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Biomedical subjects

A N Orekhov

Publications and source records attributed to A N Orekhov.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cell proliferation in normal and atherosclerotic human aorta: proliferative splash in lipid-rich lesions.

Local accumulation of cells (hypercellularity) in the intima of the arterial wall as a result of cell proliferation is recognized as one of the major manifestations of human atherosclerosis. In the present study we have used a monoclonal antibody against PCNA to identify the proliferative activity, in uninvolved intima of human aorta classified as diffuse intimal thickening, and in different types of atherosclerotic lesions: specifically, initial lesion, fatty streak, fibroatheroma and fibrous plaque. As compared with a diffuse intimal thickening, the cell number in the initial lesions, fatty streaks and in a fibrolipid plaque (fibroatheroma) was 1.5-3-fold higher, while the cellularity in a fibrous plaque (fibrotic lesion) was lower than in a fibroatheroma and comparable with the cell number in the initial lesions. Using monoclonal antibodies, inflammatory cells (T- and B-lymphocytes as well as monocytes-macrophages) have been revealed in the intima. However, most (84-93%) of the intimal cells were noninflammatory cells classified as resident cells possessing the antigens of smooth muscle cells and pericytes as well as a small number of cells unidentifiable with the antibodies used. The highest number of proliferating cells was found in a fibroatheroma (11-fold higher as compared with a diffuse intimal thickening). A significant, but lesser increase of PCNA-positive cells was revealed in other types of lesions, too. The proliferative 'splash' in lipid-rich lesions suggests a relationship between the lipid accumulation in atherosclerotic intima and the stimulation of proliferation. The highest proliferative index of resident cells (i.e. percentage of the PCNA-positive cells among the total number of resident cells) was revealed in fibrotic lesions. It was approximately eight-fold higher than in a diffuse intimal thickening. The proliferative index of inflammatory cells considerably exceeded that of resident cells. However, in all types of atherosclerotic lesions and in a diffuse intimal thickening it showed no significant differences and was similar to the proliferative index of inflammatory cells isolated from peripheral blood. This suggests that an increased number of resident cells in atherosclerotic lesions can be explained by stimulation of their proliferative activity, whereas an altered inflammatory cell number is rather a result of their penetration from the blood into the subendothelial intima with a constant proliferative index.

Adult↗
PubMed1998

Continuous subendothelial network formed by pericyte-like cells in human vascular bed.

The localization of pericyte-like cells in adult human vascular bed was studied immunocytochemically using anti-pericyte antibody--3G5. In large, medium- and small-size arteries, pericyte-like cells were identified in the inner intimal layer, predominantly in the subendothelium, in the outer layer of the media and in the adventitia in vasa vasora. In veins, pericytes were found in the subendothelial layer of the intima, in the media and in the adventitia where they were located in vasa vasora. These cells were revealed in the subendothelium of arterioles, venules and capillaries of the pia mater encephali. Examination of intimal en face preparations of various size blood vessels showed that subendothelial pericyte-like cells form a network contacting with their processes. Thus we found a continuous subendothelial network formed by pericyte-like cells in the human vascular bed.

Adult↗
PubMed1998

In vivo oxidized low density lipoprotein: degree of lipoprotein oxidation does not correlate with its atherogenic properties.

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.

Adult↗
PubMed1998

Antioxidant content in low density lipoprotein and lipoprotein oxidation in vivo and in vitro.

