Human Aortic Cells Research Articles

Effects of basic fibroblast growth factor and transforming growth factor-beta on maturation of human pediatric aortic cell culture for tissue engineering of cardiovascular structures.

Optimal in vitro conditions are necessary for the development of a strong, well structured, and functional tissue engineered cardiovascular structure eventually designed for implantation. To further optimize in vitro conditions for cell proliferation and extracellular matrix formation in tissue engineering of cardiovascular structures, in this study, ascorbic acid and growth factors as additives to standard cell culture medium were evaluated for their effect on tissue development in vitro. Biodegradable polymer patches [polyglycolic acid (PGA) coated with poly-4-hydroxybutyrate (P4HB)] were seeded with human pediatric aortic cells and cultured for 7 and 28 days. Group A was cultured with standard medium (DMEM with 10% fetal calf serum and 1% antibiotics) supplemented with ascorbic acid; group B was cultured with standard medium plus ascorbic acid and basic fibroblast growth factor (bFGF); group C was cultured with standard medium adding ascorbic acid and transforming growth factor (TGF). Analysis of the cell seeded polymer constructs included DNA assay, collagen assay, and histologic and immunohistochemical examination for cell proliferation and collagen formation. After 7 and 28 days of culture, group B and group C showed a significantly higher DNA content compared with group A. The addition of bFGF (group B) led to a markedly higher collagen synthesis after 28 days of culture compared with the additives in groups C and A. The histologic and immunohistochemical examination also revealed a more dense, organized tissue development with pronounced matrix protein formation in the tissue engineered structures in group B after 28 days of culture. When seeded on to the polymeric scaffold, human vascular cells proliferate and form organized cell tissue after 28 days of culture. The addition of bFGF and ascorbic acid to the standard medium enhances cell proliferation and collagen synthesis on the biodegradable polymer, which leads to the formation of more mature, well organized tissue engineered structures.

Mitogenic and Anti-Apoptotic Actions of Hepatocyte Growth Factor Through Erk, Stat3 and Akt in Human Aortic Endothelial Cells

P83 Background: Hepatocyte growth factor (HGF), an angiogenic growth factor, may play a pivotal role in the regulation of endothelial cells (EC), since HGF demonstrated the mitogenic and anti-apoptotic actions in endothelial cells. However, it is still unclear how HGF demonstrated those actions. Therefore, in this study, we have focused on the signal transduction systems. Methods: Human aortic EC death was induced by serum-deprived condition. Cell growth was assessed by WST assay, and thymidine incorporation and cell death was measured by LDH release and caspase 3 activity. Phosphorylation of ERK, STAT and Akt was measured by western blotting as assessed by the specific phospho-specific antibody. Results: Treatment of EC with recombinant HGF (rHGF) resulted in a significant increase in cell growth (WST: control; 0.267±0.02, rHGF 100ng/ml; 0.385±0.003, p≤0.01) and also attenuated the cell death (LDH release: control; 0.94±0.04, rHGF 100ng/ml; 0.43±0.024, p≤0.01). ERK and STAT3 (Ser727) was phosphorylated by rHGF (100 ng/ml) as assessed by the specific phospho-specific antibody, while PD98059, the inhibitor of MEK, attenuated endothelial cell growth (HGF; 0.385±0.003, HGF+PD 30uM; 0.203±0.003, p≤0.01) and the phosphorylation of STAT3 (Ser727). On the other hand, STAT3 (Tyr705), which was usually stimulated by JAK, was not phosphorylated by rHGF. In addition, Akt on the down stream of PI3 kinase was also phosphorylated by rHGF, while the specific inhibitor of PI3kinase, wortomanin and LY294002, markedly attenuated endothelial cell death assessed by LDH release and caspase3 activity. Conclusions: Overall, this study demonstrated that HGF stimulated cell growth through ERK cascade including STAT3(Ser727) activation, not JAK-STAT3(Tyr705) pathway, and inhibited endothelial cell death through PI3 kinase-Akt pathway. HGF may stimulate angiogenesis through both mitogenic and anti-cell death function in endothelial cells.

