P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development

Figures

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  Figure 1. The P2Y11 agonist NF546 restores vascular function impaired by angiotensin II stimulation. Rat aortic rings were incubated 30min with or without angiotensin II (AngII) (100 nM), NF546 (10 µM), or NF340 (10 µM), then vasomotor response was evaluated in response to increasing concentrations of phenylephrine (Phe) (1.10−8 M–5.10−7 M) and acetylcholine (Ach) (1.10−9 M–1.10−5M). (a) AngII tended to increase the vasoconstrictive response to Phe (p = 0.0547) and significantly reduced (p < 0.001,) vasorelaxation in response to Ach compared to the control (CTL) (n = 12). (b) Expression of a P2Y11-like protein was analyzed by western blot on aortic rings lysate. A band at 55 and 35 kDa could be immunodetected using anti-hP2Y11 primary antibody, and this was prevented by the use of hP2Y11 peptide (n = 2). (c) In the CTL group, NF546 or NF340 P2Y11 ligands did not show any significant effects on vascular tone (n = 12). (d) In the AngII-treated group, the P2Y11 agonist NF546 significantly restored basal vasomotor response, while the P2Y11 antagonist NF340 had no effect when used alone, but abolished the effect of NF546 when co-applied (n = 12). * p < 0.05, ** p < 0.01 and *** p < 0.001,…

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  Figure 2. P2Y11 ligands regulate vascular tone in an endothelial cell (EC)-dependent manner and through the modulation of nitric oxide (NO) bioavailability. EC-denuded aortic rings (E-) were incubated for 30 min with or without AngII (100 nM), NF546 (10 µM), or NF340 (10 µM), then vasomotor response was evaluated in response to increasing concentrations of phenylephrine (Phe) (1.10−8 M–5.10−7 M) and acetylcholine (Ach) (1.10−9 M–1.10−5M). (a) AngII incubation tended to increase the vasoconstrictive response to Phe. The P2Y11 agonist NF546 did not modulate vasoconstriction in AngII-treated groups (n = 4). (b) We observed no relaxation in response to Ach after EC denudation (n = 4). (c) Rat aortic rings were incubated 30 min with or without AngII (100 nM), NF546 (10 µM), or NF340 (10 µM), and preconstricted with Phe to reach 10% of the KCl maximal constriction level. Response to L-NAME (10 µM) was then measured. In the CTL group, P2Y11 ligands did not show any significant difference on L-NAME response. Contraction elicited by L-NAME was significantly increased in the presence of AngII incubation with NF546 preventing the AngII pro-constrictor effect, while NF340 had no effect (n = 13).…

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  Figure 3. Vascular tone regulation by ECs is modulated by P2Y11 ligands. (a) P2Y11 protein expression on HUVECs was analyzed by western blot, showing a band at 50 kDa (n = 3). (b,c) HUVECs were incubated 30 min with basal medium, AngII (100 nM) or submitted to 5 h hypoxia followed by 30 min reoxygenation (H/R), and incubated with the P2Y11 agonist NF546 (10 µM) or/and antagonist NF340 (10 µM). Endothelial nitric oxyde synthase (eNOS) and p-ser1177-eNOS protein expression were analyzed by western blot. In the CTL group, NF546 increased eNOS expression (n = 13) and p-ser1177-eNOS (n = 10). In the AngII group, eNOS expression was increased in the presence of AngII; AngII+NF546 (n = 13) and p-ser1177-eNOS was increased compared to AngII condition (n = 10); and P2Y11 receptor (P2Y11R) antagonist NF340 tended to decrease eNOS expression compared to AngII condition. In the H/R group, H/R and NF546 tended to increase eNOS expression (n = 13) and increase p-ser1177-eNOS (n = 10). (d) HUVECs were incubated 12 h with basal medium, AngII (100 nM) or submitted to 5 h hypoxia followed by 12 h reoxygenation (H/R), and addition of P2Y11R agonist NF546 (10 µM) and/or antagonist NF340 (10 µM). Endothelin-1…

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  Figure 4. P2Y11 ligands regulate vascular stress factors release. (a) H2O2 release by aortic rings was evaluated by FOX assay. Rings were incubated 30 min in FOX assay reagent with or without AngII (100 nM), NF546 (10 µM) or NF340 (10 µM). P2Y11 ligands NF546 and NF340 had no effect on H2O2 release under the basal condition. AngII incubation increased H2O2 release compared to the CTL group. NF546 incubation suppressed the effect of AngII by restoring basal H2O2 level release (n = 9). (b–d) Extracellular ATP release was measured in HUVEC supernatants. HUVECs were subjected to 5 h hypoxia and supernatants were collected after several reoxygenation durations (30 min–12 h), or subjected to AngII incubation (30 min–12 h), in presence or absence of NF546 and/or NF340. (b) Hypoxia alone tended to decrease eATP secretion. Short times of reoxygenation (30 min–1 h) significantly increased eATP secretion, which progressively returned to the basal level. (c) Short exposures to AngII (30 min–1 h) increased eATP secretion, which progressively returned to the basal level. (d) After 30 min incubation, NF546 increased eATP secretion in the CTL (n = 11), H/R group (n = 7) and in the AngII group (n = 6). * p…

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  Figure 5. P2Y11R activation reduces SMC switch towards synthetic phenotype. (a) P2Y11R expression on HCASMC was analyzed by western blot. We observed a band at 50 kda with P2Y11R antibody. (b) HCASMC proliferation was assessed by EdU incorporation during 72 h, and P2Y11R was modulated by NF546 (10 µM) and NF340 (10 µM). Under basal condition, NF546 significantly decreased HCASMC proliferation (n = 9). AngII (100 nM) incubation significantly increased HCASMC proliferation, while NF546 decreased this AngII-induced proliferation (n = 9). Previous exposure to 5 h hypoxia did not modify HCASMC proliferation, and neither NF546 nor NF340 induced differences in proliferation (n = 5). (c,d) HCASMC phenotype was determined according to the expression of α-SMA and TIMP-1 (contractile) after 24 h, evaluated by western blot. In AngII and H/R groups, NF546 increased α-SMA and TIMP-1 expression (n = 5–8). * p < 0.05 and ** p < 0.01, compared to CTL condition, if not indicated. Data given as mean ± SEM.

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  Figure 6. P2Y11R modulates growth factors secretion in the crosstalk between SMC and EC. (a) HUVECs were incubated with basal medium, AngII (100 nM) or submitted to 5 h hypoxia followed by 12 h reoxygenation (H/R), and addition of P2Y11R agonist NF546 (10 µM) and antagonist NF340 (10 µM). Supernatant (SN) was harvested after 12 h and incubated with HCASMC and EdU (10 µM) for 24 h. HCASMC proliferation was evaluated by Edu incorporation (n = 7). CTL HUVECs supernatant significantly increased HCASMC proliferation compared to the basal medium. NF546 significantly decreased this pro-proliferative effect. HUVEC AngII-conditioned medium increased HCASMC proliferation compared to the HUVEC basal conditioned medium. NF546 tended to decrease this pro-proliferative effect (p = 0.06). HUVEC H/R conditioned medium tended to increase HCASMC proliferation compared to HUVEC basal conditioned medium (p = 0.07). NF546 tended to decrease this pro-proliferative effect (p = 0.07). (b) HCASMC were incubated with basal medium, AngII (100 nM) or submitted to 5 h hypoxia followed by 24 h reoxygenation (H/R), and addition of P2Y11R agonist NF546 (10 µM) and antagonist NF340 (10 µM). VEGF secretion was evaluated in…