Aortic Intimal Smooth Muscle Cells Research Articles

Propionyl-L-carnitine Reduces Proliferation and Potentiates Bax-related Apoptosis of Aortic Intimal Smooth Muscle Cells by Modulating Nuclear Factor-κB Activity

Propionyl-l-carnitine (PLC) has been introduced among the therapeutic approaches of peripheral arterial disease, and more recently, an increase of intimal cell apoptosis has been demonstrated to contribute to its effectiveness in rabbit carotid postinjury myointimal hyperplasia prevention. How PLC mediates these effects on vascular smooth muscle cells (SMCs) remains poorly understood. We investigated the role of NF-kappaB in PLC-induced arterial remodeling. In vivo, daily PLC treatment 15 days after injury resulted in a reduction of relative rat aortic intimal volume, an increase of apoptosis, Bax up-regulation without changing the Bcl-2 level, and a reduction of NF-kappaB, vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and survivin in myointimal thickening compared with controls. In the presence of 10% serum, a reduced G(1) --> S phase progression preceded PLC-induced intimal cell apoptosis; in 0.1% serum cultures, in a dose-dependent manner, PLC rapidly induced intimal cell apoptosis and reduced p65, p50, IAP-1, and IAP-2 expression. Inhibiting NF-kappaB activation through SN50 increased apoptotic rate and Bax expression in intimal but not in medial SMCs, and successive PLC treatment failed to induce a further increase in apoptotic rate. Bax antisense oligodeoxynucleotide reduced PLC-induced intimal cell apoptosis and cytochrome c release. The PLC-induced attenuation of NF-kappaB activity in intimal cells was also due to the increase of IkappaB-alpha bioavailability, as the result of a parallel induction of IkappaB-alpha synthesis and reduction of phosphorylation and degradation. Collectively, these findings document that NF-kappaB activity inhibition contributes to PLC-induced proliferative arrest and Bax-related apoptosis of intimal SMCs.

Human plasma trans-sialidase causes atherogenic modification of low density lipoprotein

In earlier studies we have found that incubation of low density lipoprotein (LDL) with autologous blood plasma-derived serum leads to a loss of sialic acid from lipoprotein particles. In this study we demonstrated that sialic acid removed from LDL was transferred to glycoconjugates of lipoproteins, glycoproteins and sphingolipids of human serum. This showed that human serum contained the trans-sialidase activity. Gel-filtration chromatography of human blood serum demonstrated the presence of trans-sialidase activity in lipoprotein subfractions as well as in lipoprotein-deficient serum. Trans-sialidase (about 65 kDa) was isolated from lipoprotein-deficient serum using affinity chromatography carried out with Neu5Acα2-8Neu5Ac-sepharose FF-6. Optimal pH values for the trans-sialidase were 3.0, 5.0 and 7.0. Calcium and magnesium ions stimulated the enzyme activity at millimolar concentrations. Isolated enzyme can remove sialic acid from LDL, IDL, VLDL, and HDL particles (in decreasing rate order). Serum trans-sialidase transferred sialic acid from glycoconjugates of plasma proteins (fetuin, transferrin) and gangliosides (GM3, GD3, GM1, GD1a, GD1b). Sialylated glycoconjugates of human blood erythrocytes also served as substrate for serum trans-sialidase. We have found that sialic acid can also be removed from N- and O-linked glycans, sialylated Le x and Le a, oligosialic acids, and sphingolipid carbohydrate chains. The rate of sialic acid release decreased in the following order: α2,6>α2,3≫α2,8. Transferred molecule of sialic acid can form α2,6, α2,3 and to a lesser degree α2,8 linkage with galactose, N-acetyl-galactosamine or sialic acid of acceptors. The glycoconjugates of erythrocytes, lipoprotein particles, plasma proteins, neutral sphingolipids and gangliosides may serve as acceptors of transferred sialic acid. Trans-sialidase-treated native LDL becomes desialylated and then can induce cholesteryl ester accumulation in human aortic intimal smooth muscle cells. Thus, trans-sialidase may be involved in the early stages of atherogenesis characterized by foam cell formation.

Human plasma trans-sialidase effects LDL metabolism in aortic intimal smooth muscle cells

Human plasma trans-sialidase effects LDL metabolism in aortic intimal smooth muscle cells

Effects of short- and long-term exposure to linoleic acid hydroperoxide on cytosolic calcium ion level of human aortic intimal smooth muscle cells in vitro.

