T. A. Shchelkunova

Changes of lysosomes in the earliest stages of the development of atherosclerosis

One of hypotheses of atherosclerosis is based on a presumption that the zones prone to the development of atherosclerosis contain lysosomes which are characterized by enzyme deficiency and thus, are unable to dispose of lipoproteins. The present study was undertaken to investigate the characteristics and changes of lysosomes in the earliest stages of the development of atherosclerosis. Electron microscopic immunocytochemistry revealed that there were certain changes in the distribution of CD68 antigen in lysosomes along the ‘normal intima‐initial lesion‐fatty streak’ sequence. There were no significant changes found in the key mRNAs encoding for the components of endosome/lysosome compartment in initial atherosclerotic lesions, but in fatty streaks, the contents of EEA1 and Rab5a mRNAs were found to be diminished while the contents of CD68 and p62 mRNAs were increased, compared with the intact tissue. The study reinforces a view that changes occurring in lysosomes play a role in atherogenesis from the very earlier stages of the disease.

Changes in levels of gene expression in human aortal intima during atherogenesis

Changes in the contents of 36 mRNAs species related to lipid turnover, inflammation, metabolism and the action of sex hormones in samples of aortal intima along the “intact tissue — lesions of type I — lesions of type II — lesions of type Va” sequence were analyzed using quantitative PCR. The expression of several mRNAs coding for components of the vesicular transfer and lipid turnover machinery was found to be resistant to atherogenesis or even decline in the course of atherogenesis. Decrease in expression was also recorded for steroid sulfatase, androgen receptor, and low density lipoprotein receptor mRNAs. However, the contents of the majority of other mRNA species increased gradually during disease progression. The earliest changes found as early as in lesions of type I were characteristic for estrogen sulfotransferase, apolipoprotein E, scavenger receptor SR-BI, collagen COL1A2, as well as chemokine CCL18 mRNAs. The contents of several mRNAs in intact tissue and atherosclerotic injuries had gender differences. Additionally, responses of two mRNAs, for aromatase and sterol regulatory element binding protein 2, to atherosclerotic lesion were also sex-differentiated. The contents of the majority of analyzed mRNAs in peripheral blood monocyte-derived macrophages were higher than in intact aorta. The correlations found in atherosclerotic lesions between mRNA species that predominant in macrophages and those expressed at comparable levels in macrophages and intact aorta or mainly in aorta suggest that the observed rise in the content of the majority of mRNAs during atherogenesis is determined by increase in expression in resident cells. The data suggest that the revealed absence of homeostatic regulation of expression of a number of genes associated with vesicular transfer and lipid turnover can serve as one of the reasons for lysosomal function insufficiency that leads to foam cell formation in atheroma. The observed sex differences in expression of a number of mRNAs suggest that estrogens in women perform their atheroprotective effects starting with predisposition to the disease and finishing with advanced stages of the pathologic process.

Comparative contents of mRNAs of sex steroid receptors and enzymes of their metabolism in arterial walls of men

The potential role of estrogens in regulation of metabolism in arteries of men was studied. Contents of mRNAs of sex hormone receptors, of some enzymes of their metabolism, and of some potential markers of the hormone effects were determined by real-time polymerase chain reaction in fragments of 18–54-year-old men’s large arteries with and without atherosclerotic lesions. Contents of estrogen receptor alpha (ERα) and transferrin receptor mRNAs were significantly different in undamaged fragments of the aorta and of the carotid and coronary arteries. Contents of some mRNAs in the carotid artery and aorta were found to correlate, which suggested a similarly directed regulation of their expressions. The levels of ERα and aromatase mRNAs negatively correlated with the blood plasma concentration of estradiol. Levels of steroid sulfatase and aromatase mRNAs were lower and the level of estrogen sulfotransferase mRNA was higher in blood vessel fragments with atherosclerotic lesions than in undamaged fragments. It is suggested that large arteries should be different in sensitivity to estrogens and that atherosclerotic lesions could lead to local suppression of the effect of estrogen on the cells of arteries.

