In vivo vascular rarefaction and hypertension induced by dexamethasone are related to phosphatase PTP1B activation not endothelial metabolic changes

Abstract

Glucocorticoids have important anti-inflammatory and immunomodulatory activities. Dexamethasone (Dex), a synthetic glucocorticoid, induces insulin resistance, hyperglycemia, and hypertension. The hypertensive mechanisms of Dex are not well understood. Previously, we showed that exercise training prior to Dex treatment significantly decreases blood vessel loss and hypertension in rats. In this study, we examined whether the salutary effects of exercise are associated with an enhanced metabolic profile. Analysis of the NAD and ATP content in the tibialis anterior muscle of trained and non-trained animals indicated that exercise increases both NAD and ATP; however, Dex treatment had no effect on any of the experimental groups. Likewise, Dex did not change NAD and ATP in cultured endothelial cells following 24 h and 48 h of incubation with high concentrations. Reduced VEGF-stimulated NO production, however, was verified in endothelial cultured cells. Reduced NO was not associated with changes in survival or the BH4 to BH2 ratio. Moreover, Dex had no effect on bradykinin- or shear-stress-stimulated NO production, indicating that VEGF-stimulated eNOS phosphorylation is a target of Dex's effects. The PTP1B inhibitor increased NO in Dex-treated cells in a dose-dependent fashion, an effect that was replicated by the glucocorticoid receptor inhibitor, RU486. In combination, these results indicate that Dex-induced endothelial dysfunction is mediated by glucocorticoid receptor and PTP1B activation. Moreover, since exercise reduces the expression of PTP1B and normalized insulin resistance in aging rats, our findings indicate that exercise training by reducing PTP1B activity counteracts Dex-induced hypertension in vivo.

Keywords: Endothelial dysfunction; Glucocorticoid receptor; Nitric oxide; VEGF.

Figure 1.. Exercise training prevents skeletal muscle rarefaction induced by dexamethasone.

(A) capillary density, (B) capillary-to-fibers ratio, (C) histological sections from SC: sedentary control, SD: sedentary treated with DEX, TC: trained control and trained treated with DEX (TD) muscles stained with hematoxylin and eosin. The capillaries are marked by arrows (Scale bar= 100 m). Values represent the meanSEM from 6-histological slices obtained from 8–9 animals each group. ⁺p<0.05 training; *p<0.05 dexamethasone effect.

Figure 2.. Striatal muscle exercise training increases NAD, and ATP that is not changed by dexamethasone.

(A). Wistar rat tibialis anterior muscles from sedentary control (SC) and dexamethasone treated (SD) were compared to exercise trained control (TC) and dexamethasone treated (TD). (B) Direct comparison between sedentary and exercise groups regardless of treatment. All groups were exercised trained for 8 weeks prior to 14 days of treatment with dexamethasone (50 µg/kg/day, s.c. injections). Tibialis anterior muscles (30 mg) were collected and extracted with perchloric acid 5% and analyzed by HPLC as described in the Methods section. Values represent the mean±SEM from 5–10 samples in each group. ⁺p<0.05 exercise training effect.

Figure 3.. Dexamethasone induces endothelial dysfunction that is not explained by changes in tetrahydrobiopterin or energy metabolism.

(A) BAECs viability was unaffected by 24 h or 48 h incubation with increasing concentrations of dexamethasone. (B) VEGF-stimulated NO production was decreased by 24 h Dex treatment. (C) BH4 and BH2 levels were not changed by 24 h dexamethasone treatment. (D) NAD, ADP, and ATP were not changed in cells treated for 24 h or 48 h with Dex. Values represent the mean±SEM from 3–10 independent replicates. ⁺p<0.05 VEGF stimulation; *p<0.05 Dex effect.

Figure 4.. Mechanisms of endothelial cell dysfunction in dexamethasone-treated BAECs.

(A) Dex (100 µM, 24 h) did not modify bradykinin-stimulated NO release from BAECs. (B) Dex treatment (100 µM, 24 h) have no effect in NO release from shear stress-stimulated BAECs (18 dyen/cm²;4 h). Values represent the mean±SEM from 3–6 independent replicates. ⁺p<0.05 bradykinin or shear stress stimulation; ^#p<0.05 L-NNMA effect.

Figure 5.. Effects of dexamethasone on VEGF-stimulated eNOS phosphorylation.

(A) Dex (100 µM, 24 h) did not modify VEGF-induced Akt and eNOS phosphorylation. Basal levels of Akt and eNOS were confirmed in duplicate experiments (B) PTP1B inhibitor (KY226), glucocorticoid receptor inhibitor (RU486), and Dex (100 µM, 24 h) enhance VEGF-induced eNOS phosphorylation. Values represent the mean±SEM from 3 independent replicates. ⁺p<0.05 VEGF stimulation.

Figure 6.. Inhibition of PTP1B by KY226 or glucocorticoid receptor-inhibitor (RU486) ameliorate dexamethasone-induced endothelial dysfunction.

Controls are VEGF-stimulated BAECs. (A) KY226 dose-dependently rescued Dex (100 µM, 24 h) dependent NO reduction (B) The GR inhibitor (RU486, 10 µM) re-established NO release in Dex (100 µM, 24 h) treated cells. Values represent the mean±SEM from 3‒10 independent replicates. ⁺p<0.05 for VEGF stimulation, *p<0.05, VEFG+Dex, ^#p<0.05 for KY226+VEGF+Dex or RU486+VEGF+Dex.

Figure 7.

Glucocorticoid receptor mediates dexamethasone-induced endothelial dysfunction.