Increase of protooncogene c-myc mRNA levels by low density lipoprotein in vascular smooth muscle cells

The effect of low density lipoprotein (LDL) on the intracellular mRNA concentration of the protooncogene c-myc was studied in freshly isolated bovine vascular smooth muscle cells and in the rat aortic smooth muscle cell line A7r5. Northern analysis showed that LDL increased the mRNA levels of c-myc in both cell lines, the stimulation being 2-fold after 2 h incubation at a concentration of 50 micrograms LDL-protein/ml. High density lipoprotein (HDL) had no effect on c-myc mRNA levels in A7r5 cells. These results demonstrate that LDL, but not HDL, increases intracellular concentrations of c-myc in two different aortic smooth muscle cell lines.     

Low density lipoprotein enhances the thrombin-induced growth of vascular smooth muscle cells

OBJECTIVE: In the present study we investigated whether low density lipoprotein is able to enhance the growth promoting effects of thrombin in vascular smooth muscle cells. METHODS: DNA synthesis was examined by measurement of the [3H]thymidine incorporation into the cell DNA. Cell count was measured with a Neubauer cell box. Thrombin receptor mRNA was determined by Northern blotting. Ca2+ was measured by the fura 2-method. RESULTS: Thrombin (5 nmol/l), thrombin receptor activating protein (3 mumol/l) and low density lipoprotein (33 nmol/l) induce a 652 +/- 80%, 593 +/- 80% and a 316 +/- 60% increase in [3H]thymidine incorporation into DNA (mean +/- SD, n = 3), respectively. A coincubation of thrombin or thrombin receptor activating protein with low density lipoprotein led to a 1245 +/- 160% or 1200 +/- 40% increase of DNA synthesis (mean +/- SD, n = 3). Thus, coincubation of low density lipoprotein and thrombin causes a synergistic rather than an additive mitogenic effect on smooth muscle cells. Thrombin and low density lipoprotein induced a 22 +/- 8.4% and a 29% +/- 6% increase in cell number, respectively. Simultaneous treatment of vascular smooth muscle cells with thrombin and low density lipoprotein caused a 63 +/- 14% increase in cell number (mean +/- SD, n = 3). To further elucidate the underlying mechanism, we studied the effect of low density lipoprotein on the expression of thrombin receptor mRNA. Low density lipoprotein caused a 2.5-fold increase of thrombin receptor mRNA within 24 h, as assessed by Northern analysis. Preincubation of cells for 24 h with 33 nmol/l low density lipoprotein resulted in an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration from 538 +/- 54 to 923 +/- 75 nmol/l (mean +/- SD, n = 4). CONCLUSION: In summary, low density lipoprotein may enhance the mitogenic effect of thrombin probably by an up-regulation of thrombin receptor gene expression in vascular smooth muscle cells or by an elevation of the thrombin-induced increase in cytosolic free Ca2+ concentration.     

Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor

It was shown that proteins modified with advanced glycation end products (AGE) are effectively endocytosed by macrophages or macrophage-derived cells in vitro, and immunohistochemical studies involving anti-AGE antibodies demonstrated the accumulation of AGE-modified proteins (AGE-proteins) in macrophage-derived foam cells in human atherosclerotic lesions in situ, suggesting the involvement of AGE-modified LDL in the atherogenic process in vivo. To examine this suggestion, LDL was modified with glycolaldehyde, a highly reactive intermediate of the Maillard reaction. Physicochemically, glycolaldehyde-modified LDL (GA-LDL) was characterized by increases in negative charge, fluorescence intensity, and reactivity to anti-AGE antibodies, properties highly similar to those of AGE-proteins. The cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL was specifically recognized and endocytosed, followed by lysosomal degradation. The endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), and the endocytic degradation of acetyl-LDL was also competed for by GA-LDL. Furthermore, incubation of GA-LDL with these macrophages and Chinese hamster ovary cells overexpressing the macrophage scavenger receptor (MSR), but not with peritoneal macrophages from MSR-knockout mice, led to the intracellular accumulation of cholesteryl esters (CE). These results raised the possibility that AGE-modified LDL, if available in situ, is taken up by macrophages mainly via MSR and then contributes to foam cell formation in early atherosclerotic lesions.     

