Role of oxidized human plasma low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation

The effects of oxidized human plasma low density lipoproteins (Ox-LDL) on the proliferation of cultured aortic smooth muscle cells was studied, employing viable cell counting, [³H] thymidine incorporation into DNA, and the release of lactate dehydrogenase (LDH) into the medium. Oxidized LDL (prepared by incubation of LDL with copper sulfate) exerted a concentration-dependent stimulation (2 fold, compared to control) of aortic smooth muscle cell proliferation at low concentrations (0.1 µg – 10 µg/ml medium). On the other hand, at high concentrations (25–200 µg/ml), Ox-LDL produced a pronounced decrease in viable cells, a decrease in the incorporation of [³H] thymidine into DNA, and an increase in the release of LDH in the medium. In this report, the previously postulated biological roles of oxidized-LDL in atherosclerosis are discussed in view of these findings.Abbreviations Ox-LDL Oxidized human plasma Low Density Lipoproteins - SMC Smooth Muscle Cells - LDH Lactate Dehydrogenase - LPC Lysophosphatidycholine - PC Phosphatidylcholine - TNF Tumor Necrosis Factor     

Effect of LDL concentration polarization on the uptake of LDL by human endothelial cells and smooth muscle cells co-cultured

To substantiate our hypothesis that concentration polarization of low-density Upoprotein （LDL） plays an important role in the localization of atherogenesis, we investigated the effects of wall shear stress and water filtration rate （or perfusion pressure） on the luminal surface LDL concentration （cw） and the LDL uptake by human vascular endothelial cells and smooth muscle cells co-cultured on a permeable membrane using a parallel-plate flow chamber technique and a flow cyto-metry method. The results indicated that the uptake of fluorescent labeled LDL （DiI-LDL） by the co-cultured cells was positively correlated with Cw in a non-linear fashion. When cw was low, the uptake increased very sharply with increasing Cw. Then the increase became gradual and the uptake was seemingly leveled out when Cw reached beyond 160 μg/ml. The present study therefore has provided further experimental evidence that concentration polarization may occur in the arterial system and have a positive correlation with the uptake of LDLs by the arterial wall, which gives support to our hypothesis regarding the localization of atherogenesis.     

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

Summary It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3--hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.     

Minimally modified low density lipoproteins induce aortic smooth muscle cell proliferation via the activation of mitogen activated protein kinase

We have investigated the effects of modifying LDL by Cu++ and various hemoglobin preparations on aortic smooth muscle cell proliferation and on the activation of mitogen activated protein kinase. We found that at very low concentrations (10 g/ml), LDL modified by all of the above agonists markedly stimulated cell proliferation (5–10 fold). This was accompanied by a 2–3 fold stimulation in mitogen activated protein kinase (MAPK) activity. We conclude that modification of LDL under situations that are closer to those found in vivo (i.e. hypoxic conditions), may involve the activation of MAPK as a common biochemical mechanism of action. This in turn, contributes to aortic smooth muscle cell proliferation.     

Modified low density lipoproteins decrease the activity and expression of lysosomal acid lipase in human endothelial and smooth muscle cells

Lysosomal acid lipase (LAL), the only lysosomal enzyme involved in the hydrolysis of LDL-cholesteryl esters, is a key regulator of cellular cholesterol and fatty acid homeostasis and its deficiency contributes to the pathophysiology of various diseases. In this study, we questioned whether oxidized or glycated LDL, a common occurrence in atherosclerosis and diabetes, affect the activity and expression of LAL in vascular endothelial cells (EC) and smooth muscle cells (SMC). LAL activity and expression were assayed in cultured human EC and SMC exposed to oxidized LDL (oxLDL), (±)9-hydroxyoctadecadienoic acid-cholesteryl ester (HODE), glycated LDL (gLDL), or native LDL (nLDL) as control, in the presence or absence of LXR or PPAR-gamma agonists. We found that LAL activity and expression were significantly down regulated by oxLDL and HODE in EC, and by gLDL in SMC. The LXR agonist T0901317 reversed the decreased LAL expression in modified LDL- or HODE-exposed EC (P < 0.001) and in gLDL-exposed SMC, whereas PPAR-gamma agonist rosiglitazone induced a low effect only in EC. In conclusion, modified LDL down regulates LAL expression in human EC and SMC by a process involving the LXR signaling pathway. This is the first demonstration that modified LDL modulate LAL expression, in a cell specific manner.     

