Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time- and dose-dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5-hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C-activating phorbol ester, phorbol 12 myristate 13-acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.     

Intercellular communication in cultured human vascular smooth muscle cells

Intercellular communication through gap junction channelsplays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,capacitance vessel). Northern and Western blots documented the presenceof connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA incultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority ofrecords, in both vessels, single-channel activity was dominated by amain-state conductance of 120 pS, with subconducting events comprisingless than 10% of the amplitude histograms. However, some recordsshowed "atypical" unitary events that had a conductance similar toCx40 (~140-160 pS), but gating behavior like that of Cx43. Assuch, it is conceivable that the presence and coexpression of Cx40 andCx43 in IMA and SV myocytes may result in heteromeric channelformation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

     

Effect of TNF on triacylglycerol in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) isolated from bovine aorta or human saphenous vein were cultured and used to study the putative effect of recombinant human tumor necrosis factor (TNF) on lipid metabolism in vascular cells. Addition of TNF to the culture medium for 24-48 h resulted in an increase of [3H]oleic acid uptake and esterification into lipids. The effect could be seen already with 0.3 ng/ml and was maximal with 30 ng/ml. The effect of TNF was mainly on the incorporation of [3H]oleic acid into triacylglycerol which increased by 140% in the bovine cells. There was also a significant increase in [3H]cholesteryl ester. In the human SMC there was a 40% increase in [3H]oleic acid into total lipids, while the rise in [3H]triacylglycerol ranged between 60-90%. TNF did not modulate cellular triacyglycerol synthesis in cultured mouse peritoneal macrophages. Since TNF was shown to be synthesized and secreted not only by macrophages but also by smooth muscle cells, it could play an autocrine role in lipid metabolism during development of atherosclerotic lesions. The cellular population of the lesions, i.e., predominance of macrophages or smooth muscle cells, could determine the relative proportion of triacylglycerol accumulation.     

Heparin interactions with cultured human vascular endothelial and smooth muscle cells: incidence on vascular smooth muscle cell proliferation

The binding, internalization, and metabolism of [3H]-heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size-exclusion HPLC. Incubation of HUVEC with [3H]-heparin demonstrated selective binding of high-molecular-weight (MW) components (MW = 21 kd), which was followed by rapid, temperature-dependent internalization. Over the next 3 hours, this internalized [3H]-heparin was degraded to low-MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high-MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40-kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low-MW heparin fragment(s) produced in culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 micrograms/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0-20 micrograms/ml). These fragments were 10- to 15-fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.     

Fluid shear stress-induced alignment of cultured vascular smooth muscle cells

The study objectives were to quantify the time- and magnitude-dependence of flow-induced alignment in vascular smooth muscle cells (SMC) and to identify pathways related to the orientation process. Using an intensity gradient method, we demonstrated that SMC aligned in the direction perpendicular to applied shear stress, which contrasts with parallel alignment of endothelial cells under flow SMC alignment varied with the magnitude of and exposure time to shear stress and is a continuous process that is dependent on calcium and cycloskeleton based mechanisms. A clear understanding and control of flow-induced SMC alignment will have implications for vascular tissue engineering.     

Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells

We explored the hypothesis that discrepancies in the literature concerning the nature of myosin expression in cultured smooth muscle cells are due to the appearance of a new form of myosin heavy chain (MHC) in vitro. Previously, we used a very porous sodium dodecyl sulfate gel electrophoresis system to detect two MHCs in intact smooth muscles (SM1 and SM2) which differ by less than 2% in molecular weight (Rovner, A. S., Thompson, M. M., and Murphy, R. A. (1986) Am. J. Physiol. 250, C861-C870). Myosin-containing homogenates of rat aorta cells in primary culture were electrophoresed on this gel system, and Western blots were performed using smooth muscle-specific and nonmuscle-specific myosin antibodies. Subconfluent, rapidly proliferating cultures contained a form of heavy chain not found in rat aorta cells in vivo (NM) with electrophoretic mobility and antigenicity identical to the single unique heavy chain seen in nonmuscle cells. Moreover, these cultures expressed almost none of the smooth muscle heavy chains. In contrast, postconfluent growth-arrested cultures expressed increased levels of the two smooth muscle heavy chains, along with large amounts of NM. Analysis of cultures pulsed with [35S] methionine indicated that subconfluent cells were synthesizing almost exclusively NM, whereas postconfluent cells synthesized SM1 and SM2 as well as larger amounts of NM. Similar patterns of MHC content and synthesis were found in subconfluent and postconfluent passaged cells. These results show that cultured vascular smooth muscle cells undergo differential expression of smooth muscle- and nonmuscle-specific MHC forms with changes in their growth state, which appear to parallel changes in expression of the smooth muscle and nonmuscle forms of actin (Owens, G. K., Loeb, A., Gordon, D., and Thompson, M. M. (1986) J. Cell Biol. 102, 343-352). The reappearance of the smooth muscle MHCs in postconfluent cells suggests that density-related growth arrest promotes cytodifferentiation, but the continued expression of the nonmuscle MHC form in these smooth muscle cells indicates that other factors are required to induce the fully differentiated state while in culture.     

Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Specific binding sites for synthetic porcine endothelin (pET), a novel potent vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells, and its effects on cytosolic free Ca2+ concentrations ([Ca2+]i) and phosphatidylinositol (PI) response were studied in cultured rat aortic vascular smooth muscle cells (VSMC). Binding of 125I-labeled-pET to rat VSMC was time- and temperature-dependent and the cell-bound 125I-labeled-pET was resistant to dissociate. Scatchard analysis of binding studies indicated the presence of a single class of high-affinity binding sites: the apparent Kd was 2-4 X 10(-10) M and the maximal binding capacity was 11,000-13,000 sites/cell. The binding was highly specific for pET because neither well-recognized vasoconstrictors, peptide neurotoxins, nor Ca2+-channel blockers affected the binding. pET dose-dependently (10(-9)-10(-7) M) induced a transient and sustained increase in [Ca2+]i in fura-2-loaded cells of which effect was largely dependent on extracellular Ca2+, whereas it had no significant effect on PI response in 3H-myoinositol-prelabeled cells. The present data clearly demonstrates the presence of specific receptors for pET distinct from those of the well-recognized vasoconstrictors and voltage-dependent Ca2+-channels in cultured rat VSMC, and suggest that pET-induced increase in [Ca2+]i is involved in the mechanism of its vasoconstriction.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells

Cultured vascular smooth muscle cells from bovine and rat thoracic aortae and from human omental vessels have been examined for cellular responses to endothelin. In myo-[3H]-inositol-prelabelled cells endothelin induced a rapid (within 30 sec) and protracted increase of [3H]-inositol content in inositol bis- and tris-phosphates. Concomitantly, significant polyphosphoinositide hydrolysis occurred within 30 sec. Accumulation of [3H]-inositol monophosphate and hydrolysis of phosphatidylinositol were delayed. In cells prelabelled with [3H]-arachidonic acid endothelin promoted rapid production of [3H]-diacylglycerol which decayed slowly toward control values after reaching maximum levels (1-2 min). Half-maximally effective concentrations of endothelin for all these cellular responses were comparable (approximately 3-7 nM) and not significantly different between the vascular cell isolates. The involvement of the phospholipase C-signal transduction pathway in mediating endothelin-induced vasoconstriction is invoked.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Receptors for atrial natriuretic factor in cultured vascular smooth muscle cells

The presence of receptors for atrial natriuretic factor (ANF) was previously demonstrated in the mesenteric vascular bed in rats. Cultured vascular smooth muscle cells obtained from mesenteric arteries of rats were examined for binding of ANF. Saturation and competition experiments demonstrated the presence of a single class of receptors for ANF with high affinity (16 pM) and low capacity. Binding was specific. Kinetic studies showed a dissociation constant which agreed with that obtained at equilibrium in saturation and competition experiments. The exposure of the cells to unlabeled ANF for at least 24 hours showed that ANF may regulate its own receptors in smooth muscle under certain physiological conditions.     

Neuropeptide Y receptor-effector coupling mechanisms in cultured vascular smooth muscle cells

Primary cultures of rabbit pulmonary artery (RPA) vascular smooth muscle (VSM) were utilized to determine the coupling of neuropeptide Y (NPY) receptors to several effector systems in VSM. NPY inhibited forskolin-stimulated adenylate cyclase by 65%, with an EC50 of 0.3 nM. However, NPY did not stimulate phosphoinositide (PI) hydrolysis or the elevation of cytosolic calcium, (Ca+2)i, in cultured RPA-VSM cells, nor did it potentiate norepinephrine-induced PI hydrolysis or elevation of (Ca+2)i. These results suggest that NPY-induced vasocontraction is not mediated by PI hydrolysis or the modulation of (Ca+2)i.     