UNLABELLED: Human blood contains naturally occurring multiple-modified low density lipoprotein (nomLDL) capable of inducing the accumulation of cholesteryl esters in the cells of human aortic intima. NomLDL is desialylated particles of small size with an increased electronegative charge which can be separated from native low density lipoprotein (LDL) by lectin chromatography. The purpose of this study was to determine the content of antioxidants in native and nomLDL obtained from healthy subjects and from patients with coronary heart disease as well as to elucidate a possible relationship between the level of antioxidants and the degree of in vivo and in vitro LDL oxidizability. The apoB-bound cholesterol level in native and nomLDL of healthy subjects was 0.25 +/- 0.08 and 0.28 +/- 0.05 mol/mol apoB, respectively. The level of apoB-bound cholesterol in native LDL of coronary atherosclerosis patients showed no significant difference from that in healthy subjects' native lipoprotein. At the same time, the level of apoB-bound cholesterol in patients' nomLDL was 7-fold higher than in native LDL. The average duration of the lag phase of native LDL oxidation did not show a significant difference between the lipoprotein of healthy subjects and coronary atherosclerosis patients. The lag phase of nomLDL obtained from healthy subjects and patients was significantly shorter (3- and 6-fold, respectively) than for their native LDL. The latter finding points to their increased susceptibility to in vitro oxidation. Oxidizability of total LDL preparations correlated positively with their nomLDL content. The content of all the antioxidants studied (coenzyme-Q10, alpha- and gamma-tocopherols, beta-carotene and lycopene) in nomLDL was 1.5- to 2-fold lower than in native LDL. The level of apoB-bound cholesterol in nomLDL, correlated positively with the ubiquinone-10 content and showed negative correlation with ubiquinol-10 and beta-carotene levels. On the other hand, the content of apoB-bound cholesterol in native LDL correlated positively with the ubiquinol-10 level. Susceptibility of nomLDL to in vitro oxidation exhibited negative correlation with alpha-tocopherol and beta-carotene levels and a positive correlation with the ubiquinone-10 content. On the contrary, oxidizability of native LDL correlated positively with the ubiquinone-10 level. CONCLUSIONS: (a) elevated apoB-bound cholesterol level in nomLDL of coronary atherosclerosis patients indicates that peroxidation of lipids occurs in vivo; (b) in vivo lipoperoxidation in nomLDL is corroborated by increased proportion of oxidized form of coenzyme-Q10; (c) content of lipid-soluble antioxidants in nomLDL is lower than in native lipoprotein; (d) nomLDL has a higher susceptibility to in vitro oxidation than native LDL; (e) it is necessary to use isolated subfractions of native LDL and nomLDL, but not total lipoprotein preparations, to study the mechanisms of lipid peroxidation.

Adult↗
PubMed1998

The effects of antihypertensive agents on atherosclerosis-related parameters of human aorta intimal cells.

Four antihypertensive agents - amlodipine, verapamil, propranolol and perindoprilat - were studied in human cell cultures. Antiatherogenic activity was investigated using uninvolved human aortic smooth muscle intima cells and atherogenic serum obtained from patients with coronary atherosclerosis. Amlodipine and verapamil significantly inhibited serum-induced increases in cholesterol content, cell-proliferative activity and protein synthesis in the cultured cells. Propranolol increased all three parameters, while perindoprilat had no effects. In addition, amlodipine and verapamil significantly lowered the intracellular cholesterol content of smooth muscle cells derived from atherosclerotic plaque and inhibited cell proliferation and protein synthesis. Propranolol increased all of these parameters, while perindoprilat produced no effects. The antiatherogenic and antiatherosclerotic actions of verapamil and amlodipine were confirmed in an ex vivo model. These studies demonstrated a beneficial antiatherosclerotic effect of amlodipine that was greater than that of verapamil. Perindoprilat had a neutral effect on atherosclerotic parameters, while the action of propranolol appeared to be potentially detrimental.

Adult↗
PubMed1998

Optimization of the assay for sialic acid determination in low density lipoprotein.

Sialic acid level in blood plasma and circulating glycoproteins is considered to be a marker for a number of pathologic conditions, including atherosclerosis, cancer, etc. The precise measurement of sialic acid level is an important laboratory procedure to allow correct interpretation of results. Colorimetric methods commonly used for the measurement of sialic acid are not highly specific, as interfering substances may alter the results. Among these, malondialdehyde and other aldehydes play the decisive role. In the circulation, aldehydes are commonly produced during lipid peroxidation in the lipid core of lipoprotein particles, especially low density lipoprotein (LDL). To establish the impairment to the sialic acid determination in LDL introduced by interfering substances, the optimized assay based on Warren's traditional method was developed and tested in 606 LDL samples. The optimization implies the comparison of color developed using the standard Warren procedure with that due to contaminating agents, mainly thiobarbituric acid-reactive substances (TBARS). In LDL stored at 4 degreesC, the estimates obtained by the modified procedure were 41.5% or 30.1 nmol/mg lower, on average, compared to the standard procedure (n = 45, P < 0.0001). Even in LDL stored at -70 degreesC, sialic acid estimates obtained by the modified procedure were 6.6% or 3.6 nmol/mg lower, on average, compared to the standard measurement (n = 561, P < 0.005). Thus, the modified procedure avoids significant distortion of the measurement induced by the presence of interfering agents.

Colorimetry↗
PubMed1997

Antiatherosclerotic and antiatherogenic effects of a calcium antagonist plus statin combination: amlodipine and lovastatin.

