Purinergic Regulation of Endothelial Barrier Function

Abstract

Increased vascular permeability is a hallmark of several cardiovascular anomalies, including ischaemia/reperfusion injury and inflammation. During both ischaemia/reperfusion and inflammation, massive amounts of various nucleotides, particularly adenosine 5'-triphosphate (ATP) and adenosine, are released that can induce a plethora of signalling pathways via activation of several purinergic receptors and may affect endothelial barrier properties. The nature of the effects on endothelial barrier function may depend on the prevalence and type of purinergic receptors activated in a particular tissue. In this review, we discuss the influence of the activation of various purinergic receptors and downstream signalling pathways on vascular permeability during pathological conditions.

Keywords: ADP; ATP; P2X receptors; P2Y receptors; Rac1; RhoA; UTP; adenosine; endothelial permeability; oedema; peripheral actin.

Figure 1

Schematic presentation of regulators of endothelial barrier properties. Rock: Rho associated kinase

Figure 2

Key mechanisms involved in adenosine receptors-mediated endothelial barrier regulation. In lung microvasculature and macrovascular endothelium, A₂ receptor activation causes an activation of Rac1 and an inhibition of RhoA, leading to stabilisation of the endothelial barrier. On the other hand, in coronary microvascular ECs, inhibition of both RhoA and Rac1 results in disruption of endothelial cytoskeleton and barrier failure. Black arrows indicate sequence of signal transduction, broken arrow indicates involvement of multiple steps in between, and green arrows indicate increase in cellular levels of indicated second messenger. Red bocks mean inhibition. AC: adenylyl cyclase; cAMP: cyclic adenosine monophosphate; GEF: guanine exchange factor; IP3: inositol triphosphate; PKA: protein kinase A; PKC: protein kinase C; PLC: phospholipase C.

Figure 3

Effect of P2X4 receptor modulator (ivermectin; IVM) and antagonist ((5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one: 5-BDBD) on thrombin-induced endothelial hyperpermeability. HUVEC monolayers cultured on filter membranes were exposed to human thrombin (Thr, 0.3 IU/mL) in the absence (red) or presence (green) of ivermectin (IVM; 50 µM) and the flux of labelled albumin was measured as described previously [84]. In a parallel set of experiments, P2X4 receptor antagonist (5-BDBD; 10 µM) was added before the addition of ivermectin and thrombin. n = 4, * p < 0.05 vs. control, # p < 0.05 vs. Thr alone, ^§ p < 0.05 vs. IVM + Thr. For experimental details, please see methods in Supplementary File.

Figure 4

Effect of ADP and P2Y₁ antagonist (MRS2500) on thrombin-induced endothelial hyperpermeability: HUVEC monolayers cultured on filter membranes were exposed to human thrombin (Thr, 0.3 IU/mL) in the absence (red) or presence (blue) of P2Y₁ receptor agonist ADP (10 µM), and the flux of labelled albumin was measured as described previously [84]. In a parallel set of experiments P2Y₁ receptor antagonist (MRS2500; 10 µM; black) was added before the addition of ADP and thrombin. n = 4, * p < 0.05 vs. control, # p < 0.05 vs. Thr alone, ^§ p < 0.05 vs. ADP + Thr.

Figure 5

Schematic presentation of effect of various P2Y receptors’ activation on endothelial barrier function. Activation of P2Y₁ receptor stabilises while chronic activation of P2Y₂, P2Y₄, and P2Y₆ receptors results in atherosclerosis. Loss of endothelial barrier integrity is one of the early features of development of atherosclerotic plaques. The molecular mechanisms are not clear yet. P2Y₁₂ receptor is G_i-linked and its activation results in inhibition of adenylyl cyclase (AC) and reduction in intracellular cAMP content that leads to endothelial barrier destabilisation. The P2Y₁₁ is G_s-linked and its activation would lead to opposite effects and endothelial barrier stabilisation. The effects of P2Y₁₁ are hypothetical based on available information about the P2Y₁₁ receptor. Black solid arrows indicate sequence of signalling, broken arrows indicate multiple steps in between, green arrow shows increase in cellular cAMP levels, and red arrows indicate signalling via Gi leading to reduction in cAMP and endothelial barrier disruption. Red blocks mean inhibition.