摘要:

Mild homocysteinemia occurs surprisingly often in patients with premature vascular disease. We studied the possible enzymatic sources of this mild hyperhomocysteinemia and the control of homocysteine levels in plasma by treatment of patients with the cofactors and cosubstrates of homocysteine catabolism. We assessed homocysteine metabolism in 131 patients who had premature disease in their coronary, peripheral, or cerebrovascular circulation by using a standard oral methionine-load test Impaired homocysteine metabolism occurred in 28 patients. We assayed levels of the primary enzymes of homocysteine catabolism in cultured skin fibroblast extracts from 15 of these 28 patients. The patients' cystathionine 0-synthase levels (3.68±2.52 nmol/h per milligram of cell protein, mean±SD) were markedly depressed compared with those from 31 healthy adult control subjects (7.61 ±4.49,P<.001). The patients' levels of 5 -methyltetrahydrofolate: homocysteine methyltransferase were normal. While betalne: homocysteine methyltransferase was not expressed in skin fibroblasts, 24-hour urinary betaine and fyiV-dimethylglycine measurements were consistent with normal or enhanced remethylation of homocysteine by betaine: homocysteine methyltransferase in the 13 patients tested. When treated daily with choline and betaine, pyridoxine, or folic acid, there was a normalization of the postmethionine plasma homocysteine level in 16 of 19 patients. Our results indicate that mild homocysteinemia in premature vascular disease may be caused by either a folate deficiency or deficiencies in cystathionine β-synthase activity. It does not necessarily involve deficiencies of either 5-methyltetrahydrofolate: homocysteine methyltransferase or betaine: homocysteine methyltransferase. Effective treatment regimens are also defined.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Low-density (LDL) and high-density (HDLj) lipoproteins dose-dependently activate phosphoinositide turnover and elevate cytosolic free Ca2+concentrations ([Ca1+]|) in cultured vascular smooth muscle cells (VSMCs) from either human (microarterioles and aorta) or rat (aorta) sources. High-performance liquid chromatography analysis of cell extracts revealed comparable spectra of inositol phosphate isomers generated in response to either LDL, HDL, or angiotensin II (Ang II). Thus, lipoproteins and Ang II may use similar, if not identical, signal transduction pathways for the generation and metabolism of inositol phosphates and intracellular Ca2+mobilization in VSMCs. When Ang II was added in combination with either LDL or HDL], the phosphoinositide and fCa2+], responses of VSMCs were either equal to or even greater than the sum of the effects elicited by the agonists individually. This additivity/synergy between Ang II and the lipoproteins was not accompanied by alteration in the half-maximally effective dose requirements of VSMCs for either Ang II (<=>2 nmol/L, with or without lipoproteins) or lipoproteins (=50figjmhfor LDL and HDL, with or without Ang II). Neither short-term (up to 10 minutes) nor long-term (48 hours) exposure of VSMCs to lipoproteins caused desensitization of phospholipase C and intracellular Ca1+mobilization responses to either Ang II or lipoproteins. Since constant exposure of VSMCs to lipoproteins is a physiological circumstance, and because elevation of [Ca2+], and activation of phosphoinositide turnover are pivotal events for VSMC contraction and growth, we suggest that the low concentrations of lipoproteins in the vessel intlma may play an important role in regulating the response of the vasculature to Ang II.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Rapid, efficient re-endothelialization of large wounds is characterized by a specific sequence of cytoskeletal events that occur after wounding. WoundsISmm wide were created down the middle of confluent porcine aortic endothelial monolayers to study regulation of repair. The wounded cultures were incubated for short periods with cycloheximide or actinomycin D to test the hypothesis that transient inhibition of translation and transcription at the time of wounding disrupts rapid repair by interfering with centrosome redistribution to the front of the cell, an early event associated with cell migration. Although centrosome reorientation did not occur when protein synthesis was inhibited with 20 /ig/mL cycloheximide for 1 hour before and for up to 4 hours after wounding, reorientation did occur by 2 hours after