Contents of mRNAs encoding endosome/lysosome components in normal human aorta and in stage ii of atherogenesis: a hidden regulation

Contents of mRNAs encoding endosome/lysosome components EEA1, Rab5a, Lamp1, Lamp2, p62 (SQSTM1), and CD63 were measured by quantitative PCR and compared in intact fragments of human aorta and in aorta fragments with atherosclerotic lesions of stage II (fatty streaks) of the same donors. During atherogenesis an increase was detected only in the level of p62 mRNA but not in other mRNAs. Nevertheless, correlation analysis revealed a profound rearrangement of inter-gene correlations: only 30% of correlations found in the fatty streaks coincided with the correlations in normal fragments. Thus, new constellations were formed in fatty streaks concurrently with disappearance of correlations between mRNAs under study and mRNAs encoding factors of lipid accumulation, reverse cholesterol transfer, and some lipid sensors/transcription regulators of lipid metabolism.

Inheritance of androgen program of male-specific expression of unusual estrogen-binding protein by daughter hepatocytes at rat liver regeneration.

A possibility of inheritance of androgen and basic genetic programs at the level of unusual estrogen-binding protein (UEBP) by daughter hepatocytes was investigated. Liver regeneration after partial (2/3) hepatectomy or after selective poisoning of hepatocytes of the central zone of hepatic lobules with CCl4 in adult rats were used as models of total and zonal proliferation of hepatocytes, respectively. UEBP content and the pattern of its tissue expression in the course of liver regeneration were monitored by radioligand and immunocytochemical technique. In animals of all groups possessing the androgen program of UEBP expression (intact, castrated and/or hypophysectomized males, and ovariectomized females treated with androgen) UEBP content was shown to be similarly high before initiation and after completion of liver regeneration. Unlike in males, in androgenized females a transient 4-fold increase of UEBP concentration on day 4 after partial hepatectomy was observed. In animals with a basic genetic program at the level of this protein (ovariectomized females, neonatally castrated males) only trace amounts of UEBP were observed in intact as well as in regenerated liver. The data were confirmed by immunocytochemical technique. A gradient mode of distribution of UEBP-contained cells within hepatic lobules with the highest specific staining around central veins was found by immunocytochemical technique in males. Specific staining of centrolobular and periportal hepatocytes was 7- to 10-fold in intact, and 4- to 6-fold in castrated and/or hypophysectomized males. In intact females specific staining was distributed uniformly at extremely low levels similar to that in periportal hepatocytes of males. Androgen administration to ovariectomized females stimulated a significant and stable increase of UEBP content in two layers of hepatocytes surrounding the central vein. Profiles of specific staining of hepatocytes within the hepatic lobules similar to that in control animals were observed after the completion of liver regeneration of different groups of rats. The results obtained suggest all the hepatocytes to be targets for androgen programming, natural in males or experimental in females, while the extent of expression of this program depends on the position of a hepatocyte within the liver lobules and the sex of the animal.

Approaches to the Design of Selective Ligands for Membrane Progesterone Receptor Alpha

A number of progesterone derivatives were assayed in terms of their affinity for recombinant human membrane progesterone receptor alpha (mPRα) in comparison with nuclear progesterone receptor (nPR). The 16α,17α-cycloalkane group diminished an affinity of steroids for mPRα without significant influence on affinity for nPR, thus rendering a prominent selectivity of ligands for nPR. On the contrary, substitution of methyl at C10 for ethyl or methoxy group moderately increased the affinity for mPRα and significantly lowered the affinity for nPR. A similar but even more prominent effect was observed upon substitution of the 3-oxo group for the 3-O-methoxyimino group. A significant preference towards mPRα was also rendered by the 17α-hydroxy group and additional C6–C7-double bond. The data suggest that the modes of lig- and interaction with mPRα and nPR in the C3 region of the steroid molecule are different. One can speculate that combination of the above substitutions at C17, C10, C6, and C3 may give ligand(s) with high specificity towards mPRα over nPR.