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.

Evidence for tetrodotoxin-sensitive sodium currents in primary cultured myocytes from human, pig and rabbit arteries

Primary cultured human coronary myocytes express a tetrodotoxin-sensitive sodium current (I(Na)). Here, we have investigated whether I(Na) is expressed in vascular smooth muscles cells (VSMCs) isolated from other large arteries, and other mammals. VSMCs were enzymatically dissociated, kept in primary culture, and macroscopic I(Na) was recorded using the whole-cell patch-clamp technique. We found that I(Na) is expressed in VSMCs grown from human aortic (90%; n=50) and pulmonary (44%; n=19) arteries, and in the human aortic myocyte cell line HAVSMC (94%; n=27). I(Na) was also detected in pig coronary (60%; n=33), and rabbit aortic (47%; n=15), but not in rat aortic VSMCs (n=20). These different I(Na) had similar voltage thresholds for activation (approximately equal to -50 mV), and were highly sensitive to extracellularly applied tetrodotoxin. We conclude that I(Na) is expressed in VSMCs grown from various types of large arteries in humans, pig and rabbit.

Effect of insulin on human aortic endothelial nitric oxide synthase

It has recently been shown that insulin induces vasodilation in human arteries and veins in vivo. This effect of insulin has been shown to be a direct one on the human vein. In view of these observations and the fact that insulin-induced vasodilation is impaired in insulin-resistant states like type 2 diabetes and obesity, we have investigated the hypothesis that insulin may induce the expression of endothelial nitric oxide synthase (e-NOS) in endothelial cells grown from human aortae (HAECs), human lower-limb veins, and human umbilical veins (HUVECs), and microvascular endothelial cells (MVECs) from human adipose tissue. The expression of e-NOS was maximal in HAECs, and therefore, further experiments were performed on these cells. When cells reached 90% confluence, they were induced with different concentrations of insulin (0, 25, 100, and 1,000 microU/mL) for 6 days. The cells were homogenized and e-NOS expression was examined by Western blotting. A dose-dependent induction by insulin of e-NOS in the endothelial cells was clearly demonstrated. There was no detectable level of the inducible NOS isoform (i-NOS), and this effect of insulin was independent of cell proliferation. We conclude that insulin induces a dose-dependent induction of e-NOS in human aortic cells (and possibly arterial/endothelial cells), and this effect may contribute to the overall vasodilatory effect of insulin.

Neuropeptide Y induced increase of cytosolic and nuclear Ca2+in heart and vascular smooth muscle cells

It was reported that neuropeptide Y (NPY) affects cardiac and vascular smooth muscle (VSM) function probably by increasing intracellular Ca2+. In this study, using fura-2 microfluorometry and fluo-3 confocal microscopy techniques for intracellular Ca2+ measurement, we attempted to verify whether the action of NPY receptor's stimulation in heart and VSM cells modulates intracellular Ca2+ and whether this effect is mediated via the Y1 receptor type. Using spontaneously contracting single ventricular heart cells of 10-day-old embryonic chicks and the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ level and distribution, 10-10 M of human (h) NPY significantly (P < 0.05) increased the frequency of cytosolic and nuclear Ca2+ transients during spontaneous contraction. Increasing the concentration of hNPY (10(-9) M) did not further increase the frequency of Ca2+ transients. The L-type Ca2+ channel blocker, nifedipine (10(-5) M), significantly (P < 0.001) blocked the spontaneous rise of intracellular Ca2+ in the absence and presence of hNPY (10(-10) and 10(-9) M). However, the selective Y1 receptor antagonist, BIBP3226 (10(-6) M), significantly decreased the hNPY-induced (10(-10) and 10(-9) M) increase in the frequency of Ca2+ transients back to near the control level (P < 0.05). In resting nonworking heart and human aortic VSM cells, hNPY induced a dose-dependent sustained increase of basal resting intracellular Ca2+ with an EC50 near 10(-9) M. This sustained increase was cytosolic and nuclear and was completely blocked by the Ca2+ chelator EGTA, and was significantly decreased by the Y1 receptor antagonist BIBP3226 in both heart (P < 0.05) and VSM (P < 0.01) cells. These results strongly suggest that NPY stimulates the resting basal steady-state Ca2+ influx through the sarcolemma and induces sustained increases of cytosolic and nuclear calcium, in good part, via the activation of the sarcolemma membrane Y1 receptor type in both resting heart and VSM cells. In addition, NPY also increased the frequency of Ca2+ transients during spontaneous contraction of heart cells mainly via the activation of the Y1 receptor type, which may explain in part the active cardiovascular action of this peptide.