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.     

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.     

Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene

Scavenger receptor type B class I (SR-BI), initially identified as a receptor that recognizes low density lipoprotein (LDL), was recently shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters in liver and steroidogenic tissues. To evaluate effects on atherosclerosis, transgenic mice with liver-specific overexpression of SR-BI (SR-BI Tg mice) have been crossed onto LDL receptor-deficient backgrounds. To induce atherosclerosis in a setting of moderate hypercholesterolemia, heterozygous LDL receptor-deficient mice (LDLR1) were fed a high fat/cholesterol/bile salt diet, and homozygous LDL receptor knock-outs (LDLR0) were fed a high fat/cholesterol diet. LDLR1/SR-BI Tg mice showed decreases in VLDL, LDL, and HDL cholesterol and a significant 80% decrease in mean lesion area in the aortic root compared with LDLR1 mice (female LDLR1 74, 120 micrometers(2) versus LDLR1/SR-BI Tg 12, 667 micrometers(2); male 25, 747 micrometers(2)++ versus 5, 448 micrometers(2), respectively). LDLR0/SR-BI Tg mice showed decreased LDL and HDL cholesterol but increased VLDL cholesterol and no significant difference in extent of atherosclerosis compared with LDLR0 mice. Combined data analysis showed a strong correlation between atherosclerotic lesion area and the VLDL+LDL cholesterol level but no correlation with HDL level. These studies demonstrate a strong anti-atherogenic potential of hepatic SR-BI overexpression. In mice with marked overexpression of SR-BI, the protective effect appears to be primarily related to the lowering of VLDL and LDL cholesterol levels.     

Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells

Mesenchymal hamartomas of the liver are the second most common benign liver tumor of childhood. The experience with this tumor at Egleston's Children Hospital at Emory University from 1989 to 1994 is reviewed. Eight patients presented with abdominal distention or an upper abdominal mass. Six patients presented at a mean age of 8 months, and two patients presented at 17 and 23 years of age, respectively. Four patients displayed normal alpha-fetoprotein levels, whereas one patient had an elevated level. Liver function studies were normal in all patients. Abdominal ultrasonography and CT scans revealed a cystic, septated mass within the liver or on a pedicle in all patients. Five patients had simple excision of the tumor, and two had major hepatic resections. The cysts were multiloculated and lined with cuboidal bile duct epithelium surrounded by stroma containing proliferating bile ducts, blood vessels, and compressed liver tissue with no calcifications. In one patient, some pathologists favored the diagnosis of malignant myxoid fibrous histiocytoma because of similar-appearing stroma. Follow-up (mean, 35 months) revealed one symptomatic recurrence after initial resection was incomplete. There were no other recurrences and no malignant transformations. A septated, noncalcified, cystic hepatic mass in an infant with normal liver function studies and characteristic ultrasound or CT is likely a benign mesenchymal hamartoma that can be cured by total local excision.     

Regulation of mRNA encoding low density lipoprotein receptor and high density lipoprotein-binding protein in ovine corpora lutea

Three experiments were conducted to examine the regulation of steady-state concentrations of mRNA encoding ovine low density lipoprotein receptor (LDL-R) and high density lipoprotein-binding protein (HBP) in corpora lutea. In Experiment 1, corpora lutea were collected from ewes on Days 3, 6, 9, 12 and 15 (Day 0, oestrus) of the oestrous cycle. Enriched preparations of small and large steroidogenic luteal cells were also obtained on Days 6, 9, 12 and 15 of the oestrous cycle. In Experiment 2, 16 ewes were hypophysectomized on Day 5 of the oestrous cycle and received saline, luteinizing hormone (LH), growth hormone (GH) or a combination of LH+GH until collection of luteal tissue on Day 12 of the oestrous cycle. Corpora lutea were also collected from pituitary-intact control ewes on Day 5 and Day 12 of the oestrous cycle. In Experiment 3, 13 ewes on Day 11 or Day 12 of the oestrous cycle were administered prostaglandin F2 alpha (PGF2 alpha) and corpora lutea were collected 4 h, 12 h and 24 h later. Corpora lutea were also collected from 4 non-injected and 4 saline-injected (at 24 h) ewes. Results demonstrated that concentrations of mRNA encoding LDL-R did not differ throughout the oestrous cycle. Luteal tissue collected on Day 3 of the oestrous cycle had higher concentrations of mRNA encoding HBP than luteal tissue collected on any other day of the oestrous cycle. Hypophysectomy increased concentrations of mRNA encoding LDL-R but had no effect on concentrations of mRNA encoding HBP. Twelve hours following PGF2 alpha injection concentrations of mRNA encoding LDL-R were decreased but concentrations of mRNA encoding HBP were increased. Concentrations of both LDL-R and HBP mRNA were decreased 24 h following injection of PGF2 alpha. Thus, long-term positive and acute negative regulation of progesterone secretion from the corpus luteum by luteotrophic and luteolytic hormones was not mediated by changes in steady-state concentrations of mRNA encoding LDL-R or HBP.     

