Decreased arteriolar density in endothelial nitric oxide synthase knockout mice is due to hypertension, not to the constitutive defect in endothelial nitric oxide synthase enzyme

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Abstract
Hypertension in endothelial nitric oxide synthase knockout (eNOS−/−) mice is believed to be partly due to altered vasodilatation. However, nitric oxide (NO) is also known to play an important part in angiogenesis. To investigate whether capillary and arteriolar density were impaired in eNOS−/− mice, as this could account for increased vascular resistance and hypertension. Using immunohistochemistry with mouse monoclonal smooth muscle α-actin antibody to detect arterioles and rabbit polyclonal fibronectin antibody to detect capillaries, we quantified arteriolar and capillary density in the left ventricle and in the gracilis muscle from eNOS−/− mice compared with those in C57BL6J littermates (n = 6–8) in 8- and in 12-week-old mice. In a second set of experiments, we treated 8-week-old normotensive eNOS−/− mice with the antihypertensive vasodilator, hydralazine, for 1 month. Eight-week-old eNOS−/− mice were normotensive and presented similar arteriolar and capillary densities in cardiac and skeletal mucles compared with those in eNOS+/+ mice. Twelve-week-old eNOS−/− mice were hypertensive (mean arterial pressure 118 ± 21 mmHg compared with 64 ± 2 mmHg;P < 0.05). Capillary densities were similar in eNOS−/− mice and eNOS+/+ mice in the heart (4154 ± 123 and 4051 ± 247/mm2, respectively) and in skeletal muscle (961 ± 40 and 1025 ± 41/mm2, respectively). Arteriolar densities were 15% lower in skeletal muscle and in the heart in eNOS−/− mice than in the eNOS+/+ control group (P < 0.05). Hydralazine prevented hypertension and arteriolar rarefaction in eNOS−/− mice, whereas capillary density was unaffected by treatment with the vasodilator. In young non-hypertensive eNOS−/− mice, the lack of eNOS did not affect microvascular densities in either of the muscles studied. In adult hypertensive eNOS−/− mice, we observed a lower arteriolar density, but a similar capillary density compared with controls. Hydralazine prevented hypertension and arteriolar rarefaction in adult mice, suggesting a non-NO-dependent pathway. Capillary density was not affected by hydralazine.