Isolation and characterisation of the human rab18 gene after stimulation of endothelial cells with histamine

Here we report the isolation of a cDNA encoding the complete human rab18 protein from histamine-stimulated endothelial cells using differential display. The amino acid sequence showed 98% homology with the previously isolated mouse rab18 protein, which is implicated in apical/basolateral endocytosis. Northern blot analysis revealed two transcripts (2.5 kb and 1 kb) ubiquitously expressed in all examined organs, as well as in human umbilical vein endothelial and aortic cells. In blood cells rab18 transcripts were hardly detectable. The histamine-induced time-dependent increase of rab18 mRNA expression in human umbilical vein endothelial cells and peripheral blood mononuclear cells indicates for the first time a link between receptor-mediated signal transduction and the regulation of rab gene expression. This finding might also imply a role for rab proteins in inflammation.

Expression of CLCN Voltage-gated Chloride Channel Genes in Human Blood Vessels

Chloride (Cl) ion channels play a critical role in the response of both vascular smooth muscle (VSM) and endothelial (ENDO) cells to agonist stimulation. In VSM, agonist-induced Cl currents produce membrane depolarization, resulting in calcium influx through voltage-sensitive channels. ENDO cells also activate Cl currents after either agonist applicaiton or perturbation of cell volume. Although some of these currents have been characterized biophysically, the genes involved have not been identified. The CLCN family of voltage-dependent Cl channel genes comprises nine members (CLCN1–7, Ka and Kb) which demonstrate quite diverse functional characteristics while sharing significant sequence homology. We used Northern-blot analysis to study the expression of these Cl channel genes in cultured human aortic and coronary VSM cells and in aortic ENDO cells. CLCN3 is by far the most abundant CLC channel mRNA in both VSM and ENDO cells. Lower levels of expression are seen for CLCN2, CLCN4, CLCN5 and CLCN6. Expression levels were similar in VSM and ENDO cells except for CLCN4 which was more highly expressed in ENDO cells.In situhybridization was used to confirm the expression of CLCN3 in intact human fetal lung. CLCN3 message was seen in VSM and ENDO cells of both large and small pulmonary vessels, indicating that their detection by Northern blotting was not an artifact of cell culture. CLCN3 is also expressed in pulmonary epithelial and bronchial smooth muscle cells but not in chondrocytes or pulmonary interstitial cells. Recent studies suggest that CLCN3 may encode the swelling-induced Cl conductance. We used whole cell patch clamp recording to demonstrate swelling-induced Cl currents in these cultured VSM cells. This suggests that the CLCN3 protein is expressed; however, the functional role of this current in VSM remains to be determined.

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.

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.

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.

3.P.283 Inhibition of growth of human aortic vascular smooth muscle cells by a phosphodiesterase type 3 inhibitor, cilostazol, through p53-mediated apoptosis

3.P.283 Inhibition of growth of human aortic vascular smooth muscle cells by a phosphodiesterase type 3 inhibitor, cilostazol, through p53-mediated apoptosis

1.W06.5 Elevated homocyst(e)ine increases ability of human aortic cells to oxidise LDL

1.W06.5 Elevated homocyst(e)ine increases ability of human aortic cells to oxidise LDL

Design, synthesis, and evaluation of mitomycin-tethered phosphorothioate oligodeoxynucleotides.

