Low Prolactin and High 20-α-Hydroxysteroid Dehydrogenase Levels Contribute to Lower Progesterone Levels in HIV-Infected Pregnant Women Exposed to Protease Inhibitor-Based Combination Antiretroviral Therapy.

BACKGROUND: It has been reported that pregnant women receiving protease inhibitor (PI)-based combination antiretroviral therapy (cART) have lower levels of progesterone, which put them at risk of adverse birth outcomes, such as low birth weight. We sought to understand the mechanisms involved in this decline in progesterone level. METHODS: We assessed plasma levels of progesterone, prolactin, and lipids and placental expression of genes involved in progesterone metabolism in 42 human immunodeficiency virus (HIV)-infected and 31 HIV-uninfected pregnant women. In vitro studies and a mouse pregnancy model were used to delineate the effect of HIV from that of PI-based cART on progesterone metabolism. RESULTS: HIV-infected pregnant women receiving PI-based cART showed a reduction in plasma progesterone levels (P= .026) and an elevation in placental expression of the progesterone inactivating enzyme 20-α-hydroxysteroid dehydrogenase (20α-HSD; median, 2.5 arbitrary units [AU]; interquartile range [IQR], 1.00-4.10 AU), compared with controls (median, 0.89 AU; IQR, 0.66-1.26 AU;P= .002). Prolactin, a key regulator of 20α-HSD, was lower (P= .012) in HIV-infected pregnant women. We observed similar data in pregnant mice exposed to PI-based cART. In vitro inhibition of 20α-HSD activity in trophoblast cells reversed PI-based cART-induced decreases in progesterone levels. CONCLUSIONS: Our data suggest that the decrease in progesterone levels observed in HIV-infected pregnant women exposed to PI-based cART is caused, at least in part, by an increase in placental expression of 20α-HSD, which may be due to lower prolactin levels observed in these women.

Deletion of Myeloid GSK3α Attenuates Atherosclerosis and Promotes an M2 Macrophage Phenotype.

OBJECTIVE: Glycogen synthase kinase (GSK)-3α/ß has been implicated in the pathogenesis of diabetes mellitus, cancer, Alzheimer, and atherosclerosis. The tissue- and homolog-specific functions of GSK3α and ß in atherosclerosis are unknown. This study examines the effect of hepatocyte or myeloid cell deletion of GSK3α or GSK3ß on atherosclerosis in low-density lipoprotein receptor (LDLR)(-/-) mice. APPROACH AND RESULTS: We ablated GSK3α or GSK3ß expression in hepatic or myeloid cells of LDLR(-/-) mice, and mice were fed a high-fat diet for 10 weeks. GSK3α or GSK3ß deficiency in hepatic or myeloid cells did not affect metabolic parameters, including plasma lipid levels. Hepatic deletion of GSK3α or GSK3ß did not affect the development of atherosclerosis or hepatic lipid content. Myeloid deletion of GSK3α, but not of GSK3ß, reduced atherosclerotic lesion volume and lesion complexity. Mice lacking GSK3α in myeloid cells had a less inflammatory and more anti-inflammatory plasma cytokine profile. Macrophages within atherosclerotic lesions of myeloid GSK3α-deficient mice, but not of GSK3ß-deficient mice, displayed reduced expression of markers associated with M1 macrophage polarization and enhanced expression of the M2 markers. Finally, bone marrow-derived macrophages were isolated and differentiated into classical M1 macrophages or alternative M2 macrophages in vitro. GSK3α deletion, but not GSK3ß deletion, attenuated the expression of genes associated with M1 polarization while promoting the expression of genes associated with M2 polarization by modulating STAT3 and STAT6 activation. CONCLUSIONS: Our findings suggest that deletion of myeloid GSK3α attenuates the progression of atherosclerosis by promoting an M2 macrophage phenotype.

Glycogen synthase kinase 3α deficiency attenuates atherosclerosis and hepatic steatosis in high fat diet-fed low density lipoprotein receptor-deficient mice.

Studies have implicated signaling through glycogen synthase kinase (GSK) 3α/ß in the activation of pro-atherogenic pathways and the accelerated development of atherosclerosis. By using a mouse model, we examined the role of GSK3α in the development and progression of accelerated atherosclerosis. We crossed Gsk3a/GSK3α-knockout mice with low-density lipoprotein receptor (Ldlr) knockout mice. Five-week-old Ldlr(-/-);Gsk3a(+/+), Ldlr(-/-);Gsk3a(+/-), and Ldlr(-/-);Gsk3a(-/-) mice were fed a chow diet or a high-fat diet for 10 weeks and then sacrificed. GSK3α deficiency had no detectible effect on any measured parameters in chow-fed mice. High-fat-diet fed Ldlr(-/-) mice that were deficient for GSK3α had significantly less hepatic lipid accumulation and smaller atherosclerotic lesions (60% smaller in Ldlr(-/-);Gsk3a(+/-) mice, 80% smaller in Ldlr(-/-);Gsk3a(-/-) mice; P < 0.05), compared with Ldlr(-/-);Gsk3a(+/+) controls. GSK3α deficiency was associated with a significant increase in plasma IL-10 concentration and IL-10 expression in isolated macrophages. A twofold to threefold enhancement in endoplasmic reticulum stress-induced IL-10 expression was observed in Thp-1-derived macrophages that were pretreated with the GSK3α/ß inhibitor CT99021. Together, these results suggest that GSK3α plays a pro-atherogenic role, possibly by mediating the effects of endoplasmic reticulum stress in the activation of pro-atherogenic pathways.

HIV antiretroviral exposure in pregnancy induces detrimental placenta vascular changes that are rescued by progesterone supplementation.

Adverse birth outcomes are common in HIV-positive pregnant women receiving combination antiretroviral therapy (cART), especially when cART is initiated in early pregnancy. The mechanisms remain poorly understood. Using a mouse model we demonstrate that protease inhibitor based-cART exposure beginning on day 1 of pregnancy was associated with a pro-angiogenic/pro-branching shift in the placenta driven by lower Flt-1 levels and higher Gcm-1 expression. Micro-CT imaging revealed an increase in the number of arterioles in cART-treated placentas, which correlated with fetal growth restriction. Delaying initiation of cART, or supplementing cART-treated mice with progesterone, prevented the pro-angiogenic/pro-branching shift and the associated placenta vascular changes. In agreement with our mouse findings, we observed an increase in the number of terminal-villi capillaries in placentas from HIV-positive cART-exposed women compared to HIV-negative controls. Capillary number was inversely correlated to maternal progesterone levels. Our study provides evidence that cART exposure during pregnancy influences placenta vascular formation that may in turn contribute to fetal growth restriction. Our findings highlight the need for closer investigation of the placenta in HIV-positive pregnancies, particularly for pregnancies exposed to cART from conception, and suggest that progesterone supplementation could be investigated as a possible intervention to improve placenta function in HIV-positive pregnant women.

Quantitative analysis and characterization of atherosclerotic lesions in the murine aortic sinus.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.