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. 2018 Jun;175(11):2063-2076.
doi: 10.1111/bph.14200. Epub 2018 Apr 25.

Heat shock protein-27 and sex-selective regulation of muscarinic and proteinase-activated receptor 2-mediated vasodilatation: differential sensitivity to endothelial NOS inhibition

Vivek Krishna Pulakazhi Venu  1   2 , Mahmoud Saifeddine  1 , Koichiro Mihara  1 , Mahmoud El-Daly  1   3 , Darrell Belke  2 , Jonathan L E Dean  4 , Edward R O'Brien  2 , Simon A Hirota  1 , Morley D Hollenberg  1   5
Affiliations

Heat shock protein-27 and sex-selective regulation of muscarinic and proteinase-activated receptor 2-mediated vasodilatation: differential sensitivity to endothelial NOS inhibition

Vivek Krishna Pulakazhi Venu et al. Br J Pharmacol. 2018 Jun.

Abstract

Background and purpose: Previously, we demonstrated that exogenous heat shock protein 27 (HSP27/gene, HSPB1) treatment of human endothelial progenitor cells (EPCs) increases the synthesis and secretion of VEGF, improves EPC-migration/re-endothelialization and decreases neo-intima formation, suggesting a role for HSPB1 in regulating EPC function. We hypothesized that HSPB1 also affects mature endothelial cells (ECs) to alter EC-mediated vasoreactivity in vivo. Our work focused on endothelial NOS (eNOS)/NO-dependent relaxation induced by ACh and the coagulation pathway-activated receptor, proteinase-activated receptor 2 (PAR2).

Experimental approach: Aorta rings from male and female wild-type, HSPB1-null and HSPB1 overexpressing (HSPB1o/e) mice were contracted with phenylephrine, and NOS-dependent relaxation responses to ACh and PAR2 agonist, 2-furoyl-LIGRLO-NH2 , were measured without and with L-NAME and ODQ, either alone or in combination to block NO synthesis/action. Tissues from female HSPB1-null mice were treated in vitro with recombinant HSP27 and then used for bioassay as above. Furthermore, oestrogen-specific effects were evaluated using a bioassay of aorta isolated from ovariectomized mice.

Key results: Relative to males, HSPB1-null female mice exhibited an increased L-NAME-resistant relaxation induced by activation of either PAR2 or muscarinic ACh receptors that was blocked in the concurrent presence of both L-NAME and ODQ. mRNAs (qPCR) for eNOS and ODQ-sensitive guanylyl-cyclase were increased in females versus males. Treatment of isolated aorta tissue with HSPB1 improved tissue responsiveness in the presence of L-NAME. Ovariectomy did not affect NO sensitivity, supporting an oestrogen-independent role for HSPB1.

Conclusions and implications: HSPB1 can regulate intact vascular endothelial function to affect NO-mediated vascular relaxation, especially in females.