Lipid Regulators during Atherogenesis: Expression of LXR, PPAR, and SREBP mRNA in the Human Aorta

Transcription factors LXRs, PPARs, and SREBPs have been implicated in a multitude of physiological and pathological processes including atherogenesis. However, little is known about the regulation of these transcription factors at different stages of atherosclerosis progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to compare the contents of mRNAs in pairs intact-injured aorta fragments taken from the same donors. Only minor changes in LXRα, LXRβ, PPARα, PPARγ, SREBP1, and SREBP2 mRNA levels were found in initial lesions as compared with intact non-diseased tissue. The contents of all mRNAs but SREBP2 mRNA were found to be progressively up-regulated in fatty streaks and fibrous lipoid plaques. These changes were only partially reproduced in cultured macrophages upon lipid loading. Wave-shaped changes in abundance of correlations between given group of mRNAs and 28 atherosclerosis-related mRNA species in the course of atherogenesis were observed. The impact of specific mRNA correlations on the total correlations also significantly varied between different lesion types. The study suggests that the extent and forms of LXR/PPAR/SREBP participation in intima functions vary nonlinear in individual fashion in atherogenesis. We speculate that the observed changes in mRNAs expression and coupling reflect shifts in lipid ligands availability and cellular composition in the course of atherosclerosis progression.

Effect of sex hormones on levels of mRNAs coding for proteins involved in lipid metabolism in macrophages

The effects of sex hormones estradiol (E2), testosterone (Te), and 5α-dihydrotestosterone (DT) on cholesterol accumulation induced by modified low density lipoproteins (LDL) in macrophages differentiated from human peripheral blood monocytes and on the levels of mRNAs coding for proteins involved in lipid metabolism have been studied. All three hormones at physiological concentrations (1 nM) are capable of reducing cholesterol accumulation in cells. The treatment of cells with modified and native (not inducing cholesterol accumulation) LDL results in similar alterations in the expression of several mRNAs aimed primarily at homeostatic regulation of lipid metabolism. These alterations depend on the sex of macrophage donors and in some cases are even reversed in cells obtained from male and female donors. The cells not treated with modified LDL have no significant gender differences in the expression of the examined mRNAs. Hormones, either independently or in combination with the modified LDL, influence the levels of some mRNAs, and each hormone shows an individual range of effects. Correlation analysis of changes in mRNA content in the cells showed that the hormones may interfere with coordination of gene expression. Hormone action leads to: (1) reduced coupling of the content of individual mRNAs with their initial levels in the control cells; (2) reduced coupling of different mRNA levels; (3) regrouping of mRNAs between the clusters; and (4) changes in the number of factors that determine the correlation links between mRNAs. The data show that sex hormones may have impact on the level of expression of certain genes and, in particular, on the coordination of gene expression in macrophages.

Selection of progesterone derivatives specific to membrane progesterone receptors

The search of selective agonists and antagonists of membrane progesterone receptors (mPRs) is a starting point for the study of progesterone signal transduction mechanisms mediated by mPRs, distinct from nuclear receptors. According to preliminary data, the ligand affinity for mPRs differs significantly from that for classical nuclear progesterone receptors (nPRs), which might indicate structural differences in the ligand-binding pocket of these proteins. In the present work, we analyzed the affinity of several progesterone derivatives for mPRs of human pancreatic adenocarcinoma BxPC3 cell line that is characterized by a high level of mPR mRNA expression and by the absence of expression of nPR mRNA. The values were compared with the affinity of these compounds for nPRs. All tested compounds showed almost no affinity for nPRs, whereas their selectivity towards mPRs was different. Derivatives with an additional 19-hydroxyl group and removed 3-keto group had the highest selectivity for mPRs. These results suggest these compounds as the most selective progesterone analogs for studying the mechanisms of progestin action via mPRs.

Purification of specific rat liver estrogen-binding protein by affinity chromatography on estradiol-sepharose

Specific e s t r o g e n , binding pro te in (SEBP) of r a t l ive r d i f fers essent ia l ly in ce r ta in p r o p e r t i e s f r o m known r e c e p t o r s and t r a n s p o r t p ro te ins [1, 5, 7]. Besides aspec ts of regulat ion of the level of this sex-dependent p r o t e in in r a t s a l ready known, new aspec t s of mul t i fac tor ia l control of de te rmina t ion and regulat ion of gonad-de pendent p r o c e s s e s have been d i scovered [2, 3, 8].