High affinity saturable uptake of oxidized low density lipoprotein by macrophages from mice lacking the scavenger receptor class A type I/II

Oxidation of low density lipoproteins (LDL) has been implicated as a causal factor in the pathogenesis of atherosclerosis. Oxidized LDL has been found to exhibit numerous potentially atherogenic properties in vitro, including receptor-mediated uptake by macrophages. Oxidized LDL is a ligand for the class A scavenger receptor type I/II (SR-AI/II), but cross-competition studies with cultured macrophages suggested that there is an additional receptor(s) that is specific for oxidized LDL and that does not interact with acetyl LDL or other chemically modified LDL. A number of macrophage membrane proteins, including CD36, FcgammaRII-B2, scavenger receptor BI, and macrosialin/CD68, have been found to bind to oxidized LDL in vitro and have been proposed as candidate oxidized LDL receptors. However, because of overlapping ligand specificity with the SR-AI/II, it has been difficult to evaluate the relative importance of these proteins in the uptake of oxidized LDL by macrophages. In the present report, we have studied the uptake and degradation of oxidized LDL by macrophages from mice in which the SR-AI/II gene had been disrupted. The uptake of acetyl LDL was reduced by more than 80% in macrophages from scavenger receptor knockout mice, confirming that most of the uptake of acetyl LDL by macrophages can be attributed to this receptor. In contrast, the uptake of extensively oxidized LDL was reduced by only 30% and showed high affinity, saturable uptake with apparent Km of about 5 microg/ml, similar to that of the SR-AI/II. This indicates that about 70% of the uptake of oxidized LDL in macrophages is attributable to an alternate oxidized LDL receptor(s). In contrast to findings reported with CD36, mildly oxidized LDL was internalized much more slowly than extensively oxidized LDL. Unlabeled oxidized LDL, polyinosinic acid, phosphatidylserine-rich liposomes, and LDL or bovine albumin modified by fatty acid oxidation products were effective competitors for the uptake of radioiodinated oxidized LDL by macrophages from knockout mice, whereas acetyl LDL and malondialdehyde-modified LDL were relatively poor competitors. This ligand specificity differs from that of CD36-related (class B) scavenger receptors but is similar to the reported specificity of macrosialin/CD68 in ligand blots. However, the rate of uptake of oxidized LDL by knockout macrophages was not increased by phorbol ester or in thioglycollate-elicited macrophages, both of which are expected to increase the amount of macrosialin on the cell surface. In macrophages from SR-AI/II knockout mice, ligand blots of membrane proteins with iodinated, oxidized, or acetylated LDL revealed several bands, with apparent molecular size on SDS-polyacrylamide gel electrophoresis of 60, 94, 124, and 210 kDa, but none of the bands were specific for oxidized LDL. These results provide direct evidence that a receptor other than SR-AI/II is responsible for most of the uptake of oxidized LDL in murine macrophages, but further studies are needed to identify the receptor(s) involved.     