Properties of endothelial cell and smooth muscle cell cultured in ambient pressure

Summary Cultures of umbilical vein endothelial cells and smooth muscle cells were studied in a constant pressure chamber. The following results were obtained: (a) Endothelial cell growth was maximal at 80 mmHg and minimal at 0 mmHg (atmospheric pressure) for the first 2 d of incubation. However, these growth rates were reversed during the following 6 d because of steady increase in growth at 0 mm Hg and a decrease in growth at higher pressures. A degeneration of endothelial cells began at 120 mmHg and marked degeneration was noted at 160 mmHg. Growth of Smooth muscle cells was not influenced by ambient pressure and a steady increase in labeled nuclei continued throughout the period of culture. (b) Elastin, stainable with tannic acid, was noted electronmicroscopically in both endothelial and smooth muscle cells. (c) Production of prostacyclin by endothelial cells was maximal at 0 mmHg and minimal at 80 mmHg, in contrast to the growth pattern of these cells. Production of thromboxane B₂ by endothelial cells and prostacyclin and thromboxane B₂ by smooth muscle cells was very slight and not significantly different. Although it is not known at present what mechanism acts on the vascular cells when cultured in ambient pressure, these results may indicate a new concept of the behavioral relationship between endothelial cell, smooth muscle cell, and blood pressure in vivo.     

Incorporation of [3H]glucosamine into glycosaminoglycans in different cell culture conditions by rabbit aortic smooth muscle cells

This study sought to elucidate the optimal cell culture conditions for studies concerned with the incorporation of [³H]glucosamine into glycosaminoglycans by rabbit aortic smooth muscle cells. The incorporation of radioactivity into extracellular sulphated glycosaminoglycans was linear for at least 72 h and that into pericellular sulphated glycosaminoglycans for up to 24 h. The incorporation of radiolabel into hyaluronic acid was linear only up to 12 h. In the exponential growth phase the incorporation of [³H]glucosamine into sulphated glycosaminoglycans and hyaluronic acid proved to be less marked than in the stationary growth phase, but the highest values were nevertheless obtained immediately after trypsinisation. When studied in the stationary growth phase, cell density and incorporation of [³H]glucosamine were positively correlated in the case of hyaluronic acid, but in the case of sulphated glycosaminoglycans there was a negative correlation. The serum concentration of the incubation medium and the incorporation of radioactivity into hyaluronic acid were positively related. With sulphated glycosaminoglycans this was the case only after a 7-day preincubation in the different serum concentrations. when incorporation was studied without preincubation, the incorporation of radioactivity into sulphated glycosaminoglycans proved to be negatively associated with the serum concentration of the medium. The environmental pH of the cells was associated with the incorporation of radioactivity into hyaluronic acid and sulphated glycosaminoglycans in that between pH values 6.8 and 7.9 the incorporation of radioactivity increased when the pH of the medium was raised.     

Characterization and modulation of fibroblast/endothelial cell co-cultures for the in vitro preformation of three-dimensional tubular networks

