Metyrapone Treatment Protects Low-Density Lipoprotein Receptor Knockout Mice against Hypercorticosteronemia Development without Changing Atherosclerosis Susceptibility.

The steroid 11beta-hydroxylase inhibitor metyrapone is able to effectively reverse the hypercortisolemia detected in human Cushing's Syndrome patients. In this current preclinical study, we investigated whether metyrapone monotherapy can also reverse the hypercortisolemia-associated increase in atherosclerotic cardiovascular disease risk. In this instance, female low-density lipoprotein receptor knockout mice fed a cholic acid-containing high cholesterol/high fat diet to induce the development of hypercorticosteronemia and atherosclerotic lesions were treated twice daily with 100 mg/kg metyrapone for 4 weeks. Metyrapone effectively protected against hypercorticosteronemia development with endpoint plasma corticosterone levels remaining 43% lower than in controls (p < 0.01). Gene expression analysis in livers and adrenals validated that glucocorticoid receptor signaling was also reduced. Importantly, metyrapone treatment did not impact plasma cholesterol levels or alter atherosclerotic plaque areas or lesional collagen contents. However, metyrapone induced significant systemic lymphocytopenia as evident from marked decreases in splenic white pulp contents and thymus weights (-48% and -41%, respectively; p < 0.001). In conclusion, we have shown that treatment with metyrapone diminishes hypercorticosteronemia without affecting atherosclerosis susceptibility in cholic acid-containing high cholesterol/high fat diet-fed low-density lipoprotein receptor knockout mice. These preclinical findings highlight that restoring plasma glucocorticoid levels to normal is not necessarily sufficient to overcome the cardiovascular co-morbidities associated with human Cushing's disease.

Prolactin receptor antagonism uncouples lipids from atherosclerosis susceptibility.

The pituitary-derived hormone prolactin has been suggested to stimulate the development of atherosclerosis and cardiovascular disease through its effects on metabolism and inflammation. In this study, we aimed to challenge the hypothesis that inhibition of prolactin function may beneficially affect atherosclerosis burden. Hereto, atherosclerosis-susceptible LDL receptor (Ldlr) knockout mice were transplanted with bone marrow from transgenic mice expressing the pure prolactin receptor antagonist Del1-9-G129R-hPRL or their non-transgenic littermates as control. Recipient mice expressing Del1-9-G129R-hPRL exhibited a decrease in plasma cholesterol levels (-29%; P<0.05) upon feeding a Western-type diet (WTD), which could be attributed to a marked decrease (-47%; P<0.01) in the amount of cholesterol esters associated with pro-atherogenic lipoproteins VLDL/LDL. By contrast, Del1-9-G129R-hPRL-expressing mice did not display any change in the susceptibility for atherosclerosis after 12 weeks of WTD feeding. Both the absolute atherosclerotic lesion size (223 ± 33 × 10(3) µm(2) for Del1-9-G129R-hPRL vs 259 ± 32 × 10(3) µm(2) for controls) and the lesional macrophage and collagen contents were not different between the two groups of bone marrow recipients. Importantly, Del1-9-G129R-hPRL exposure increased levels of circulating neutrophils (+91%; P<0.05), lymphocytes (+55%; P<0.05), and monocytes (+43%; P<0.05), resulting in a 49% higher (P<0.01) total blood leukocyte count. In conclusion, we have shown that prolactin receptor signaling inhibition uncouples the plasma atherogenic index from atherosclerosis susceptibility in Ldlr knockout mice. Despite an associated decrease in VLDL/LDL cholesterol levels, application of the prolactin receptor antagonist Del1-9-G129R-hPRL does not alter the susceptibility for initial development of atherosclerotic lesions probably due to the parallel increase in circulating leukocyte concentrations.

Leukocytosis and enhanced susceptibility to endotoxemia but not atherosclerosis in adrenalectomized APOE knockout mice.

Hyperlipidemic apolipoprotein E (APOE) knockout mice show an enhanced level of adrenal-derived anti-inflammatory glucocorticoids. Here we determined in APOE knockout mice the impact of total removal of adrenal function through adrenalectomy (ADX) on two inflammation-associated pathologies, endotoxemia and atherosclerosis. ADX mice exhibited 91% decreased corticosterone levels (P<0.001), leukocytosis (WBC count: 10.0 ± 0.4 x 10E9/L vs 6.5 ± 0.5 x 10E9/L; P<0.001) and an increased spleen weight (P<0.01). FACS analysis on blood leukocytes revealed increased B-lymphocyte numbers (55 ± 2% vs 46 ± 1%; P<0.01). T-cell populations in blood appeared to be more immature (CD62L+: 26 ± 2% vs 19 ± 1% for CD4+ T-cells, P<0.001 and 58 ± 7% vs 47 ± 4% for CD8+ T-cells, P<0.05), which coincided with immature CD4/CD8 double positive thymocyte enrichment. Exposure to lipopolysaccharide failed to increase corticosterone levels in ADX mice and was associated with a 3-fold higher (P<0.05) TNF-alpha response. In contrast, the development of initial fatty streak lesions and progression to advanced collagen-containing atherosclerotic lesions was unaffected. Plasma cholesterol levels were decreased by 35% (P<0.001) in ADX mice. This could be attributed to a decrease in pro-atherogenic very-low-density lipoproteins (VLDL) as a result of a diminished hepatic VLDL secretion rate (-24%; P<0.05). In conclusion, our studies show that adrenalectomy induces leukocytosis and enhances the susceptibility for endotoxemia in APOE knockout mice. The adrenalectomy-associated rise in white blood cells, however, does not alter atherosclerotic lesion development probably due to the parallel decrease in plasma levels of pro-atherogenic lipoproteins.