Mitomycin C (1) is the prototypical bioreductive alkylating agent. Studies have shown that mitomycin C and its derivatives selectively alkylate guanine residues within di- and trinucleotide DNA sequences. This investigation sought to improve the selective DNA bonding properties of the mitomycins by coupling them with antisense oligodeoxynucleotides. Two procedures were developed that allowed the attachment of a phosphorothioate oligodeoxynucleotide containing a hexylamino spacer at the 5' terminus with a C(10)-activated mitomycin. In the first procedure, decarbamoylation of 1 (NaOCH3/ benzene) afforded 10-decarbamoylmitomycin C (10), which was treated with either dimethyl sulfate or methylthiochloroformate and base to yield 10-decarbamoylporfiromycin (11) and N(1a)-[(methylthio)-carbonyl]-10-decarbamoylmitomycin C (12), respectively. Activation of the C(10) site in 11 and 12 with 1,1'-carbonyldiimidazole or with 1,1'-thiocarbonyldiimidazole provided the N(1a)-substituted mitomycin 10-decarbamoyl-10-O-carbonylimidazoles (5, 7) and 10-decarbamoyl-10-O-thiocarbonylimidazoles (6, 8), respectively. Compounds 5-8 were reacted with glycine methyl ester hydrochloride (17) and base in both methylene chloride and aqueous buffered solutions to determine the ease and efficiency in which these C(10)-activated mitomycin derivatives coupled to amines. It was found that 5-8 all reacted with 17 in methylene chloride to give the coupled products 18-21 but that improved amine coupling yields in water were observed for the 10-decarbamoyl-10-O-thiocarbonylimidazoles 6 and 8 as compared with the 10-decarbamoyl-10-O-carbonylimidazoles 5 and 7. This finding led to the coupling of the phosphorothioate oligodeoxynucleotide, H2N(CH2)6-P(S)(OH)-GGCCCCGTG-GTGGCTCCAT (22) to 8. Compound 22 complemented a 19-base sequence in the translation initiation region of the human A-raf-1 gene. Use of excess 8 (28 equiv) with 22 gave only a 36% yield of the coupled product 23, which proved difficult to separate from 22. In the second procedure, phosphorothioate oligodexynucleotides that contained a hexylamino spacer at the 5'termini were coupled to 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in four steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 11 gave the C(10) mesylate 13, which was then treated with NaN3 (dimethylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfiromycin (14). Catalytic reduction (PtO2, H2) of 14 in pyridine afforded C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate provided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chloride and aqueous buffered solutions to give 25. Use of the 5'hexylaminophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave the conjugated adducts 28-31, respectively, in a 12% to near-quantitative yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the coding region of the human FGFR1 gene. One adduct, 29, reduced the number of FGFR1 receptors in human aortic smooth cells for bFGF on the cell surface, which suggested down-regulation of FGFR1 gene expression. Further, 29 inhibited cultured human aortic smooth muscle cell proliferation and was less cytotoxic than porfiromycin (2). The biological assay data suggest that the phosphorothioate oligodexynucleotide porfiromycin conjugates may be more target selective and less toxic than either mitomycin or porfiromycin and thus be promising therapeutic agents.

Effect of lipoprotein(a) on lipid metabolism of cultured human intimal aortic cells