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [¹²⁵I]rAM revealed the presence of a single class of high-affinity (K_d1.3 × 10⁻⁸ M) binding sites for rAM in VSMC. The apparent K_i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10⁻⁷ M. Affinity labeling of VSMC membranes with [¹²⁵I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC₅₀ of 10⁻⁸ M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.     

Carbon monoxide effects on calcium levels in vascular smooth muscle

Previously we showed that carbon monoxide (CO) relaxes vascular smooth muscle in the working heart and thoracic aorta preparations perfused with hemoglobin-free, Krebs-Henseleit (KH) solution. The CO-induced relaxation was not caused by hypoxia, nor was it mediated by adrenergic influences, adenosine, or prostaglandins. In these studies the effect of CO on calcium (Ca++) concentrations in vascular smooth muscle was determined using 45Ca as a tracer. Isolated rat thoracic aorta segments were incubated with 45Ca and gassed with O2, N2, or CO for 60 min. Verapamil was used to verify the effectiveness of the test system. Ca++ concentrations were 488 +/- 35 and 515 +/- 26 mM/g tissue (X +/- SE) in aortic rings gassed with O2 and N2, respectively. CO reduced Ca++ concentrations significantly (P less than 0.01) by 29% to 369 +/- 18 mM/g tissue. Verapamil treatment reduced Ca++ concentrations by 40% to 314 +/- 23 mM/g tissue. These results suggest that CO relaxes vascular smooth muscle and dilates blood vessels by decreasing Ca++ concentrations in vascular smooth muscle.     

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells

This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.     

Characterization of phosphatidylinositol-specific phospholipase C from cultured vascular smooth muscle cells.

Phosphoinositide-specific phospholipase C (PLC) activities have been partially purified from cultured vascular smooth muscle cells and analyzed for substrate specificity, calcium and pH requirements, and molecular weight. The purification procedure involved DEAE-cellulose and heparin-Sepharose chromatographies followed by Mono Q and size exclusion high performance liquid chromatography. This technique resolves multiple peaks of activity using phosphatidylinositol (PI) and PI 4,5-bisphosphate (PIP2) as substrates. The major peak was purified to near homogeneity as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PLC activity in vascular smooth muscle cells can be divided into two types based on their calcium and pH requirements, substrate preferences, and molecular weights. The low molecular weight PLC hydrolyzes both PI and PIP2, has a molecular mass of 58 kDa, requires the most calcium for full activation, and has a PI-pH profile that shifts slightly with calcium concentration. Screening a cDNA library with oligonucleotides directed against several of the known PLCs identified a highly expressed PLC cDNA that is 99% homologous to PLC-alpha, suggesting that this low molecular weight peak in fact corresponds to PLC-alpha. The high molecular mass peak (157 kDa) shows much greater activity against PI than PIP2, is active at lower calcium concentrations, and has a PI-pH optimum of 5.0 regardless of calcium concentration. Each of the PIP2 PLC activities is strongly dependent on the relative levels of calcium and pH in the assay buffer. These observations suggest that vascular smooth muscle contains both a high and low molecular weight PLC whose activities are affected markedly by the changes in calcium and pH accompanying hormonal stimulation of the cell.     

Calcitonin gene-related peptide receptor in cultured vascular smooth muscle and endothelial cells

Using 125I-labeled-Tyr0-rat(r)-calcitonin gene-related peptide (CGRP), a potent vasodilatory neuropeptide, we have identified and characterized specific binding sites for CGRP in cultured rat vascular smooth muscle cells (VSMC) and bovine endothelial cells (EC). rCGRP and human (h) CGRP equipotently inhibited 125I-rCGRP binding to both cells, but human calcitonin (hCT) was less potent and other unrelated polypeptides were ineffective. Both rCGRP and hCGRP, but not hCT, equally stimulated intracellular cAMP generation in both cells distinct from beta-adrenergic receptor-mediated mechanism, although they had no effect on cGMP generation in either cell or synthesis of prostacyclin in EC. Autoradiograph of affinity-labeled cell membranes revealed that 125I-rCGRP interacts with a single binding component of almost identical molecular size (approximately 60-kDa) in both cells under reducing and nonreducing conditions. The present study demonstrates for the first time the presence of CGRP receptors in cultured VSMC and EC, functionally coupled to adenylate cyclase system distinct from beta-adrenergic receptors. It is suggested that CGRP-induced vasorelaxation may be mediated partly by cAMP-dependent and/or endothelium-dependent mechanism.