Thrombin induces thrombomodulin mRNA expression via the proteolytically activated thrombin receptor in cultured bovine smooth muscle cells

We propose that a long-term cure for the recalcitrant chronic venous ulcer must involve a dual surgical approach including (1) wide excision of the ulcer and surrounding liposclerotic tissue bed, and (2) replacement by a free flap containing multiple, competent microvenous valves with a normal microcirculation. Advantages of free flaps over skin grafting include improvement of the underlying pathophysiology; increase in blood supply to the area; ability to cover exposed bone, joint, or tendon; and a lower incidence of recurrence. During the past 8 years, 20 consecutive muscle free flaps were performed in 18 patients for 19 recalcitrant venous ulcers (two "sequential" flaps to the ipsilateral leg in 1 patient and a repeat flap after initial failure in 1 patient). Twelve males and 6 females ranged in age from 17 to 76 years (mean, 44 years). Nontraumatic, nonosteomyelitic venous ulcers had been present for an average of 3.5 years (range, 1-10 years) and failed an average of 2.4 skin grafts (range, 0-6 grafts). Defects ranged from 100 to 600 cm2 (mean, 238 cm2). Donor tissues included rectus abdominis (N = 13), latissimus dorsi (N = 5), gracilis (N = 1), and serratus (N = 1) muscles. Recipient vessels included posterior tibial (N = 12), anterior tibial (N = 6), and peroneal (N = 2). In all instances except one, only one vein, usually one of the venae comitantes, was anastomosed in end-to-end fashion. Successful free tissue transfer was accomplished in 18 of 20 flaps (90%). Complications included infection with partial flap and/or skin graft loss (three flaps), and partial skin graft loss (two flaps). There were no recurrences within the flaps; however, breakdown occurred at the junction between the flap and residual adjacent liposclerotic skin in 1 patient. Follow-up average 32.7 months (range, 8-65 months); 3 patients were lost to follow-up. Free muscle transfer can provide a long-term cure for the recalcitrant venous ulcer by replacing the diseased tissue bed with healthy tissue containing multiple, competent microvenous valves and a normal microcirculation. This can be accomplished in one reconstructive procedure with excellent long-term results.     

The expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) on human vascular smooth muscle cells and monocytes and its down-regulation by lovastatin

Accumulation of oxidatively modified low-density lipoprotein (oxLDL) in the vascular wall is a characteristic feature of atherosclerosis. oxLDL can be taken up into monocytes, smooth muscle cells, and endothelial cells by several known scavenger receptors such as scavenger receptor class A I and II, CD36, and CD68. A new lectin-like oxLDL receptor (LOX-1) was recently found in bovine and human endothelial cells. We studied whether LOX-1 is also expressed in other cells present in the atherosclerotic lesion and whether its expression can be modified. We found LOX-1 expression in human blood monocytes, umbilical smooth muscle and endothelial cells, and 3T3 fibroblasts. LOX-1 mRNA expression in monocytes could be significantly suppressed by lovastatin. Thus, LOX-1 expression is not restricted to endothelial cells and its down-regulation by HMG-CoA reductase inhibitors could contribute to the clinical benefits of these drugs.     

Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats

We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by measuring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5 +/- 2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3 +/- 0.6 nmol/L and 1.4 +/- 0.5 mumol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2 alpha showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.     

Transforming growth factor beta1 induces IL-1 receptor antagonist production and gene expression in rat vascular smooth muscle cells

BACKGROUND: We compared long-term results of coronary artery bypass grafting between 1976 and 1988 in 176 patients 40 years old or younger with a matched control group of 176 patients 25 to 30 years older. METHODS: Mean age was 37.4 +/- 2.7 years (+/- standard deviation) in the study group and 64.2 +/- 2.9 years in the control group. Matching criteria were age, sex, left ventricular ejection fraction, number of bypass grafts, and year of operation. RESULTS: The study group had more smokers (p = 0.000) and more patients with hypercholesterolemia (p = 0.026), unstable angina (p = 0.003), and preoperative myocardial infarction (p = 0.009); fewer patients had hypertension (p = 0.000) and diabetes (p = 0.005) in this group than in the control group. The internal mammary artery was used in 31% of the study patients and in 30% of the controls. The actuarial survival rates after 5, 10, and 15 years were 92%, 86%, and 72% in the study group and 92%, 86%, and 66% in the control group (p = 0.202). Young age was a predictor of cardiac reoperation. CONCLUSIONS: Late survival is similar for young and older patients, but the reintervention rate is higher in the younger group. The absence of unstable angina, a left ventricular ejection fraction greater than 0.45, and the use of internal mammary artery grafts increase survival in all patients.