Objective
To determine whether oxidative stress plays a role in the development of hypertension using a mouse model of fetal programming induced by endothelial nitric oxide synthase deficiency.
Study Design
Homozygous nitric oxide synthase knockout and wild type mice were cross-bred producing maternal (endothelial nitric oxide synthase +pat/−mat ) and paternal (endothelial nitric oxide synthase +mat/−pat ) heterozygous offspring. RNA from liver and kidney tissues of female pups were obtained at 14 weeks of age. Relative expression of the heat shock protein-B6, peroxiredoxin-3, superoxide dismutase-1, peroxisome proliferator-activated receptor gamma, nitric oxide synthase-1 and -2 were determined.
Results
In the kidneys, expression of nitric oxide synthase-2, peroxiredoxin-3, heat shock protein-B6, and superoxide dismutase-1 was up-regulated in endothelial nitric oxide synthase +pat/−mat but not in endothelial nitric oxide synthase +mat/−pat compared with wild type offspring. In the liver, there were no significant differences in the expression of nitric oxide synthase-1, nitric oxide synthase-2, peroxiredoxin, superoxide dismutase-1, or peroxisome proliferator-activated receptor gamma; however, heat shock protein-B6 was down-regulated in both heterozygotes offspring compared with wild type.
Conclusion
The intrauterine environment alters oxidative pathways gene expression in the kidneys of offspring, which may be a mechanism in the development of adult hypertension.
The intrauterine environment has long-term consequences that affect individuals well beyond the intrauterine life. This process, known as “developmental origin of adult diseases,” implies that insults during critical periods of development lead to long-lasting adaptive changes in the fetal structure, growth, and physiologic functions. Those changes lead to long-term consequences such as cardiovascular disease, hypertension, and metabolic syndrome. Oxidative stress is one such insult that may be operating during fetal development in an adverse uterine environment.
Oxidative stress is defined as the sustained increase in the levels of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion radical, and other free radicals. It is believed to play a role in the pathogenesis of many chronic diseases such as atherosclerosis, heart failure, hypertension, and diabetes. ROS are normal substrates of aerobic metabolism and derived from the reduction of oxygen. They are essential parts of the inter- and intracellular signaling systems when present at normal concentrations. Under these physiologic conditions, ROS are contained by the antioxidant defense system. However, imbalance between ROS production and clearance leads to pathologically elevated levels of ROS, which cause cellular dysfunction by oxidizing various biochemical structures such as DNA, lipids, and protein. The end result is what is commonly referred to as oxidative stress damage.
In cells with aerobic metabolism, numerous intracellular proteins contribute to maintaining the cellular redox status, including heat shock protein (HSP, B6), peroxiredoxin 3 (PeriRedox), mitochondria-specific H 2 O 2 scavenger or superoxide dismutase (SOD-1), peroxisome proliferator-activated receptor gamma (PPAR-γ), neuronal (nNOS or NOS1), and inducible nitric oxide synthase (iNOS or NOS2). These enzymes serve different functions in mediating (or protecting against) the oxidative stress damage; and their role in the kidneys and liver has been linked to several chronic diseases as atherosclerosis, diabetes, hypertension, and metabolic syndrome.
To study the mechanisms responsible for fetal programming of adult vascular disease, we have used a transgenic animal model of vascular programming induced by endothelial nitric oxide synthase (e-NOS or NOS3) deficiency. NOS-3 is the enzyme responsible for the generation of nitric oxide (NO) in endothelial cells. NO is a potent smooth muscle relaxant and 1 of the primary modulators of vascular tone, particularly in pregnancy. It plays a vital role in maintaining adequate uteroplacental perfusion, and inhibition of its synthesis leads to development of hypertension and fetal growth restriction in pregnancy.
Using this animal model, we have previously shown that heterozygous offspring born to knockout (KO) mothers (and wild-type [WT]father) are born smaller and develop abnormal vascular responses and higher blood pressure as adults compared with offspring born to WT mothers (and KO fathers) as well as altered protein expression, especially among those involved in oxidative stress and vascular homeostasis, in their vasculature later in life. Therefore, our objective was to study whether the effects of the intrauterine environment on the development of hypertension in adult offspring in this murine model of fetal vascular programming may be in part mediated by altered expression of genes involved in the oxidative stress pathways in the offspring.
Materials and Methods
Mature cycling female and male mice (4-6 weeks old) homozygous for disruption of the NOS3 gene (NOS3-knockout, strain B6.129P2-Nos3 tm1Unc , stock no. 002684, NOS3 −/−KO ) and their age-matched WT controls (NOS3-WT, strain C57BL/6J, stock no. 000664, NOS3 +/+WT ) were purchased from Jackson Laboratory (Bar Harbor, ME). Animals were maintained and bred in the animal care facility at the University of Texas Medical Branch. They were housed separately in temperature- and humidity-controlled quarters with constant 12-hour light:dark cycles and provided with food and water ad libitum. Regular maintenance and care was provided by certified personnel and veterinary staff according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch. All surgical procedures were carried out by trained personnel according to the IACUC guidelines.
The details of the model have been previously described, but briefly homozygous NOS3 KO (NOS3 −/−KO ) and WT mice (NOS3 +/+WT ) were cross-bred to produce paternally (NOS3 +mat/−pat ) and maternally derived (NOS3 +pat/−mat ) heterozygous offspring ( Figure 1 ). These offspring are genomically similar but they developed in normal mothers or mothers lacking NOS-3, respectively. At 14 weeks of age, these heterozygous pups were sacrificed by the CO 2 inhalation method per the IACUC and the American Veterinary Medical Association guidelines. In addition, we killed NOS3 +/+WT offspring to be used as a control group. A total of 6-10 female mice were obtained in each group.
