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The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells.

Sathanoori R, Swärd K, Olde B, Erlinge D - PLoS ONE (2015)

Bottom Line: Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2.The effect of the antagonists was confirmed with siRNA knockdown of the receptors.In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Clinical Sciences, Lund University, Lund, Sweden.

ABSTRACT
Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase. These findings support a novel role for P2X7 and P2X4 coupled to induction of inflammatory molecules in modulating high glucose and palmitate-induced endothelial cell activation and dysfunction.

No MeSH data available.


Related in: MedlinePlus

Knockdown of P2X7 and P2X4 inhibit high glucose and palmitate-induced expression of inflammatory genes.siRNA knockdown of P2X7 and P2X4 in the HUVECs inhibits high glucose and palmitate-induced (24 h) gene expression of IL-1β (A), IL-6 (B), PTGS2 (C), IL-8 (D), ICAM-1 (E), and VCAM-1 (F). Cells transfected with the negative control siRNA (Neg. Ctl. siRNA) was used as controls and PPIA was used as the housekeeping gene to normalize all transcript levels. n = 3 independent experiments each done in replicates; *p ≤ 0.05.
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pone.0125111.g003: Knockdown of P2X7 and P2X4 inhibit high glucose and palmitate-induced expression of inflammatory genes.siRNA knockdown of P2X7 and P2X4 in the HUVECs inhibits high glucose and palmitate-induced (24 h) gene expression of IL-1β (A), IL-6 (B), PTGS2 (C), IL-8 (D), ICAM-1 (E), and VCAM-1 (F). Cells transfected with the negative control siRNA (Neg. Ctl. siRNA) was used as controls and PPIA was used as the housekeeping gene to normalize all transcript levels. n = 3 independent experiments each done in replicates; *p ≤ 0.05.

Mentions: Given the observation that conditions of high glucose and palmitate increased the levels of P2X7 and P2X4 in HUVECs, we first used antagonists to test if they were involved in the induction of inflammatory genes. Antagonizing P2X7 with 100 nmol/L AZ11645373 [33] in HUVECs exposed to high glucose and palmitate, inhibited the upregulation (Fig 2) of CASP1 (21.6±1.8%; p < 0.0001; Fig 2A), IL-1β (66.7±7.8%; p = 0.0002; Fig 2B), IL-6 (23.5±6%; p < 0.0008; Fig 2C), PTGS2 (27.2±6%; p = 0.0002; Fig 2D), and IL-8 (40.6±5.3%; p < 0.0001; Fig 2E). Moreover, PSB-12253 (1 μmol/L), a previously characterized and validated P2X4 antagonist [29,34], had a significant inhibitory effect on CASP1 (10.5±4%; p = 0.001; Fig 2A), IL-1β (59.7±10.2%; p = 0.0003; Fig 2B), IL-6 (36±8.7%; p < 0.0001; Fig 2C) and PTGS2 (36.7±4.8%; p < 0.0001; Fig 2D) mRNA. Furthermore, we observed similar inhibitory effects using another P2X7 antagonist (50 μmol/L A438079) (S1 Fig). Next, we used siRNA knockdown of P2X7 and P2X4 to determine their specific role in mediating high glucose and palmitate-induced expression of inflammatory markers in HUVECs. Both P2X4 [29] and P2X7 (S2 Fig; 86% decrease in P2X7 mRNA) siRNA were validated in the HUVECs and the effects observed with these siRNA correlated well with that of the antagonists (Fig 3A–3F). We therefore used AZ11645373 and PSB-12253 for the rest of the study.


The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells.

Sathanoori R, Swärd K, Olde B, Erlinge D - PLoS ONE (2015)

Knockdown of P2X7 and P2X4 inhibit high glucose and palmitate-induced expression of inflammatory genes.siRNA knockdown of P2X7 and P2X4 in the HUVECs inhibits high glucose and palmitate-induced (24 h) gene expression of IL-1β (A), IL-6 (B), PTGS2 (C), IL-8 (D), ICAM-1 (E), and VCAM-1 (F). Cells transfected with the negative control siRNA (Neg. Ctl. siRNA) was used as controls and PPIA was used as the housekeeping gene to normalize all transcript levels. n = 3 independent experiments each done in replicates; *p ≤ 0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4418812&req=5

pone.0125111.g003: Knockdown of P2X7 and P2X4 inhibit high glucose and palmitate-induced expression of inflammatory genes.siRNA knockdown of P2X7 and P2X4 in the HUVECs inhibits high glucose and palmitate-induced (24 h) gene expression of IL-1β (A), IL-6 (B), PTGS2 (C), IL-8 (D), ICAM-1 (E), and VCAM-1 (F). Cells transfected with the negative control siRNA (Neg. Ctl. siRNA) was used as controls and PPIA was used as the housekeeping gene to normalize all transcript levels. n = 3 independent experiments each done in replicates; *p ≤ 0.05.
Mentions: Given the observation that conditions of high glucose and palmitate increased the levels of P2X7 and P2X4 in HUVECs, we first used antagonists to test if they were involved in the induction of inflammatory genes. Antagonizing P2X7 with 100 nmol/L AZ11645373 [33] in HUVECs exposed to high glucose and palmitate, inhibited the upregulation (Fig 2) of CASP1 (21.6±1.8%; p < 0.0001; Fig 2A), IL-1β (66.7±7.8%; p = 0.0002; Fig 2B), IL-6 (23.5±6%; p < 0.0008; Fig 2C), PTGS2 (27.2±6%; p = 0.0002; Fig 2D), and IL-8 (40.6±5.3%; p < 0.0001; Fig 2E). Moreover, PSB-12253 (1 μmol/L), a previously characterized and validated P2X4 antagonist [29,34], had a significant inhibitory effect on CASP1 (10.5±4%; p = 0.001; Fig 2A), IL-1β (59.7±10.2%; p = 0.0003; Fig 2B), IL-6 (36±8.7%; p < 0.0001; Fig 2C) and PTGS2 (36.7±4.8%; p < 0.0001; Fig 2D) mRNA. Furthermore, we observed similar inhibitory effects using another P2X7 antagonist (50 μmol/L A438079) (S1 Fig). Next, we used siRNA knockdown of P2X7 and P2X4 to determine their specific role in mediating high glucose and palmitate-induced expression of inflammatory markers in HUVECs. Both P2X4 [29] and P2X7 (S2 Fig; 86% decrease in P2X7 mRNA) siRNA were validated in the HUVECs and the effects observed with these siRNA correlated well with that of the antagonists (Fig 3A–3F). We therefore used AZ11645373 and PSB-12253 for the rest of the study.

Bottom Line: Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2.The effect of the antagonists was confirmed with siRNA knockdown of the receptors.In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Clinical Sciences, Lund University, Lund, Sweden.

ABSTRACT
Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase. These findings support a novel role for P2X7 and P2X4 coupled to induction of inflammatory molecules in modulating high glucose and palmitate-induced endothelial cell activation and dysfunction.

No MeSH data available.


Related in: MedlinePlus