Various assays of different complexity are used in research on angiogenesis in health and disease. The results of these assays increasingly impact the field of tissue engineering because preformed microvascular networks may connect and conduct to the vascular system of the host, thereby helping us to support the survival of implanted cells and tissue constructs. An interesting model that supports the formation of EC (endothelial cells) tubular structures in vitro is based on co-culturing them with fibroblasts. Our initial multilayer approach was recently transferred into a three-dimensional spheroid model using HUVEC (human umbilical vein endothelial cells) as model cells. The aim of the present study is to further characterize, extend and validate this fibroblast/EC spheroid co-culture system. We have evaluated the model with a maximum size of 600-650 μm attained on day 3 from inoculation of 4×104 fibroblasts with 1×104 EC. Cell count and spheroid diameter significantly decreased as a function of time, but the EC network that developed over a period of 14 days in culture was clearly visible and viable, and central cell death was excluded. We successfully included HMVEC (human microvascular endothelial cells) of dermal origin in the system and replaced FBS (fetal bovine serum) with human AB serum, which positively impacted the EC network formation at optimized concentrations. The need for exogenous growth factors [VEGF (vascular endothelial growth factor), EGF (epithelial growth factor), bFGF (basic fibroblast growth factor) and IGF-1 (insulin-like growth factor-1)] routinely added to classical EC media was also assessed. The behaviour of both fibroblasts and EC in response to a combination of these exogenous growth factors differed critically in fibroblast/EC spheroid co-cultures compared with the same cells in the multilayer approach. VEGF was the most relevant exogenous factor for EC network formation in fibroblast/EC multilayers, but was ineffective in the spheroid system. IGF-1 was found, in general, to be dispensable; however, while it had a negative impact on EC networking in the presence of bFGF and EGF in the multilayer, it did not in the spheroid approach. We conclude that the critical determinants of EC network formation and cell survival are not universal, but have to be specifically optimized for each culture model.     

Simultaneous isolation of endothelial and smooth muscle cells from human umbilical artery or vein and their growth response to low-density lipoproteins

In the pathogenesis of atherosclerosis the interplay of endothelial cells (ECs) and smooth muscle cells (SMCs) is disturbed. Oxidatively modified low-density lipoproteins (oxLDLs), important stimulators of atherosclerotic plaque formation in vessels, modify the growth response of both cell types. To compare growth responses of ECs and SMCs of the same vessel with oxLDLs, we developed a method to isolate both cell types from the vessel walls of umbilical cords by enzymatic digestion. The method further allowed the simultaneous isolation of venous and arterial cells from a single umbilical cord. In culture, venous ECs showed an elongated appearance compared with arterial ECs, whereas SMCs of artery and vein did not look different. Smooth muscle cells of both vessel types responded to oxLDLs (60 microg/ml) with an increase in their [(3)H]-thymidine incorporation into DNA. On the contrary, ECs of artery or vein decreased [(3)H]-thymidine incorporation and cell number in the presence of oxLDLs (60 microg/ml) of increasing oxidation grade. Thus, human umbilical SMCs and ECs of the same vessel show a disparate growth response toward oxLDLs. But the physiologically more relevant minimal oxLDLs did not decrease proliferation in venous ECs but only in arterial ECs. This difference in tolerance toward minimal oxLDLs should be taken into account while using venous or arterial ECs of umbilical cord for research in atherosclerosis. Further differences of venous and arterial ECs in tolerance toward minimal oxLDLs could be of clinical relevance for coronary artery bypass grafts.     

Influence of low density lipoproteins on vascular smooth muscle cell growth and motility: modulation by extracellular matrix.

Low density lipoproteins (LDL) are thought to play a major role in cardiovascular diseases such as atherosclerosis. Much remains to be done to understand the cellular effects of LDL and how the extracellular matrix (ECM) influences these effects. We found that LDL produced a dose dependent increase in vascular smooth muscle cell (SMC) proliferation. The ECM altered the proliferative response of SMC to LDL: on collagen I there was a 66% inhibition, endothelial cell derived-ECM a 2-fold increase, and collagen IV no difference in proliferation compared to paired controls. LDL affected SMC motility (cell area and shape factor) but the extent and direction of the effect depended on whether the cells were cultured on uncoated or coated dishes. LDL treated cultures had a 5-fold lower migration rate but net movement was not different, suggesting that LDL decreased SMC random movement. There was a dose-dependent accumulation of lipid by SMC incubated with LDL and, subsequently, cytoplasmic lipid droplets were observed. Cells cultured on uncoated plates showed an increased cholesterol content as a function of LDL concentration. In contrast, cells cultured on a collagen IV matrix showed no net change in cholesterol content over the range of LDL concentrations studied. Hence, the uptake of LDL cholesterol appears to be completely inhibited by this matrix. These studies indicate that the influence of LDL on several SMC parameters is modulated by ECM components.     