The effect of Lp(a) on lipid metabolism in cells cultured from unaffected human aortic intima has been investigated. Lp(a) isolated from the blood of healthy subjects failed to alter intracellular neutral lipid content. On the other hand, Lp(a) obtained from coronary atherosclerosis patients induced a 1.5- to 2.5-fold increase in intracellular levels of free and esterified cholesterol and triglycerides. The sialic acid content of patients' Lp(a) was 2.5-fold lower as compared with that of healthy subjects' Lp(a). Healthy donors' Lp(a) in vitro desialylated with neuraminidase were able to accumulate lipids within the cells. Using lectin-chromatography on Ricinus Communis agglutinin-agarose, Lp(a) was divided into subfractions differing by sialic acid content. Desialylated (sialic acid-poor) Lp(a), but not sialylated lipoproteins, were capable of increasing the intracellular content of total cholesterol. Desialylated but not sialylated Lp(a) formed aggregates during incubation under cell culture conditions. Isolated aggregates of desialylated Lp(a) induced lipid accumulation in cells. Elimination of Lp(a) aggregates from cultural medium prevented the increase of intracellular lipids. Complexes of Lp(a) with the matrix components and antibodies increased lipid level in cultured cells. We assume that formation of large particles of desialylated Lp(a) aggregates or Lp(a)-containing complexes is a necessary step for lipid accumulation in human intimal cells.

Modified low density lipoprotein from diabetic patients causes cholesterol accumulation in human intimal aortic cells

Fifty-five serum samples from 99 Type 1 and 71 serum samples from 81 Type 2 diabetic patients (56% and 88%, respectively) brought about a 1.5–3.5-fold increase in total cholesterol content of cultured human intimal aortic cells. This atherogenic effect did not correlate with patient's age, diabetes duration or plasma lipid levels, and was mainly due to low density lipoprotein (LDL). Cholesterol accumulation in cells incubated with LDL highly correlated with that in cells exposed to corresponding patient's serum ( r = 0.872 and r = 0.811, P < 0.0001, in Type 1 and Type 2 diabetic patients, respectively). In LDL from diabetic patients the sialic acid content was decreased by an average of 30% ( P < 0.05), as compared with healthy subjects, and the fructosyl lysine content was increased by an average of 25% ( P < 0.05). Atherogenic effect of patients' LDL significantly correlated with their fructosyl lysine content ( P < 0.0001) and negatively correlated with sialic acid content ( P < 0.0001). Two LDL fractions were further separated from the total LDL preparation by affinity chromatography on Ricinus communis agglutinin-agarose. The bound (desialylated) LDL fraction was characterized by an increased fructosyl lysine content and the alfered neutral lipid and phospholipid composition, while non-bound (sialylated) LDL fraction did not differ from normal LDL. Desialylated, but not sialylated, LDL fraction induced massive cholesterol accumulation in cultured cells. In conclusion, the cholesterol accumulating effect of diabetic patients' blood sera is mainly related to atherogenic low density lipoprotein fraction, which is modified in various ways — by increased non-enzymatic glycosylation, desialylation and alterations in lipid composition. This multiple-modified LDL may contribute to the premature atherosclerosis development in diabetes mellitus.

A new antiinflammatory compound, leumedin, inhibits modification of low density lipoprotein and the resulting monocyte transmigration into the subendothelial space of cocultures of human aortic wall cells.

Addition of leumedin, N-[9H-(2,7-dimethylfluorenyl-9-methoxy) carbon]-L-leucine at 30-60 microM together with LDL almost completely prevented the induction of monocyte chemotactic protein mRNA, reduced monocyte chemotactic protein 1 levels by 84%, and inhibited monocyte migration into the subendothelial space of cocultures of human aortic wall cells by < or = 98%. LDL incubated with leumedin formed a stable complex that remained intact even after refloating in an ultracentrifuge. Leumedin at 50 microM did not change conjugated diene formation during coculture modification of LDL or Cu++ catalyzed oxidation of LDL. Unlike LDL from control rabbits, LDL isolated from rabbits that were injected with 20 mg/kg leumedin was remarkably resistant to modification by the coculture and did not induce monocyte migration to a significant degree. Moreover, HDL isolated from rabbits injected with leumedin was far more effective in protecting against LDL modification by the artery wall cocultures than HDL from control rabbits. We conclude that leumedins can associate with lipoproteins in vivo, rendering LDL resistant to biological modification and markedly amplifying the protective capacity of HDL against in vitro LDL oxidation by artery wall cells.

Carbohydrate composition of protein and lipid components in sialic acid-rich and -poor low density lipoproteins from subjects with and without coronary artery disease.