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Figures

Figure 1
Figure 1
Concentration–response curves for the contractile action of phenylephrine (PE) in the absence and presence of L‐NAME or ODQ acting alone or in combination. Increased tension (g) in response to increasing concentrations of PE was measured in the absence or presence of L‐NAME (0.1 mM) as outlined in the Methods for tissues from female (A–C) and male (D–F) wild‐type (A, D), HSPB1‐null (B, E) and HSPB1‐overexpressing (C, F) mice. Lines represent four parameter logistic curves, which calculate the variables available on GraphPad PRISM. Variables were compared by one‐way ANOVA followed by post hoc tests. *P < 0.05, PE alone versus + L‐NAME alone; ODQ alone and in combination with L‐NAME and ODQ.
Figure 2
Figure 2
ACh‐mediated relaxant responses observed in the absence or presence of L‐NAME and ODQ acting alone or in combination. PE‐constricted aorta tissues from female (A–C) and male (D–F); wild‐type (wt/wt) (A, D), HSPB1‐null (−/−) (B, E) and HSPB1‐overexpressing (o/e) mice (C, F) were treated with increasing concentrations of ACh either in the absence or presence of 0.1 mM L‐NAME or 1 μM ODQ acting either alone or in combination (L‐NAME plus ODQ), and the relaxant responses were measured as outlined in the Methods. Lines represent four parameter logistic curves which calculate the variables available on GraphPad PRISM. Variables were compared by one‐way ANOVA followed by post hoc tests. *P < 0.05 PE alone versus + L‐NAME alone; #P < 0.05, + L‐NAME alone and ODQ alone versus +L‐NAME and ODQ.
Figure 3
Figure 3
2‐fLI/PAR2‐mediated relaxant responses observed in the absence or presence of L‐NAME and ODQ acting either alone or in combination. PE‐constricted aorta tissues from female (A–C) and male (D–F); wild‐type (wt/wt) (A, D), HSPB1‐null (−/−) (B, E) and HSPB1‐overexpressing (o/e) mice (C, F) were treated with increasing concentrations of 2‐fLI either in the absence or presence of 0.1 mM L‐NAME or 1 μM ODQ acting either alone or in combination (L‐NAME plus ODQ), and the relaxant responses were measured as outlined in the Methods. Lines represent four parameter logistic curves, which calculate the variables available on GraphPad PRISM. Variables were compared by one‐way ANOVA followed by post hoc tests. *P < 0.05 PE alone versus + L‐NAME alone; #P < 0.05, + L‐NAME alone versus ODQ and L‐NAME and ODQ in combination.
Figure 4
Figure 4
Concentration–response curves for relaxation induced by ACh, 2‐fLI/PAR2 and SNP in aorta tissue obtained from ovariectomized mice. PE‐constricted aorta tissues from ovariectomized females: wild‐type (wt/wt), HSPB1‐null (−/−) and HSPB1‐overexpressing (o/e) mice were treated with increasing concentrations of ACh (A), 2‐fLI (B) and SNP (C) either in the absence or presence of 0.1 mM L‐NAME, and the relaxant responses were measured as outlined in the Methods. Lines represent four parameter logistic curves which calculate the variables available on GraphPad PRISM. Variables were compared by one‐way ANOVA followed by post hoc tests. *P < 0.05, untreated tissues versus tissues + L‐NAME.
Figure 5
Figure 5
Aorta tissues from female mice have increased eNOS and guanylyl cyclase alpha‐subunit mRNAs relative to male‐derived tissues. (A–C) The histograms show the qPCR analyses of mRNA isolated from male and female aorta tissue to quantify the eNOS, Gucy1a2 and Gucy1a1 mRNA levels. Increased mRNA levels for all of the mRNAs were observed in female compared with male derived tissues. The qPCR measurements were normalized to signals for the TATA‐box binding protein (TBP) levels. Data were then normalized to males and expressed as a relative abundance (fold change, compared to males) (mean ± SD: *P < 0.05 male vs. female mRNA levels).
Figure 6
Figure 6
HSPB1/HSP27 treatment augments L‐NAME sensitivity for blocking vasodilatation mediated by muscarinic receptors and PAR2 in female HSPB1‐null aorta tissue. Female HSPB1‐null aortic ring tissues cultured in euglycaemic serum‐free medium (DMEM: 10 mM glucose) for 3 h at 37°C were untreated or were treated with 50 μg·mL−1 (2.2 μM) recombinant HSP27 and were then evaluated for ACh (A) or 2‐fLI (B)‐mediated vasodilatation in the absence or presence of 0.1 mM L‐NAME as outlined in the Methods. L‐NAME was able to eliminate vasodilatation completely in the HSPB1‐treated tissues . Concentration–response data represent four parameter logistic curves calculated using GraphPad PRISM. Variables were compared by one‐way ANOVA followed by post hoc tests. *P < 0.05, for HSP27‐treated tissues or untreated tissues versus L‐NAME alone or HSP27 along with L‐NAME. #P < 0.05, for untreated tissues plus L‐NAME versus HSP‐27‐treated tissues plus L‐NAME.
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