Endothelial dysfunction and reduced antioxidant protection in an animal model of the developmental origins of cardiovascular disease

Abstract

Endothelial dysfunction underlies cardiovascular disease (CVD) in humans and is reported in animal models of developmental origins of such disease. We have investigated whether impaired antioxidant defences and NO generation underlie the genesis of endothelial dysfunction and operate as part of the normal processes of developmental plasticity regulating the induction of phenotype in the offspring. Female Wistar rats were fed either a control (C, 18% protein) or protein-restricted (PR, 9% protein) diet throughout pregnancy. Dams and pups were returned to standard laboratory chow post partum. In male offspring, PR resulted in a reduced endothelial responsiveness to acetylcholine (P < 0.05) in resistance arteries, with vascular remodelling evident from a reduction in smooth muscle content. mRNA expression of endothelial NO synthase (eNOS) was increased (P < 0.05) but there was no change in mRNA levels of manganese superoxide dismutase (MnSOD) or glutamate cysteine ligase (GCL) expression. Interestingly, expression of the antioxidant enzyme haem oxygenase-1 (HO-1) was reduced in the liver (P < 0.05). Female PR offspring also showed a reduced endothelial responsiveness but exhibited no changes in expression of eNOS, iNOS, soluble guanylate cyclase (sGC) or antioxidant genes. Thus, in this model of the developmental origins of CVD, the structure and function of resistance arteries in offspring is altered in complex ways which cannot simply be explained by attenuation in vascular eNOS or in antioxidant protection afforded by GCL or MnSOD. The dysfunction in male offspring may partially be counteracted by an up-regulation of eNOS expression; however, PR does lead to reduced HO-1 expression in these offspring, which may affect both their growth and vascular function. Our findings have established that PR induces significant phenotypic changes in male offspring that may be indicative of an adaptive response during development.

Figure 1. Growth of male (A) and female (B) offspring of C (◯, n= 6) and PR (•, n= 6) dams

***P < 0.001 two-way ANOVA.

Figure 2

Systolic blood pressure in male (A) and female (B) offspring at 28 ± 1, 56 ± 1 and 112 ± 1 days of age from C (□, n= 8–11) and PR (▪, n= 8–11) dams

Figure 3. Cumulative additions of the endothelium-dependent vasodilator ACh to mesenteric arteries of male (A) and female (B) offspring from C (◯, n= 3–10) and PR (•, n= 5–8) dams

**P < 0.01 pEC₅₀ C versus PR. *P < 0.05 pGC₅₀ C vs PR

Figure 4. Cumulative additions of the endothelium-dependent vasodilator ACh in the absence (◯, n= 8–10) or presence of the SOD mimetic tempol (1 mm; ▪, n= 8–9) to mesenteric arteries from male offspring from C (A) or PR (B) dams

*P < 0.05 max response naïve versus tempol.

Figure 5

A, representative cross-section of 2nd order mesenteric artery stained with Miller's Elastin and Van Gieson's stain; yellow computer masks of collagen (B), elastin (C) and smooth muscle (D) staining. E, percentage of media + intima thickness of vascular smooth muscle, elastin and collagen in mesenteric arteries from the offspring of control (□, n= 6) and PR (▪, n= 8) dams, means ±s.e.m., **P < 0.01 C versus PR by Student's t test.

Figure 6

A and B, heptatic mRNA levels of 28S, 18S and β-actin from male (A) and female (B) offspring from C (□) or PR (▪) dams. *P < 0.05, **P < 0.01 and ***P < 0.0001 PR versus C diet, Student's t test. C and D, mesenteric artery mRNA levels of MnSOD, GCL and HO-1 from male (C) and female (D) offspring of C (□) or PR (▪) dams. E and F, hepatic mRNA levels of MnSOD, GCL and HO-1 from male (E) and female (F) offspring of C (□) or PR (▪) dams. *P < 0.05 C versus PR, Student's t test.

Figure 7. Correlation between hepatic HO-1 expression and PM weight in the offspring of C (◯, n= 10) and PR (•, n= 10) dams

R²= 0.27.