When cultured intimal smooth muscle cells from human aorta were exposed for a short period of time (25 sec) to linoleic acid hydroperoxide (100 nmol/ml), influx of calcium ions into the cytosol of these cells was provoked, and a temporal increase in cytosolic calcium ion level was observed. Two calcium channel blockers inhibited this influx. Long-term exposure (4 min) of these cells to the hydroperoxide also provoked the influx of calcium ions, which resulted in a longer time of the calcium ion increase. In the latter case, however, the calcium channel blocker inhibited the initial influx, but then the influx started and continued even in the presence of the blocker. Such difference in exposure time-dependent effects should be taken into account in considering pathological roles of lipid hydroperoxides.

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.

Emergence and distribution of intimal smooth muscle cells in the postnatal rat aorta

The distribution of aortic intimal smooth muscle cells in the normal rat during postnatal development was studied by electron microscopy and by staining with fluorescence-labeled phalloidin. The phenotypes of intimal and medial smooth muscle cells were almost identical at first; however, during development, the former remained synthetic, whereas the latter became contractile. Confocal laser scanning microscopy was utilized to observe intimal and medial cells separately. Intimal smooth muscle cells were rarely observed in neonatal rats, but appeared by 10 days of age and increased during postnatal development. A combination of confocal and conventional fluorescent microscopy clearly demonstrated that the intimal smooth muscle cells were preferentially distributed in: (1) the right-lateral and dorsal wall of the upper thoracic aorta, (2) the left-lateral and ventral wall of distal two-thirds of the descending aorta, and (3) the downstream side of branch orifices. Intimal smooth muscle cells in group (1) were oriented randomly, whereas most in group (2) ran longitudinally. Intimal smooth muscle cells at branches in group (3) ran obliquely from the edges at the downstream side in an upstream direction. They tended to accumulate in regions of the aortic wall considered to be under high tensile stress.

A Possible Mechanism for the Stimulation of Metalloproteinase Production in Human Aortic Intimal Smooth Muscle Cells by Linoleic Acid Hydroperoxide

To approach the mechanism of the stimulating effect of linoleic acid hydroperoxide on the production of matrix metalloproteinases (MMPs) in human aortic intimal smooth muscle cells (ISMC), we investigated the effect of the hydroperoxide on the cytosolic level of Ca2+. Linoleic acid hydroperoxide provoked an increase in the cytosolic Ca2+level, but it had no effect on the level of inositol phosphates (IPs) in these cells, in contrast with the effect of platelet-derived growth factor (PDGF), which elevated the level of both Ca2+and IPs in these cells. A23187, a calcium ionophore, stimulated ISMC to produce matrix prometalloproteinase 1. These results indicate that linoleic acid hydroperoxide stimulates the production of MMPs in ISMC by elevation of the cytosolic Ca2+level without the intervention of IPs. In addition, we found that the hydroperoxide has no effect on the binding of PDGF to its specific receptor on ISMC.

Ambient pressure stimulates immortalized human aortic endothelial cells to increase DNA synthesis and matrix metalloproteinase 1 (tissue collagenase) production.

In the present study, we investigated the effect of ambient pressure on [3H]-thymidine incorporation and on the production of matrix metalloproteinase 1 (tissue collagenase/proMMP-1) using human aortic endothelial cells immortalized with simian virus 40 (SE-1). Incubation of cells at ambient pressures of 50 and 100 mmHg for 24 h slightly increased [3H]-thymidine incorporation when directly compared with normal culture conditions. The amount of [3H]-thymidine incorporated in SE-1 reached a maximum at 150 mmHg, while a further increase in pressure to 200 mmHg decreased incorporation. The same ambient pressure slightly stimulated human aortic intimal smooth muscle cells (SMC) to increase [3H]-thymidine incorporation but not medial SMC. Immunoblot analysis also showed that ambient pressure, ranging from 50 to 200 mmHg, like 12-O-tetradecanoyl-phorbol-13-acetate stimulated SE-1 to produce proMMP-1, an effect not seen with either intimal or medial SMC. The amount of proMMP-1 produced also reached a maximum level at 150 mmHg. We postulate that human endothelial cells are ambient pressure sensitive and that relatively lower ambient pressures play an important role in the growth of endothelial cells, while higher pressures injure endothelial cells, resulting in the initiation of atherosclerosis. This cell line may prove useful in the investigation of both the physiological and pathological roles of blood pressure on endothelial cell function.

