Upregulation of heme oxygenase-1 combined with increased adiponectin lowers blood pressure in diabetic spontaneously hypertensive rats through a reduction in endothelial cell dysfunction, apoptosis and oxidative stress

Abstract

This study was designed to investigate the effect of increased levels of HO-1 on hypertension exacerbated by diabetes. Diabetic spontaneously hypertensive rat (SHR) and WKY (control) animals were treated with streptozotocin (STZ) to induce diabetes and stannous chloride (SnCl(2)) to upregulate HO-1. Treatment with SnCl(2) not only attenuated the increase of blood pressure (p<0.01), but also increased HO-1 protein content, HO activity and plasma adiponectin levels, decreased the levels of superoxide and 3-nitrotyrosine (NT), respectively. Reduction in oxidative stress resulted in the increased expression of Bcl-2 and AKT with a concomitant reduction in circulating endothelial cells (CEC) in the peripheral blood (p<0.005) and an improvement of femoral reactivity (response to acetylcholine). Thus induction of HO-1 accompanied with increased plasma adiponectin levels in diabetic hypertensive rats alters the phenotype through a reduction in oxidative stress, thereby permitting endothelial cells to maintain an anti-apoptotic environment and the restoration of endothelial responses thus preventing hypertension.

Keywords: Heme oxygenase; adiponectin; apoptosis; diabetes; hypertension; oxidative stress.

Figure 1.

A) Western blot and densitometry analysis of HO-1 and HO-2 proteins in kidney from WKY rats or SHRs. Rats were treated with SnCl₂ (once a week 50 mg/kg body weight for 4 weeks, S.C.), STZ (once a week 65 mg/kg body weight for 4 weeks i.v. for diabetic models) or both SnCl₂ and STZ. Immunoblots were performed with antibodies against rat HO-1 and HO-2. Data are representative of 4 separate experiments. B) Mean band density normalized relative to β-actin (n=4, * p<0.05 vs. WKY control rats, * * p<0.05 vs. WKY DM (diabetic models), # p<0.05 SHR control, ^† p<0.05 vs. SHR DM). C) HO activity in kidney from WKY rats or SHRs (n=4, * p<0.05 vs. corresponding control rats).

Figure 2.

Plasma adiponectin levels of WKY rats and SHRs (* p<0.05 vs. WKY control and SHR control respectively, # p<0.01 vs. WKY control and SHR DM + SnCl₂ respectively, ** p<0.05 vs. WKY control and SHR DM + SnCl₂, respectively).

Figure 3.

O₂⁻ production levels of kidney and western blot analysis of nitrotyrosine in SHRs (* p<0.05 vs. SHR control, # p<0.05 vs. SHR DM).

Figure 4.

A and B. Blood pressure was measured by the tail cuff method after the treatment (* p<0.05 vs. WKY DM, ** p<0.05 vs. WKY control, # p<0.05 vs. SHR control, ^† p < 0.05 vs. SHR DM).

Figure 5.

A) The ring segments from femoral arteries were exposed to phenylephrine (PE) in a dose-dependent manner (10⁻⁸ − 10⁻⁴ M). PE-induced contraction of SHRs was significantly increased as compared to WKY rats (*p<0.05 vs. WKY control rats). B) Femoral arteries were precontracted with phenylephrine (PE) and then exposed to acetylcholine (Ach) in a dose dependent manner (10⁻⁹ − 10⁻⁵ M). Vessels from SHRs demonstrated a decreased response compared to WKY rats. C) The ring segments from femoral arteries were exposed to phenylephrine (PE) in a dose-dependent manner (10⁻⁸ − 10⁻⁴ M). PE-induced contraction of WKY rats treated with STZ was significantly increased as compared to control rats, this increase was fully reversed in WKY rats treated with STZ and SnCl₂ (*p<0.05 vs. WKY control rats). D) Femoral arteries were precontracted with phenylephrine (PE) and then exposed to acetylcholine (Ach) in a dose dependent manner (10⁻⁹ − 10⁻⁵ M). Vessels from WKY rats treated with STZ demonstrated a decreased response compared to control rats significantly, which was fully reversed by SnCl₂ treatment (*p<0.05 vs. WKY control rats, # p<0.05 vs.WKY DM rats).

Figure 6.

A) The ring segments from femoral arteries were exposed to phenylephrine (PE) in a dose-dependent manner (10⁻⁸ − 10⁻⁴ M). PE-induced contraction of SHRs treated with STZ was significantly increased as compared to control rats. With SnCl₂ administration, the contraction in SHRs treated with STZ was more decreased compared to control rats (*p, ^†p<0.05 vs. SHR control, #p<0.05 vs. DM rats). B) Femoral arteries were precontracted with phenylephrine (PE) and then exposed to acetylcholine (Ach) in a dose dependent manner (10⁻⁹ − 10⁻⁵ M). Vessels from SHRs treated with STZ demonstrated a decreased response compared to control rats significantly, which was reversed by SnCl₂ administration (*p<0.05 vs. SHR control, #p<0.05 vs. DM rats).

Figure 7.

Number of circulating endothelial cells in control and diabetic rats was measured. Number of cells in SHR controls increased significantly compare to WKY control rats. STZ treatment increased the cells significantly in WKY rats and SHRs relative to controls. Administration of SnCl₂ decreased circulating endothelial cells in WKY DM rats, SHR control and SHR DM, but had no significant effect in WKY control rats (*p<0.05 vs. WKY control rats, * * p<0.05 vs. WKY DM rats, #p < 0.05 vs. SHR control rats, ^†p < 0.05 vs. SHR DM).

Figure 8.

A and B. Western blot and densitometry analysis of anti-apoptotic Bcl-2 in kidney from WKY rats (A, * P<0.05 vs. WKY DM rats) or SHRs (B, * p<0.05 vs. SHR control, #p<0.05 vs. SHR DM). Mean band density normalized relative to β-actin.

Figure 9.

Potential mechanisms underlying the chemoprotective actions of HO-1 in diabetes and hypertension.