Nifedipine induces apoptosis in cultured vascular smooth muscle cells from spontaneously hypertensive rats

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7-10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca2+ channel antagonist, nifedipine (5 X 10(-5) M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 x 10(-7) M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.     

Prostaglandin D2 synthase inhibits the exaggerated growth phenotype of spontaneously hypertensive rat vascular smooth muscle cells

Lipocalin-type prostaglandin D2 synthase (L-PGDS) has recently been linked to a variety of pathophysiological cardiovascular conditions including hypertension and diabetes. In this study, we report on the 50% increase in L-PGDS protein expression observed in vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR). L-PGDS expression also increased 50% upon the differentiation of normotensive control cells (WKY, from Wistar-Kyoto rats). In addition, we demonstrate differential effects of L-PGDS treatment on cell proliferation and apoptosis in VSMCs isolated from SHR versus WKY controls. L-PGDS (50 microg/ml) was able to significantly inhibit VSMC proliferation and DNA synthesis and induce the apoptotic genes bax, bcl-x, and ei24 in SHR but had no effect on WKY cells. Hyperglycemic conditions also had opposite effects, in which increased glucose concentrations (20 mm) resulted in decreased L-PGDS expression in control cells but actually stimulated L-PGDS expression in SHR. Furthermore, we examined the effect of L-PGDS incubation on insulin-stimulated Akt, glycogen synthase kinase-3beta (GSK-3beta), and ERK phosphorylation. Unexpectedly, we found that when WKY cells were pretreated with L-PGDS, insulin could actually induce apoptosis and failed to stimulate Akt/GSK-3beta phosphorylation. Insulin-stimulated ERK phosphorylation was unaffected by L-PGDS pretreatment in both cell lines. We propose that L-PGDS is involved in the balance of VSMC proliferation and apoptosis and in the increased expression observed in the hypertensive state is an attempt to maintain a proper equilibrium between the two processes via the induction of apoptosis and inhibition of cell proliferation.     

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.     

Binding of synthetic beta-human atrial natriuretic peptide to cultured rat vascular smooth muscle cells

We have studied the effects of synthetic beta-human atrial natriuretic peptide (beta-hANP), an antiparallel dimer of alpha-hANP, on receptor binding and cGMP generation in cultured rat vascular smooth muscle cells and compared the effects with those of alpha-hANP. Characteristics of temperature-dependent binding and degradation of 125I-beta-hANP were similar to those of 125I-alpha-hANP. Scatchard analysis indicated a single class of binding sites for beta-hANP with a maximal binding capacity one-half that of alpha-hANP. Parallel and antiparallel dimers were equipotent in inhibiting the binding and stimulating intracellular cGMP formation, of which the maximal effect was about one-half that of alpha-hANP. Reverse-phase high performance liquid chromatography revealed that most of beta-hANP added to cells was converted to a small molecular mass component corresponding to alpha-hANP after incubation. These data suggest that the less potent effect of beta-hANP in receptor binding and cGMP generation may be partly accounted for by the possible conversion of beta-hANP to alpha-hANP at the site of target cells.     

Electrophysiological recordings from spontaneously contracting reaggregates of cultured vascular smooth muscle cells from chick embryos

Single cells were trypsin-dispersed from blood vessels (great vessels near the heart and mesenteric vessels) of 10–20 day chick embryos, and induced to reaggregate into small spheres (0.1–0.5 mm ) either by gyration or by plating on cellophane. Many reaggregates contracted spontaneously or in response to electrical stimulation during culture periods of up to 6 weeks. When the spherical reaggregates were allowed to adhere to a glass substrate, cells emigrated from the spheres to form aprons of monolayered cells which continued to contract. Thick and thin myofilaments (mean diameters of 146 and 65 Å, respectively) were observable in a large fraction of cells studied in electron micrographs. Vascular smooth muscle (VSM) cells were identified in the reaggregates by recording resting potentials of −40 to −60 mV, and by action potential generation. The action potentials were preceded by pacemaker potentials, had slow rates of rise (<20 V/sec), and were insensitive to tetrodotoxin (TTX). Although the action potentials depend on an inward slow current, D-600 did not block the action potentials of the VSM cells. Reaggregates of atrial cells, produced at the same time for comparison, had larger resting potentials (up to −80 mV), less automaticity, fast rates of rise (mean of about 85 V/sec), and complete TTX sensitivity, thus indicating dependence on fast Na⁺ channels. These findings indicate that identifiable VSM cells can be successfully maintained in primary culture for several weeks, and these cells retain electrical and contractile properties similar to those of smooth muscle cells in intact adult blood vessels. This preparation provides a convenient system for electrophysiological and pharmacological studies of VSM cells.     

Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.     

Electron-microscopic studies on reaggregate cultures of vascular smooth muscle cells from normotensive and spontaneously hypertensive rats

Summary Vascular smooth muscle cells were taken from the aortae of the WKY (normotensive) and SHR (spontaneously hypertensive) strains of rat by enzymatic dispersion and put into reaggregate culture. Initially the cells became individual spheroids having average diameters of 10 m and surfaces that were either rough or smooth. The cells were far more complex than they appeared on their surfaces; after one day in culture, there was considerable internal variation in these cells. All the cells, whether WKY or SHR, lost the bulk of their cytoplasmic contents (including myofilaments, many mitochondria, and vesicular structures) in the early stages of culture and eventually became flattened. After 14 days in culture, these modified cells collected to form reaggregates that were commonly roughly spherical and several hundred m in diameter. These reaggregates consisted of peripheral regions made up of several layers of flattened cells overlying cores formed by glia-like networks of cells similar in cytological appearance to the cells at the periphery. The meshes formed in this way contained cellular debris derived from dead cells or extrusion of cellular contents. It appears that SHR cells are quicker to form reaggregates than are WKY cells. Yet the SHR cells retained a rounded conformation after five days, whereas the WKY cells were more flattened and formed a more discrete aggregate at this stage of culture. However, by the fourteenth day of culture, differences between the two cell strains were not so pronounced, as far as could be judged by observations made with scanning and transmission electron microscopy. Both WKY and SHR cells at 14 days appeared highly secretory, possessing large Golgi systems as well as numerous ER cisternae and mitochondria. SHR cells produced greater amounts of connective tissue at all stages of culture than did WKY cells, indicating that a similar difference may contribute to the hypertension which develops naturally in situ in SHR animals.     

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

Summary Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar¹,Ile⁸-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.     

Impaired expression of PPAR gamma protein contributes to the exaggerated growth of vascular smooth muscle cells in spontaneously hypertensive rats

Peroxisome proliferator-activated receptor gamma (PPAR gamma), a member of the nuclear receptor family, has been implicated in the regulation of vascular smooth muscle cell (VSMC) growth; however, the underlying mechanisms are still not fully understood. We hypothesized that PPAR gamma functional deficiency may contribute to the enhanced proliferation of VSMC associated with hypertension in spontaneously hypertensive rats (SHR). We observed that PPAR gamma mRNA level in SHR VSMC was 3 approximately 4 fold higher than that from Wistar-Kyoto rats (WKY), but the protein expression levels of PPAR gamma are significantly lower in SHR than WKY VSMC, suggesting an impaired control of PPAR gamma protein expression in SHR VSMC. The deficiency of PPAR gamma protein expression in SHR VSMC was demonstrated by PPAR gamma reporter gene assays. Furthermore, the exaggerated growth of SHR VSMC was markedly attenuated by adenoviral PPAR gamma overexpression. Taken together, our results provided the first direct evidence that impaired expression of PPAR gamma protein contributes to the exaggerated growth of SHR VSMC.     