Isolation of a morphologically and functionally distinct smooth muscle cell type from the intimal aspect of the normal rat aorta. Evidence for smooth muscle cell heterogeneity

Recent studies indicate that the neointima of injured rat arteries is composed of a subpopulation of smooth muscle cells (SMCs) distinct from medial smooth muscle cells. However, SMC diversity in normal adult aorta has remained elusive. This study characterizes two morphologically and functionally distinct SMC types isolated from different anatomic regions of the normal rat aorta. Rat aortic medial smooth muscle cells (MSMCs) were isolated from the media after removal of the intimal and adventitial cells. Rat aortic intimal smooth muscle cells (ISMCs) were isolated from the intimal aspect of everted rat aortas. The two cell types were characterized morphologically and immunohistochemically and were compared for their capacity to contract collagen gels in response to endothelin-1. MSMCs were spindle-shaped and grew in hills and valleys showing features previously described for vascular SMCs. Conversely, ISMCs displayed a polygonal and epitheloid shape, grew mainly as a monolayer, and had a higher proliferative rate. Both cell types expressed alpha-smooth muscle actin and were negative for Factor VIII-RAg. ISMCs produced large amounts of a laminin and type IV collagen-rich extracellular matrix which had a characteristic pericellular distribution. ISMCs, but not MSMCs, rapidly contracted collagen gels in response to endothelin-1. This study indicates that the normal rat aorta contains two types of SMCs located in anatomically distinct regions of the vessel wall. Because of their functional characteristics, the SMCs isolated from the intimal aspect of the aorta may play an important role in physiologic as well as pathologic conditions.

Patent Evaluation: Novel inhibitors of atherosclerotic intimal thickening

SummaryNovelty: Novel inhibitors of atherosclerotic intimal thickening which contain pyridine derivatives are disclosed. These agents have strong inhibitory effects against HMG-CoA reductase, against proliferation of aortic intimal smooth muscle cells, migration of aortic medial smooth muscle cells and adhesion of blood cells to endothelial cells.Biology: Inhibitory effects on migration of aortic smooth muscle cells were determined in vitro using aortic slices from male Sprague Dawley rats. The specified compound demonstrated 100% inhibition of PDGF and SMC-CM at 10-5M. The inhibitory effects on the proliferation of aortic intimal and medial smooth muscle cells was also demonstrated. The specified compound demonstrated 100% inhibition at 10-5 and 10-6M. Inhibitory effects on [3H]-thymidine uptake adhesion of leukaemic cells and adhesion of J774 macrophages are also reported.Chemistry: Three compounds are specifically claimed including 3,5-dihydroxy-7-[6′-cyclopropyl-3′-ethyl-4′-(4″-fluorophenyl)-2′-methylthieno[2,3-b]pyridin-5′-yl]hept-6-enoic acid.

Plasmenylethanolamine is the major storage depot for arachidonic acid in rabbit vascular smooth muscle and is rapidly hydrolyzed after angiotensin II stimulation.

The present study demonstrates that rabbit aortic intimal smooth muscle cells contain the majority of their endogenous arachidonic acid mass in plasmenylethanolamine molecular species. To demonstrate the potential significance of these plasmenylethanolamines as substrates for the smooth muscle cell phospholipases that are activated during agonist stimulation, aortic rings were prelabeled with [3H]arachidonic acid and stimulated with angiotensin II. Although the specific activities of the choline and inositol glycerophospholipid pools were similar after the labeling interval, ethanolamine glycerophospholipids had a specific activity of only 20% of the specific activity of choline and inositol glycerophospholipids. Despite the marked disparity in the specific activities of these three phospholipid classes, angiotensin II stimulation resulted in similar fractional losses (35-41%) of [3H]arachidonic acid from vascular smooth muscle choline, ethanolamine, and inositol glycerophospholipid classes. Reverse-phase HPLC demonstrated that greater than 60% of the [3H]arachidonic acid released from ethanolamine glycerophospholipids after angiotensin II stimulation originated from plasmenylethanolamine molecular species. Taken together, the results demonstrate that the major phospholipid storage depot for arachidonic acid in vascular smooth muscle cells are plasmenylethanolamine molecular species which are important substrates for the phospholipase(s) that are activated during agonist stimulation.