Low density lipoprotein (LDL) from patients with coronary heart disease (CHD) caused 78-286% increase in accumulation of cholesterol in human aortic subendothelial cells compared to 2-17% caused by LDL from normal subjects. Ricin-Sepharose affinity chromatography was used to separate LDL into two subfractions, one sialic acid-rich (SAR) and the other sialic acid-poor (SAP). SAP-LDL from CHD patients caused 156-307% increase in accumulation of cellular cholesterol, whereas SAR-LDL from these patients caused only 14-21% increase. SAP-LDL from normal healthy subjects caused 50-86% increased accumulation, whereas their SAR-LDL induced only 2-12% increase. Carbohydrate analysis of SAP-LDL protein isolated from four CHD patients revealed mean values of 59, 25, 61, and 11 nmoles of N-acetyl glucosamine, galactose, mannose, and sialic acid per mg protein, respectively. Mean values for SAR-LDL protein from these patients were 59, 31, 77, and 24 nmol/mg protein, respectively. Analysis of SAP-LDL protein from four normal healthy subjects indicated respective mean values of 58, 29, 72, and 22 nmol/mg, whereas SAR-LDL protein from normals contained 59, 29, 72, and 29 nmol/mg. The carbohydrate content of LDL lipids represents about 25% of the total carbohydrate present in the lipoprotein. The mean values for SAP-LDL lipids from four CHD patients were about 2, 2, 18, 18, and 2 nmol/mg protein for N-acetyl galactosamine, N-acetyl glucosamine, galactose, glucose, and sialic acid, respectively. The mean values for SAR-LDL lipids from these patients were 3, 4, 34, 41, and 5 nmol/mg, respectively. Analysis of SAP-LDL lipids from four normal healthy subjects indicated respective mean values of 4, 6, 30, 31, and 3 nmol/mg, whereas SAR-LDL lipids from these subjects contained 6, 9, 41, 46, and 7 nmol/mg. These results suggest that the different biological properties of SAR-LDL and SAP-LDL are related to their different carbohydrate compositions.

Lipid accumulation in the subendothelial cells of human aortic intima impairs cell-to-cell contacts: A comparative study in situ and in vitro

We have modified our technique used to examine the three-dimensional cytoarchitecture of human aorta. Using this modification we confirmed the disruption of the contacts among subendothelial cells in the fatty streaks. Thinning and arborization of contact-forming cellular processes were also demonstrated. The incubation of cultured human aortic subendothelial cells with desialylated low-density lipoproteins (LDL), LDL immobilized on latex, and LDL-free latex microspheres induced the alterations in cell-to-cell contacts similar to those occurring in a fatty streak in situ. It is proposed that these alterations may be associated with lipid accumulation and activation of phagocytosis and/or intracellular accumulation of large insoluble particles.

Immortalization of human aortic smooth muscle cells with origin-minus simian virus 40 DNA.

We established two cell lines of human smooth muscle cells (SMC) by transfection of cells from the aortic intima and aortic media with origin-minus simian virus 40 (ori-minus SV40) DNA. Ori-minus SV40 DNA very efficiently immortalized human smooth muscle cells in culture. Proteins that these cell lines produced included type I, III, IV, and V collagens, fibronectin, and human matrix metalloproteinases (MMP)-1 (tissue collagenase), -2 ("type IV collagenase"), and -3 (stromelysin). The protein production in these cell lines generally mimicked that of normal SMC, but the immortalization stimulated the cell line of medial SMC to produce excessive MMP-2 and to secrete MMP-9 (92-kDa gelatinase). However, since these cell lines did not show a fully malignant phenotype, we concluded that, in addition to the degradation of extracellular matrix macromolecules, including basement membrane components by MMP-2, -3, and/or -9, some additional factors must be involved for the malignancy of fully transformed cells and that these immortalized human aortic SMC, which share many characteristics with normal SMC, will prove useful to study the role(s) of metalloproteinases in atherosclerosis.