Loss-of-function N178T variant of the human P2Y4 receptor is associated with decreased severity of coronary artery disease and improved glucose homeostasis.

Human P2Y4 is a UTP receptor, while in mice it is activated by both ATP and UTP. P2Y4 knockout (KO) in mice protects against myocardial infarction and is characterized by increased adiponectin secretion by adipocytes, and decreased cardiac inflammation and permeability under ischemic conditions. The relevance of these data has, however, not been explored to date in humans. In a population study comprising 50 patients with coronary artery disease (CAD) and 50 age-matched control individuals, we analyzed P2RY4 mutations and their potential association with CAD severity and fasting plasma parameters. Among the mutations identified, we focused our attention on a coding region polymorphism (rs3745601) that results in replacement of the asparagine at residue 178 with threonine (N178T) located in the second extracellular loop of the P2Y4 receptor. The N178T variant is a loss-of-function mutation of the human P2Y4 receptor and is encountered less frequently in coronary patients than in control individuals. In coronary patients, carriers of the N178T variant had significantly reduced jeopardy and Gensini cardiac severity scores, as well as lower resting heart rates and plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP). Regarding fasting plasma parameters, the N178T variant was associated with a lower concentration of glucose. Accordingly, P2Y4 KO mice had significantly improved glucose tolerance and insulin sensitivity compared with their WT littermate controls. The improvement of insulin sensitivity resulting from lack of the P2Y4 receptor was no longer observed in the absence of adiponectin. The present study identifies a frequent loss-of-function P2Y4 variant associated with less severe coronary artery atherosclerosis and lower fasting plasma glucose in coronary patients. The role of the P2Y4 receptor in glucose homeostasis was confirmed in mouse. P2Y4 antagonists could thus have therapeutic applications in the treatment of myocardial infarction and type 2 diabetes.

Corrigendum: Central role of PD-L1 in cardioprotection resulting from P2Y4 nucleotide receptor loss.

[This corrects the article DOI: 10.3389/fimmu.2022.1006934.].

Central role of PD-L1 in cardioprotection resulting from P2Y4 nucleotide receptor loss.

A better understanding of the immune function of pericardial adipose tissue is essential to adapt treatments after myocardial infarction. We showed previously that inactivation of mouse P2Y4 nucleotide receptor induces adiponectin overexpression and protection against myocardial infarction. We investigated here the inflammatory state of pericardial adipose tissue in ischemic P2Y4-deficient mice. We demonstrated that P2Y4-deficient mice displayed adipocyte beiging with increased PD-L1 expression and a higher number of regulatory leukocytes in their pericardial adipose tissue after left anterior descending artery ligation, compared to wild type mice. Effectively, a higher level of anti-inflammatory M2c macrophages and regulatory T cells was observed in pericardial adipose tissue of P2Y4 KO mice and correlated with reduced post-ischemic expansion of fat-associated lymphoid clusters. Interestingly, the anti-inflammatory effects observed in P2Y4 KO mice, were no more observed in P2Y4/adiponectin double KO ischemic mice. Finally, the reduction of T cell infiltration and cardiac fibrosis observed in P2Y4-deficient heart was lost after injection of anti-PD-L1 blocking antibody in ischemic mice. The present study defines P2Y4 as a regulator of PD-L1 and adiponectin, and as a potential target for anti-inflammatory therapies to improve myocardial infarction outcome. The combined effect of P2Y4 loss on adipocyte beiging and regulatory leukocyte increase highlights this nucleotide receptor as an important player in post-ischemic cardiac response.

UTP Regulates the Cardioprotective Action of Transplanted Stem Cells Derived From Mouse Cardiac Adipose Tissue.

Adipose tissue is a source of stem cells with a high potential of differentiation for cell-based regenerative therapies. We previously identified mouse P2Y2, an ATP and UTP nucleotide receptor, as a regulator of adipogenic and endothelial differentiation of cardiac adipose-derived stem cells (cADSC). We investigated here the potential involvement of P2Y2 receptor in the cardioprotective action of undifferentiated cADSC transplantation in mouse ischemic heart. Transplantation of cADSC was realized in the periphery of the infarcted zone of ischemic heart, 3 days after left anterior descending artery ligation. A strong reduction of collagen stained area was observed 14 days after cADSC injection, compared to PBS injection. Interestingly, loss of P2Y2 expression totally inhibits the ability of transplanted cADSC to reduce cardiac fibrosis. A detailed gene ontology enrichment analysis was realized by comparing RNA-sequencing data obtained for UTP-treated wild type cASDC and UTP-treated P2Y2-null cASDC. We identified UTP target genes linked to extracellular matrix organization such as matrix metalloproteinases and various collagen types, UTP target genes related to macrophage chemotaxis and differentiation into pro-fibrotic foam cells, and a significant number of UTP target genes linked to angiogenesis regulation. More particularly, we showed that UTP regulated the secretion of CCL5, CXCL5, and CCL12 chemokines and serum amyloid apolipoprotein 3, in the supernatants of UTP-treated cADSC. Interestingly, CCL5 is reported as a key factor in post-infarction heart failure and in the reparative and angiogenic action of transplanted ADSC on ischemic tissue. We investigated then if a UTP-pretreatment of cADSC amplifies their effect on cardiac revascularization in mouse ischemic heart. Transplantation of cADSC was able to increase peri-infarct capillary density, 14 days after their injection. This beneficial effect on cardiac revascularization was enhanced by a UTP-pretreatment of cADSC before their transplantation, and not observed using P2Y2-null cADSC. Our data support that the efficacy of transplanted cADSC can be regulated by the release of inflammatory mediators such as extracellular nucleotides in the ischemic site. The present study highlights the P2Y2 receptor as a regulator of cADSC cardioprotective action, and as a potential target for the therapeutic use of undifferentiated cADSC in post-ischemic cardiac ischemia.

P2Y2 Nucleotide Receptor Is a Regulator of the Formation of Cardiac Adipose Tissue and Its Fat-Associated Lymphoid Clusters.

The formation of pericardial adipose tissue (PAT) and its regulatory function in cardiac inflammation are not well understood. We investigated the potential role of the ubiquitous ATP/UTP nucleotide receptor P2Y2 in the PAT by using P2Y2-null mice. We observed that P2Y2-null mice displayed a lower mass of PAT and a reduced density of its fat-associated lymphoid clusters (FALCs) and, more particularly, B cells. Loss of P2Y2 receptor in pericardial preadipocytes decreased their adipogenic differentiation and maturation abilities in vitro. Gene profiling identified P2Y2 target genes in PAT linked to immunomodulation. These data led to the identification of an increase of M2c anti-inflammatory macrophages correlated with increased apoptosis of B lymphocytes in P2Y2-null pericardial fat. In addition, follicular helper T cells, which contribute to B cell expansion in germinal centers, were dramatically decreased. The effect of P2Y2 loss was also investigated after ischemia-mediated expansion of FALCs in a model of myocardial infarct. Loss of P2Y2 led to reduced expansion of B and neutrophil populations in these clusters, whereas density of M2c anti-inflammatory macrophages was increased. Our study defines the P2Y2 nucleotide receptor as a regulator of the formation and inflammatory status of pericardial fat. The P2Y2 receptor could represent a therapeutic target in the regulation of PAT function before and during cardiac ischemia.