forum contributions. In a commentary entitled ‘Immunology and atherosclerosis: is there an individual ID which defines our susceptibility?’, Iñigo Lozano from the Hospital Universitario Cabueñes from Gijon, Spain and colleagues comment on the recent publication ‘Subclinical atherosclerosis and accelerated epigenetic age mediated by inflammation: a multi-omics study’ by Fátima Sánchez-Cabo from the Centro Nacional de Investigaciones Cardiovasculares (CNIC) in Madrid, Spain.14,22 Sánchez-Cabo et al. respond in a separate comment.23 The editors hope that this issue of the European Heart Journal will be of interest to its readers. Dr. Crea reports speaker fees from Abbott, Amgen, Astra Zeneca, BMS, Chiesi, Daiichi Sankyo, Menarini outside the submitted work. With thanks to Amelia Meier-Batschelet, Johanna Huggler, and Martin Meyer for help with compilation of this article.