Essential role of the low density lipoprotein receptor-related protein in vascular smooth muscle cell migration

The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor highly expressed in human aortic smooth muscle cells. In the present study, we used the short interfering RNA (siRNA) technique to explore the role of LRP in smooth muscle cell migration. We identified an LRP-specific siRNA that selective silences LRP expression in human aortic smooth muscle cells. As a consequence, LRP-mediated ligand degradation was significantly reduced. More important, we found that platelet-derived growth factor-dependent cell migration was inhibited in cells transfected with LRP siRNA. These results demonstrate an important role of LRP in smooth muscle cell migration.     

Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.     

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.     

Collagen synthesis and accumulation in long-term rabbit aortic smooth muscle cell cultures

Summary This study describes the ability of aortic smooth muscle cells to synthesize and accumulate collagen with time in culture. Inasmuch as smooth muscle cell cultures multilayer and continue to divide, albeit slowly, and can be maintained in the same vessels where seeded for extended periods of time, a long-term aging study from a single subcultivated population of cells was carried out. This is different from the usual cell-culture aging achieved by an increase in cell population doublings obtained by repeated subcultivations. The latter process, which is trypsin induced, involves a changing cellular environment including the extracellular matrix that is produced by the cells in culture. Second subcultures of weanling rabbit, aortic media, smooth muscle cells maintained for different periods of time up to 14 wk displayed decreasing hydroxyproline formation with time. Proline hydroxylation was determined by pulsing these second-passage cells with [¹⁴C]proline for 24 h at various times during the 14 wk period. The cell layer and medium were evaluated separately for radioactive proline and hydroxyproline and the medium for bacterial collagenase-susceptible protein as well. The percent of hydroxylation in the medium decreased from >31% within 1 wk after plating to 15.2% after 14 wk in culture. The percent of collagenase-susceptible protein in the medium decreased in a comparable manner. The DNA levels increased during the entire period although initially somewhat more rapidly. Accumulation of protein in the extracellular matrix continued during the 14-wk span. The accumulation of hydroxyproline in the extracellular matrix also continued to increase throughout the culture period, but it did slow down significantly. Yet the cells appear not to have lost their ability to accumulate connective tissue and protein in the insoluble cell layer. The data suggest clearly that the percent collagen synthesis relative to total protein synthesis decreases in the older cultures; total protein synthesis also decreases as expected. This study was supported by NIH Program Projects AG00001 and HL 13262.     

The various effects of fractionated oxidized low density lipoproteins on the growth of smooth muscle cells in culture

The effects of fractionated oxidized low density lipoproteins (oxidized LDL) on the growth of vascular smooth muscle cells (VSMC) and their relationship to the formation of lysophosphatidylcholine (lyso-PC) as well as the activation of protein kinase C (PKC) were studied. VSMC were isolated from porcine aorta by explant culture. LDL was isolated from porcine blood by sequential ultracentrifugation and oxidized LDL was obtained by incubating LDL with 5 µM CuSO₄ at 37° C for various lengths of time. Our results showed that LDL oxidized for 12 h and eluted from fast protein liquid chromatography at 43 min inhibited the growth of VSMC, and that LDL oxidized for longer than 48 h and eluted at 48 min stimulated the growth of VSMC. The formation of lyso-PC in the oxidized LDL correlated well with its stimulatory effect, suggesting that lyso-PC is responsible for the mitogenic effect of oxidized LDL. This stimulatory effect of oxidized LDL was inhibited by staurosporine, a PKC inhibitor. Treatment with oxidized LDL increased the activity of membrane PKC, but it decreased that of cytosolic PKC, suggesting the translocation of PKC from cytosol to the membrane in the presence of oxidized LDL. These results suggested that the oxidized LDL-stimulated VSMC growth was mediated by the formation of lyso-PC and the activation of PKC.     