cycloheximide was washed out The times taken for the wound to close for cyclohexlmide-treated and control cells did not differ (60±l.l vs 60±0.8 hours). When transcription was inhibited with 0.25 /ig/mL actinomycin D for 1 hour before and for 1 hour after wounding, re-endothelialization was dramatically reduced. The time taken for the wound to close was almost five times longer (288 ±5.3 hours) than for control cells. The cells moved very slowly, maintaining a flattened, spread-out shape, as opposed to being elongated. The centrosomes did not reorient to the front of the cell throughout the entire period. However, addition of actinomycin D for 2 hours when centrosomes had already moved to the front of the cells (4 hours after wounding) did not reduce subsequent wound repair (60±13hours). This study supports our hypothesis that centrosome redistribution is essential for efficient wound repair and suggests that redistribution is regulated by transcription of essential gene(s) that is induced immediately after wounding by an unknown short-lived signal. Two possible signals are the loss of cell contact and/or a soluble substance released from the cells at the time of wounding. When the signal is unable to induce transcription, dysfunctional repair occurs by a very slow centrosome-independent process.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Hepatic lipase-deficient subjects in the Ontario kindred are compound heterozygotes for hepatic lipase mutations (Ser$-$Phe and Thr$$Met). Cholesteryl ester-rich 0-very-low-density lipoprotein (β- VLDL) accumulates in plasma and such subjects have premature atherosclerosis. To determine a possible mechanism, we hypothesized that hepatic lipase-deficient 0-VLDL, homozygous for apolipoprotein (apo) E3, would cause cholesteryl ester accumulation and foam cell formation in macrophages. β-VLDL and pre-β-VLDL were isolated by Pevikon electrophoresis and incubated with J774 macrophages, cells that do not secrete apoE. 0-VLDL increased cellular cholesteryl ester content 13-fold, whereas pre-β-VLDL increased cholesteryl ester sevenfold. 0-VLDL increased acyl CoA: cholesterol acyltransferase activity fourfold (measured as [MC]oleate incorporation into cholesteryl ester). Preincubation of hepatic lipase-deficient β-VLDL with the anti-apoE monoclonal antibody 1D7, which inhibits binding of apoE to low-density lipoprotein receptors, inhibited cellular cholesteryl ester accumulation by 75%, whereas the anti-apoB blocking monoclonal antibody 5E11 failed to inhibit cellular cholesteryl ester accumulation. In contrast to hepatic lipase deficiency, β-VLDL from type in subjects (E2/E2) failed to increase cellular cholesteryl ester or acyl CoA: cholesterol acyltransferase more than 1.5-fold. Thus, hepatic lipasedeficient 0-VLDL readily induces cholesteryl ester accumulation in J774 macrophages, a process mediated by functional apoE3. This may explain the premature atherosclerosis observed in this kindred.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Pathology laboratories in nine cooperating centers collected arteries from 1532 persons 15 through 34 years of age who died of external causes, principally homicides, accidents, and suicides. A central laboratory stained the arteries and evaluated the atherosclerotic lesions. All of the aortas and about half of the right coronary arteries in the youngest age group (15 through 19 years) had lesions. The mean percent intimal surface involved by lesions, in 5-year age groups, increased from 15 through 34 years. Raised lesions increased with age in extent and prevalence in the aorta and the right coronary artery. Black subjects had more extensive fatty streaks than white subjects in all three arterial segments. Young women had more extensive fatty streaks in the abdominal aorta; young men had more in the thoracic aorta. Male subjects had more extensive and a higher prevalence of raised lesions than did female subjects in the right coronary artery. White and black subjects did not differ significantly in the extent of raised lesions. Among the three arterial segments, the right coronary had the least percentage of intimal surface involved with all types of lesions but had the highest proportion of raised lesions among total lesions. These results confirm the origin of atherosclerosis in childhood and show that the prevalence and extent of fatty streaks and fibrous plaques increase rapidly during the 15- through 34-year age span.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