Recently, the Regression Growth Evaluation Statin Study (REGRESS) demonstrated the synergistic, antiatherogenic effect of lipid-lowering therapy with pravastatin in combination with calcium antagonists. This combination retarded the progression of stenosis and reduced the number of new lesions more effectively than did statin therapy alone. In the present study, our objective was to elucidate the mechanism of this more pronounced effect of the statin-calcium antagonist combination on the atherosclerotic lesion. Smooth muscle cells cultured from the subendothelial intima of the human aorta were incubated with whole blood serum or with low-density lipoprotein (LDL) taken from patients cotreated with lovastatin and amlodipine. Serum added to the cells cultured from the atherosclerotic lesion reduced cell cholesterol. Such an antiatherosclerotic effect of cotreatment with amlodipine-lovastatin was revealed in this study and was more pronounced than the effect of treatment with either amlodipine or lovastatin alone. LDL isolated from atherogenic plasma stimulated cell cholesterol accumulation. Treatment with amlodipine alone and the amlodipine-lovastatin combination ameliorated the atherogenic effect of LDL. As compared with amlodipine alone, the combination demonstrated a considerably higher antiatherogenic effect on LDL atherogenicity. Amlodipine-lovastatin cotreatment increased sialic acid and decreased the susceptibility of LDL to oxidation more effectively than amlodipine alone. In addition, combination therapy reduced the LDL negative charge, while amlodipine alone was impotent. These findings may serve as an explanation of the more pronounced antiatherogenic effect at the lipoprotein level of amlodipine-lovastatin combined therapy compared with amlodipine therapy alone.

Amlodipine↗
PubMed1997

Lack of correlation between degree of human plasma low density lipoprotein oxidation and its atherogenic potential.

We have recently found that adducts of lipids, particularly cholesterol, with apolipoprotein B (apoB) are stable markers of human plasma low density lipoprotein (LDL) oxidation [7]. In this study we attempt to assess the relationship between the degree of plasma LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intima smooth muscle cells, i.e., LDL atherogenic potential. LDL samples of 32 out of 39 healthy subjects did not increase cholesterol content in cells cultured from grossly normal intima of human aorta. Most of LDL preparations isolated from coronary atherosclerosis patients with (34 out of 43) or without (35 out of 45) hypercholesterolemia stimulated intracellular cholesterol accumulation by 32-302%. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.10, n = 127). These results suggest that atherogenicity of LDL circulating in human plasma does not depend on the degree of lipid peroxidation in LDL particles.

Adult↗
PubMed1997

Metabolism of native and naturally occurring multiple modified low density lipoprotein in smooth muscle cells of human aortic intima.

The subfraction of low density lipoprotein (LDL) with low sialic acid content that caused accumulation of cholesterol esters in human aortic smooth muscle cells has been found in the blood of coronary atherosclerosis patients. It was demonstrated that this subfraction consists of LDL with small size, high electronegative charge, reduced lipid content, altered tertiary structure of apolipoprotein B, etc. LDL of this subfraction is naturally occurring multiple-modified LDL (nomLDL). In this study we compared the binding, uptake and proteolytic degradation of native LDL and nomLDL by smooth muscle cells cultured from human grossly normal intima, fatty streaks, and atherosclerotic plaques. Uptake of nomLDL by normal and atherosclerotic cells was 3.5- and 6-fold, respectively, higher than uptake of native LDL. Increased uptake of nomLDL was due to increased binding of this LDL by intimal smooth muscle cells. The enhanced binding is explained by the interaction of nomLDL with cellular receptors other than LDL-receptor. Modified LDL interacted with the scavenger receptor, asialoglycoprotein receptor, and also with cell surface proteoglycans. Rates of degradation of nomLDL were 1.5- and 5-fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. A low rate of nomLDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased compared with normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol, or metallo-proteinase inhibitors had partial effect. This fact reveals that cathepsin D is involved in initial stages of apoB degradation by intimal smooth muscle cells. Obtained data show that increased uptake and decreased lysosomal degradation of nomLDL may be the main cause of LDL accumulation in human aortic smooth muscle cells, leading to foam cell formation.

Adult↗
PubMed1997

In vitro effect of garlic powder extract on lipid content in normal and atherosclerotic human aortic cells.