Co-culture of endothelial cells and smooth muscle cells affects gene expression of angiogenic factors

Endothelial cells (EC) are in contact with the underlying smooth muscle cells (SMC). The interactions between EC and SMC in the vessel wall are considered to be involved in the control of growth and function of blood vessels. A co-culture system of EC and SMC and a method for separation of these cells was developed in order to investigate whether the presence of physical contact between EC and SMC affected the gene expression of angiogenic factors. Human EC and SMC were prepared from the great saphenous veins. Autologous EC were added on top of the confluent layer of SMC. After 72 h in co-culture, the EC were magnetically separated from SMC with the use of superparamagnetic beads. RT-PCR products for bFGF, bFGFR, VEGF, PDGF-AA, PDGF-BB, TGF-beta, and beta-actin were analyzed to study the mRNA expressions. The protein level of selected factors was studied by ELISA technique. In co-cultured SMC there was a statistically significant higher gene expression of VEGF, PDGF-AA, PDGF-BB, and TGF-beta and significant lower gene expression of bFGF and its receptor than in single cultured SMC. The protein level of PDGF-BB and TGF-beta was also significantly higher in co-cultured SMC. In co-cultured EC there were no significant differences in gene expression of PDGF-AA, PDGF-BB, and TGF-beta compared with single cultured EC. The gene expression and protein synthesis of VEGF was significantly higher in co-cultured EC. The findings from the present study suggest that cell-cell interactions of EC and SMC affect the gene and protein expression of angiogenic factors.     

内皮细胞生长状态对血管平滑肌细胞增生迁移的影响

实验通过建立细胞共培养体系,探讨内皮细胞生长状态对血管平滑肌细胞增生迁移的影响及机制。检测指标包括~3H-TdR掺入、细胞周期、细胞迁移计数和α-SM-actin mRNA表达。结果显示,融合生长内皮使平滑肌细胞~3H-TdR掺入量明显降低,增加平滑肌细胞停留在G_0/G_1期的比例,上调平滑肌细胞α-SM-actin mRNA表达;而对数生长内皮细胞使平滑肌细胞~3H-TdR掺入量明显升高,促进平滑肌细胞由 G_0/G_1期进入G_2/M和S期,下调平滑肌细胞α-SM-actin mRNA表达。对照组平滑肌细胞在基础状态下存在少量迁移,对数增殖内皮细胞组平滑肌迁移数比对照组增高约4倍(P<0.01),而融合生长内皮细胞组平滑肌迁移数仅为对照组的0.5倍(P<0.05)。结果提示内皮细胞生长状态不同,对平滑肌细胞生物学特性的影响也不同,增殖期内皮明显促进平滑肌细胞增生迁移、下调平滑肌细胞α-SM-actin mRNA表达。     

Estimation of Chinese hamster ovary cell density in packed-bed bioreactor by lactate production rate

A method is described for estimating recombinant Chinese hamster ovary (rCHO) cell density in a packed-bed bioreactor by lactate production rate. The lactate production rate, which depended on both the cell numbers and cell growth rate, was modeled by segregating the cell population into two parts: one growing at a maximum specific growth rate and another non-growing. The individual cell in each part had the same lactate production rate. The established rate equation of lactate production matched the experimental data reasonably well and could be used to estimate the cell growth in the batch culture with microcarriers. Furthermore, in the perfusion culture of rCHO cells in a packed-bed bioreactor, the final cell density, 1.3×10¹⁰ cells l^–1, estimated by lactate production rate, was comparable to the direct sample counting of 1.2×10¹⁰ cells l^–1, showing that lactate production rate method would be useful in tracing the cell growth in packed-bed bioreactors.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.