In this article, we describe a 46-year-old man with severe high-density lipoprotein (HDL) deficiency and his kindred. In the proband, HDL cholesterol and apolipoprotein (apo) A-I levels were 5 and 4.5 mg/dL, respectively. Xanthomata, xanthelasma, arcus corneae, and hepatosplenomegaly were not present The proband had coronary artery disease, but it was impossible to state whether the HDL deficiency cosegregated with premature coronary artery disease in this kindred. Pedigree analysis was suggestive of a codominant familial disease. Poiymerase chain reaction amplification of the apoA-I gene of the proband, followed by subcloning and sequencing, did not reveal any mutation in either the coding regions or intron-exon junctions. A kinetic study using deuterated leucine to endogenoushy label apoA-I was performed to elucidate the metabolic basis of the apoA-I deficiency. We demonstrated marked hypercatabolism of apoA-I in the proband, with a fractional catabolic rate more than 10 times faster than normal; the plasma residence time of apoA-I in the proband was only 038 day compared with 4.10 days in a control subject The apoA-I production rate was also substantially decreased in the proband. The association of a normal apoA-I gene sequence with marked hypercatabolism of apoA-I is similar to that described in Tangier disease. However, except for the presence of mild, diffuse, corneal deposits, this patient had no evidence of the reticuloendothelial cholesterol deposition characteristic of Tangier disease. This study establishes that a form of severe hypoalphalipoproteinemia distinct from Tangier disease can be caused by marked hypercatabolism of a normal A-I apolipoprotein.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Although the accumulation of cholesterol in macrophages appears to be an initial step in atherogenesis, low-density lipoprotein (LDL), a major risk factor for atherosclerosis, does not promote cholesterol accumulation in macrophages in its native form. On the other hand, apolipoprotein (apo) A-I-containing lipoprotein removes cholesterol from cholesterol-loaded macrophages (foam cells) and prevents cholesterol from accumulating in the cells. We examined the effect of LDL on cholesterol removal by two species of apoA-I-containing lipoproteins, one containing only apoA-I (LpA-I) and the other containing apoA-I and apoA-II (LpA-1/A-II). When foam cells were incubated with LpA-I or LpA-I/A-H, cellular cholesterol mass was reduced. In contrast, when LDL was added, the cholesterol-reducing capacities of these lipoproteins were dose-dependently inhibited by LDL. In the presence of LDL, LpA-I and LpA-I/A-II removed free cholesterol preferentially from LDL rather than from the plasma membrane of foam cells. In addition, a fair amount of cellular cholesterol was directly moved to LDL rather than to LpA-I or LpA-I/A-II. The cellular cholesterol that moved to LDL was completely compensated for by the cholesterol influx from LDL to foam cells. Thus, net cholesterol efflux (a combination of influx and efflux) from foam cells was inhibited by LDL. These results, taken together, indicate that LDL may accelerate foam cell formation by inhibiting cholesterol removal from the cells and that elevated levels of plasma LDL may become a risk factor for atherosclerosis by inhibiting the function of LpA-I and LpA-I/A-II at the cellular level.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The differentiation-dependent expression of purinergic receptors for metaboiicaiiy stable analogues of adenosine and ATP was studied in human mononuclear phagocytes (MNPs). Iigands of these receptors are able to modulate cellular cholesterol metabolism. In addition, the intracellular signal transduction pathways of the purinergic receptor system were examined. ATPyS, the metabolic stable analogue of ATP, was used as a P2ligand, and 2-p-(2-carboxyethyl)phenylethylamino-5′-Ar-ethylcarboxamido adenosine (CGS 21680) and 5′-(JV-ethylcarboxamido)adenosine (NECA) were used as P, Iigands in binding studies. Binding of [$S] ATPyS to MNPs at 4°C revealed saturable low-affinity binding sites with aKdof 868±52 nmol/L and B $ of 73±0.4 pmol per 10* cells in 1-day cultured human MNPs and aKtof 780±30 nmol/L and B ml of 14.0±0.8 pmol per 10′ cells in 7-day cultured human MNPs. The characterization of the P, receptors on 1- and 7-day cultured human MNPs showed that they are expressed only on 7-day cultured human MNPs. The specific binding