Purinergic Receptor – A Target for The Treatment of Cardiovascular Disease?

By Kelly Macdonald-Ramsahai 

Cardiovascular Diseases (CVDs) describe pathologies involving the heart, and its supplying and peripheral blood vessels including, but not limited to, stroke, atherosclerosis (AS), and coronary heart disease. Commonly these diseases present low-grade chronic inflammation and vascular dysfunction. Their manifestation is multi-factorial, involving genetic, environmental and behavioural risk factors such as physical activity, diet and exposure to tobacco. CVDs are considered the leading cause of mortality worldwide – The World Health Organisation (WHO) report CVDs cost 17.3 million lives in 2008, which is projected to increase to 23 million by 2030 (World Health Organization, 2020). Additionally, CVD patients display increased risk of developing physical and psychological co-morbidities including Insulin Resistance (IR), type 2 diabetes mellitus (T2D), depression, and eating disorders (EDs). Therefore, from a translational perspective, investigating CVDs at a cellular and molecular level are fundamental to provide novel insights regarding its pathogenesis, and offer new avenues for pharmacological intervention. Importantly, this could decrease mortality rates, improve individual quality of life and overall public health, and relieve demand for healthcare services.

Purinergic receptors (purinoceptors) describe a family of plasma membrane (PM) bound receptors which are ubiquitously expressed in mammalian cells (Burnstock, 1976). Purinoceptors, which bind extracellular nucleotide analogues, are branched into two families: P1 metabotropic which bind adenosine; and P2 receptors which bind ATP. The latter P2 ATP receptors are further sub-classified into P2X inotropic receptors and P2Y G protein-coupled receptors (GPCRs). P2X and P2Y purinoreceptors are highly expressed in the central nervous system (CNS), where they facilitate well-characterised biological functions including cell maturation and synaptic transmission. Due to the influence of CNS in smooth muscle, purinoreceptors regulate the physiology of peripheral organs including the arteries, bladder and gut (Tozaki-Saitoh, Makoto Tsuda and Inoue, 2011). Additionally, extracellular release of nucleotides, such as ATP, are Damage Associated Molecular Patterns (DAMPs) or ‘danger molecules’ which increase following cellular stress or injury. Therefore, purinergic signalling confers fundamental roles in pathophysiological initiation and development (Garcia-Martinez, Shaker, Mehal, 2015). Considering this, purinergic signalling – with particular focus on the delicate lines between its beneficial roles and its disease contributions – are areas of interest when investigating CVD pathogenesis.

The P2X7 receptor (P2X7R) – an example of a P2X inotropic receptor subtype – is activated upon exposure to extracellular ATP (eATP) (>100uM) (Chen et al, 2018). Similarly to all purinoreceptors, its physiological roles in vasculature are ambiguous, however, P2X7R is highly expressed in the cardiac developmental progenitor epicardium derived cells. Additionally, nine-month-old P2X7R-/- mice have 13% increase in heart size compared to wild-type (WT) mice (Beaucage et al, 2013). Therefore, P2X7R may regulate early cardiac development. In adulthood, P2X7R signalling facilitates the entry of cardio-protective agents to cardiomyocytes following myocardial infarction (MI), which may promote post-infarction recovery. Supporting this, P2X7R inhibition increased the infarct size and left ventricular developed pressure (LVDP) in rats cardiomyocytes indicating P2X7R signalling is important during recovery to MI (Vessey, LI and Kelley, 2011). 

CVDs are regarded as chronic low-grade inflammatory diseases because patients display a sustained and up-regulated inflammatory profile. Therefore, targeting anti-inflammatory pathways may be an effective treatment. Whilst physiological roles of P2X7R in adult vascular physiology remain ambiguous, mounting evidence suggests P2X7R signalling contributes to the development of inflammatory-related CVDs: P2X7R is up-regulated at a gene and protein level in CVD patients and models of vascular disease including AS, myocardial ischemic repercussion injury (M/IR), and renal failure models (Chen et al, 2018). Of significance, eATP-P2X7R signalling facilitates the activation of an intracellular multi-domain molecular complex, the NLRP3 inflammasome, in macrophages. Inflammasomes are intracellular multi-domain molecular platforms for caspase-1 activation. In turn, caspase-1 induces the maturation and section of pro-inflammatory cytokines IL-1β and IL-18 which promote inflammation and immune recruitment; and Gasdermin-D which induces pyroptosis (an inflammatory form of cell death) (Garcia-Martinez, Shaker and Mehal, 2015). Therefore, inappropriate and sustained activation of NLPR3 inflammasome, as a result of increased eATP-P2X7R signalling, may contribute to vascular dysfunction during CVD pathogenesis. P2X7R ablation in cells, through genetic manipulation or pharmacological inhibition, ameliorates pro-inflammatory marker expression and an inflammatory status in vascular disease models (Granata et al, 2015). Considering chronic inflammation is toxic in vasculature, P2X7R may be a promising target to hinder CVD progression, as its signalling axis clearly promotes inflammation in vascular diseases.

The eATP-P2X7R axis further promotes vascular vulnerability by promoting cell death. For example, P2X7R signalling increases expression of pro-apoptotic factors (Hsp-70 and caspase-8) (Granado et al, 2015). Under normal physiological conditions, the pro-survival microRNA (miR)-150, which is considered to have a protective function in vasculature, targets the 3’untranslated region (UTR) of p2x7r thereby negatively regulating its expression via the RNA interference (RNAi) mechanism. During CVD pathogenesis, miR-150 is down-regulated, which increases P2X7R signalling and exaggerates cell death. In accordance, miR150-/- mice have increased myocardial necrosis, which is reversed upon over-expression of miR-150 (Tang et al, 2015). Additionally, retinal capillary and pericyte cell death via calpain-dependent apoptotic pathways is attenuated upon supplementation with P2X7R antagonists in DR-rats and human retinal pericytes, supporting this notion (Shibata et al, 2018; Plantania et al, 2017). 

Intracellular Endothelial Cell (EC) junction protein expression is commonly perturbed in CVD patients, which implicates their permeability, integrity and contractility. Pharmacological inhibition of P2X7R in ATP-stimulated ECs increased trans-EC resistance, reflecting an improvement in its integrity (Plantania et al, 2019). This indicates P2X7R signalling during CVD may implicate EC barrier function and integrity in vasculature, which can contribute to its vulnerability. Lastly, P2X7R-mediated IL-1ß release activates EMMPRIN via MAPK, which up-regulates MMP9 expression. MMP9 increases extracellular matrix gelatinolysis in AS carotid plaques causing instability, rupture and thrombosis. P2X7R blockade in human monocytic cells reduces MAPK activation and MMP9, confirming P2X7R as a pathological agent during late stages-AS (Piscopiello et al, 2013; Lin et al, 2018). 

In summary, P2X7R is an important biological molecule with multiple pathophysiological roles. For example, increased eATP-P2X7R signalling, as a result of up-regulated P2X7R expression, facilitates inflammation and vascular vulnerability in CVDs. P2X7R is a promising molecular target for CVD treatment. However, the roles of P2X7R signalling in normal vasculature remain ambiguous, which hinders the clinical use of P2X7R inhibitors for diseased vasculature. Therefore, additional research must be undertaken to confidently determine the consequences of down-regulating P2X7R in the vasculature system, before P2X7R inhibitors can be considered for CVD treatment.

References:

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