In the present study, the mechanism of the in vitro effect of garlic powder extract (GPE) on lipid content of cultured human aortic cells was investigated. The addition of GPE abolished atherogenic blood serum-induced accumulation of free cholesterol, triglycerides, and cholesteryl esters in smooth muscle cells derived from uninvolved (normal) intima. In cells isolated from atherosclerotic plaque, GPE lowered these lipids. GPE inhibited lipid synthesis both in normal and atherosclerotic cells. It inhibited acyl-CoA:cholesterol acyltransferase activity that participates in the cholesteryl ester formation and stimulated cholesteryl ester hydrolase that degrades cholesteryl esters. This may explain the lipid reduction caused by GPE in atherosclerotic cells. GPE inhibited the uptake of modified low density lipoprotein and degradation of lipoprotein-derived cholesteryl esters, thus considerably reducing the intracellular accumulation of cholesteryl esters. This suggests the mechanism responsible for the prevention of lipid accumulation in aortic cells caused by atherogenic blood serum.

Adult↗
PubMed1997

Collagen-synthesizing cells in initial and advanced atherosclerotic lesions of human aorta.

Accumulation of extracellular matrix, fibrosis, is regarded to be one of the major manifestations of atherosclerosis. Collagen type I is the predominant matrix component in human atherosclerotic plaques. In this work we have demonstrated procollagen type I expressing cells (PCI-cells) and studied their localization in grossly normal human aorta and atherosclerotic lesions: initial lesions, fatty streaks, fibrolipid lesions (fibrolipid plaque, fibroatheroma), fibrotic lesions (fibrous plaque). PCI-cells were revealed immunocytochemically using SPI.D8 monoclonal antibody against human procollagen type I. We failed to detect PCI-cells in the areas of grossly normal aorta and media underlying atherosclerotic lesions. Positively stained cells were shown in the areas of initial lesions, fatty streaks, fibrolipid and fibrous plaques. The largest amount of PCI-cells was revealed in fatty streaks. These cells were predominantly localized in the preluminal proteoglycan-rich intimal sublayer. Intimal cells in grossly normal regions formed a common cellular network contacting each other with their processes. The cellular network is found to be partly disintegrated in atherosclerotic lesions, which leads to the appearance of isolated cells. The share of isolated PCI-cells localized outside the intimal cellular network was higher in advanced lesions than in the areas of early atherosclerotic lesions. In initial lesions most of PCI-cells were identified as smooth muscle cells using antibodies to smooth muscle alpha-actin. In fatty streaks PCI-expressing smooth muscle cells were fewer in number. Much fewer cells double-stained with anti-alpha-actin and anti-PCI antibodies were found in fibrolipid and fibrous plaques. The proportion of these double stained cells was higher among total number of PCI-cells involved in the cellular network versus PCI-cells outside the network. The results of the study demonstrated that the most active de novo synthesis of interstitial collagen takes place in the regions of atherosclerotic lesions characterized by lipid deposition, which may lead to the further progression of atherosclerotic lesions.

Aorta↗
PubMed1997

Subendothelial smooth muscle cells of human aorta express macrophage antigen in situ and in vitro.

Cells bearing a smooth muscle cell marker--alpha-actin and a macrophage marker--CD68 antigen were immunocytochemically identified on 'en face' preparations of human aortic intima. Cells, expressing smooth muscle alpha-actin, macrophage CD68 antigen and both markers, i.e. smooth muscle cells possessing the macrophage antigen, were identified both in grossly normal aortic areas and in atherosclerotic lesions (fatty streaks and atherosclerotic plaques). CD68-positive smooth muscle cells were most common in the lipid-rich areas: fatty streaks and atherosclerotic plaque shoulders. Cells expressing smooth muscle alpha-actin and CD68 were also revealed in primary cultures prepared from grossly normal and atherosclerotic intima. Cells expressing both antigens were found in all examined cultures. The proportion of these cells in cultures from grossly normal areas and atherosclerotic plaques was similar: 14.5 +/- 4.1 and 14.6 +/- 4.8%, respectively. Cultures from fatty streaks had a higher content of cells expressing both antigens: 25.1 +/- 7.0%. Modified low density lipoprotein-induced intracellular lipid accumulation in cells cultured from grossly normal intima led to a three-fold increase in the number of cells sharing alpha-actin and CD68 antigen. Accumulation of latex beads by phagocytosis had a similar effect. It was suggested that in atherosclerotic lesions intracellular lipid accumulation and other stimulators of phagocytosis may provoke the expression of macrophage-associated antigen CD68 in settled cells of the subendothelial intima of human aorta.

Adult↗
PubMed1996

Characteristics of low density lipoprotein isolated from circulating immune complexes.