curve of the adenosine A2receptor agonist [3H]CGS 21680 was biphaslc, with aKuof 33±15 nmol/L and aK& of 90±10 nmol/L and withBmMX, of 0.19±0.06 pmol per 10′ cells and B, $ of 0.41 ±0.09 pmol per 10* cells, whereas NECA did not exhibit specific binding. The typical agonists for probing A, receptor subtypes did not bind to 1- and 7-day cultured human MNPs, indicating that only A2receptors are expressed on 7-day cultured human MNPs. ATPyS enhanced [Ca2+]| in 1- and 7-day cultured human MNPs in a concentration-dependent manner, whereas the Pi Iigands, adenosine and CGS 21680, induced Ca2+flux only in 7-day cultured MNPs. All three drugs increased intracellular cAMP levels in 7-day cultured human MNPs at a concentration of 10~5mol/L, whereas no effect was observed in 1-day cultured human MNPs. The uptake of fluorescently labeled acetylated low-density lipoprotein (LDL) in 7-day cultured human MNPs was inhibited by adenosine, CGS 21680, ATP, and ATPyS. No significant influence of these compounds was measured on the uptake of LDL, acetylated LDL, and high-density lipoprotein, in 1-day cultured MNPs. Our investigations indicate that the expression of P2jand A2receptors is increased during differentiation of blood monocytes to macrophages. It is shown that both purinergic Iigands, ATPyS and CGS 21680, inhibit the uptake of acetylated LDL in a concentration-dependent manner, which might impair foam cell formation.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Elevated blood levels of homocysteine represent an independent risk factor for premature arterial vascular disease and thrombosis. We investigated whether homocysteine could induce tissue factor (TF) procoagulant activity in cultured human endothelial cells. Homocysteine increased cellular TF activity in a timeand concentration-dependent manner. Low concentrations of homocysteine (0.1 to 0.6 mmol/L), similar to those found in the blood of patients with homocystinuria, enhanced TF activity by 25% to 100%. Other sulfur-containing amino acids (cystine, homocystine, cysteine, and methionine) induced less TF activity than did homocysteine; however, β-mercaptoethanol and dithiothreitol were more effective than homocysteine in increasing TF activity. Preincubation of homocysteine with a sulfhydryl inhibitor such as JV-ethylmaleimide prevented homocysteine induction of TF activity. A quantitative polymerase chain reaction method indicated that homocysteine increased TF mRNA in endothelial cells. These results indicate that an atherogenic amino acid, homocysteine, can initiate coagulation by the TF pathway through a mechanism involving the free thiol group of the amino acid and by TF gene transcription. These data support the hypothesis that perturbation of vascular coagulant mechanisms may contribute to the thrombotic tendency seen in patients with homocystinuria.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Uptake of modified lipoproteins by resident arterial monocytes/macrophages is believed to be a key event in the formation of foam cells and thus in the early phases of atherosclerosis. Low-density lipoproteins (LDLs) that undergo oxidative changes become suitable for uptake by macrophages through a specific scavenger receptor that leads to cholesteryl ester accumulation. Because the interaction of other oxidized lipoproteins with macrophages has been poorly investigated, we studied the effect of oxidatively modified high-density lipoproteins (HDLs) on the sterol metabolism of J774-A1 macrophages. Unlike native HDLs, oxidized HDLs caused a concentration-dependent accumulation of unesterified cholesterol and decreased [14C]oleate incorporation into steryl esters. Oxidized HDLs also decreased [I4C]acetate incorporation into newly synthesized sterols. Cell surface binding ofl25I-oxidlzed HDLs to the macrophages was saturable, with an apparent dissociation constant (A$) of 0.96 nmol/mL. Both oxidized and acetylated LDLs but not native lipoproteins could compete for binding of '"I-oxidized HDL. The data support the conclusion that the effects elicited by oxidized HDLs on the sterol metabolism of macrophages are significantly different from those of native HDLs. The binding of oxidized HDLs to macrophages occurs at sites that are likely the same as those for modified LDLs. We speculate that, if occurring in vivo, HDL oxidation would generate modified lipoproteins capable of modulating the cholesterol homeostasis of macrophages.

ISSN:1049-8834    

出版商:OVID    

年代:1993

数据来源: OVID