Circulating immune complexes (CIC) containing low density lipoprotein (LDL) were recently found in the blood of patients with coronary atherosclerosis. In the present study, we investigated the chemical composition and physical characteristics of the lipoprotein constituents of these CIC. CIC were isolated from the blood of atherosclerotic patients by affinity chromatography using anti-human immunoglobulin G-agarose. Low density lipoprotein of these complexes (CIC-LDL) was obtained by ultracentrifugation. CIC-LDL was compared with free circulating LDL isolated from the blood plasma of the same patients. Plasma LDL was fractionated by lectin-chromatography on RCA120-agarose to obtain desialylated LDL (atherogenic) and sialylated LDL (nonatherogenic). Both CIC-LDL and desialylated LDL, but not native (sialylated) lipoprotein, induced a 1.8- to 3-fold increase in the intracellular contents of free and esterified cholesterol of cells cultured from grossly normal areas of human aorta. The sialic acid level in CIC-LDL was 1.3- and 2.1-fold lower than in desialylated or native LDL, respectively. The neutral lipid and phospholipid contents of CIC-LDL and desialylated LDL were reduced as compared to native LDL. The levels of lipid-oxidation products, thiobarbituric acid-reactive substances and hydroperoxides, were similar in all lipoprotein preparations. However, desialylated LDL and CIC-LDL had an elevated oxysterol content. Gradient ultracentrifugation revealed that CIC-LDL particles had a higher density than native LDL. The mean diameters of native, desialylated and CIC-LDL accounted for 24.0, 21.3 and 19.5 nm, respectively. Like desialylated LDL, CIC-LDL displayed a higher electrophoretic mobility compared with that of native LDL. Thus, LDL obtained from circulating immune complexes appears to be a multiple-modified lipoprotein possessing many similarities to desialylated LDL. It was also found that the LDL content of circulating immune complexes correlates well with the desialylated LDL level in human plasma but not with the total LDL concentration. We believe that desialylated LDL predominately interacts with antibodies forming immune complexes. Taken together, our findings suggest that multiple-modified desialylated LDL is the circulating autoantigen for anti-LDL autoantibodies.

Adult↗
PubMed1996

Atherogenic modified LDL in diabetes.

This paper summarizes the recent findings on LDL atherogenic modifications in diabetic patients. LDL from diabetic patients, unlike LDL from healthy subjects, caused a significant increase in cholesterol content of cells cultured from unaffected human aortic intima, i.e., produced a direct atherogenic effect. LDL was divided into two fractions (nonbound and bound) by affinity chromatography on Ricinus communis agglutinin-agarose. The amount of bound LDL was significantly higher in diabetic patients compared with healthy subjects. Bound LDL was characterized by significantly lowered sialic acid content and a significantly increased fructosyl lysine level compared with unbound LDL, i.e., was desialylated and nonenzymatically glycosylated lipoprotein. The bound (desialylated), but not unbound (sialylated), LDL subfraction induced cholesterol accumulation in cultured cells. Bound LDL was also characterized by a significantly lowered content of neutral lipids and demonstrated increased electrophoretic mobility on agarose gel electrophoresis. Bound and nonbound LDL differed significantly in hydrated density and particle size, as was determined by density gradient ultracentrifugation and native polyacrylamide gradient gel electrophoresis. The results of this study have shown that the in vivo modified atherogenic LDL subfraction in the blood of diabetic patients is represented by small, dense, more electronegative, desialylated, and glycated LDL.

Arteriosclerosis↗
PubMed1996

Similarity between naturally occurring modified desialylated, electronegative and aortic low density lipoprotein.

The sialic acid content of electronegative low density lipoprotein (LDL) and LDL isolated from human aortic intima was measured. Sialic acid level in electronegative LDL of healthy subjects was 1.7-fold lower than in native LDL. Sialic acid content in electronegative LDL of coronary atherosclerosis patients was 3-fold lower than in native LDL. Lipoproteins isolated from grossly normal human aortic intima and from fatty streaks contained 20-56% less sialic acid as compared to blood plasma LDL. A negative correlation was established between the ability of electronegative and aortic LDL to stimulate lipid accumulation in cells cultured from uninvolved human aortic intima and lipoprotein sialic acid content. The results obtained indicate that electronegative and aortic LDLs have a low sialic acid content, i.e., are desialylated lipoproteins. Considered together with the fact that all known atherogenic LDLs have similar characteristics, our findings suggest that modified LDLs are the same lipoprotein particles subjected to multiple modification.

Adult↗