Characterization of insulin binding sites in cultured smooth muscle cells of rat aorta

Insulin receptors could be demonstrated in cultured smooth muscle cells of rat aorta. The specific binding of 125I-insulin was time-, temperature- and pH-dependent. The optimal temperature for our studies was 12 degrees C. At this temperature maximal specific binding was 0.5% of total counts at 120 min incubation. The pH-optimum for the binding process was between 7.5 and 8. Degradation of 125I-insulin at 12 degrees C was 14%, no degradation of binding sites could be measured at this temperature. Dissociation of 125I-insulin was rapid. 50% of the labeled hormone remained associated with the cells. Half-maximal inhibition of 125I-insulin binding was produced by insulin at 4 X 10(-11) mol/l. Scatchard-analysis gave curvilinear plots, that may suggest negative cooperativity. Specificity of binding was studied in competition experiments between 125I-insulin, insulin, proinsulin, insulin-like growth factors and human growth hormone. Half-maximal inhibition of 125I-insulin binding was produced by proinsulin at 2 X 10(-9) mol/l and by insulin-like growth factors at 9 X 10(-9) mol/l. Human growth hormone had no significant effect on the insulin binding.     

Decreased sensitivity of spontaneously hypertensive rat aortic smooth muscle to vasorelaxation by atriopeptin III

The effects of a synthetic form of Atrial Natriuretic Factor (ANF) on spontaneously hypertensive rat aortic smooth muscle were investigated using either an alpha-adrenoceptive agonist (phenylephrine) or an agent which partially depolarized the plasma membrane (20mM KCl) as a contractile agent. The relaxant response was studied under conditions resembling normal physiological calcium ion levels (1.5mM) as well as over a range of calcium ion concentrations (0.1-2.5mM). The results demonstrate a hyporesponsiveness of hypertensive aorta to vasorelaxation induced by synthetic ANF, which is more apparent when the tissue is contracted with KCl. The results also suggest that ANF, which has been shown previously to inhibit intracellular and receptor operated calcium channel mobilization only, may additionally work through a mechanism which is related to the voltage induced calcium flux across the membrane, which also is inhibited less in hypertensive smooth muscle.     

Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP)

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal laminalike material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

Reduced levels of cyclic AMP contribute to the enhanced oxidative stress in vascular smooth muscle cells from spontaneously hypertensive rats

We have earlier shown that aortic vascular smooth muscle cells (VSMC) from 12-week-old spontaneously hypertensive rats (SHR) exhibited enhanced production of superoxide anion (O(2)(-)) compared with Wistar-Kyoto (WKY) rats. This production was attenuated to control levels by losartan, an angiotensin II (Ang II) AT(1)-receptor antagonist, suggesting that the AT(1) receptor is implicated in enhanced oxidative stress in SHR. Since AT(1) receptor activation signals via adenylyl cyclase inhibition and decreases cAMP levels, it is possible that AT(1) receptor-mediated decreased levels of cAMP contribute to the enhanced production of O(2)(-) in SHR. The present study was undertaken to investigate this possibility. The basal adenylyl cyclase activity as well as isoproterenol and forskolin-mediated stimulation of adenylyl cyclase was significantly attenuated in VSMC from 12-week-old SHR compared with those from WKY rats, whereas Ang II-mediated inhibition of adenylyl cyclase was significantly enhanced by about 70%, resulting in decreased levels of cAMP in SHR. NADPH oxidase activity and the levels of O2- were significantly higher (about 120% and 200%, respectively) in VSMC from SHR than from WKY rats. In addition, the levels of p47(phox) and Nox4 proteins, subunits of NADPH oxidase, were significantly augmented about 35%-40% in VSMC from SHR compared with those from WKY rats. Treatment of VSMC from SHR with 8Br-cAMP, as well as with cAMP-elevating agents such as isoproterenol and forskolin, restored to control WKY levels the enhanced activity of NADPH oxidase and the enhanced levels of O(2)(-), p47(phox), and Nox4. Furthermore, in the VSMC A10 cell line, 8Br-cAMP also restored the Ang II-evoked enhanced production of O(2)(-), NADPH oxidase activity, and enhanced levels of p47(phox) and Nox4 proteins to control levels. These data suggest that decreased levels of cAMP in SHR may contribute to the enhanced oxidative stress in SHR and that increasing the levels of cAMP may have a protective effect in reducing oxidative stress and thereby improve vascular function.     

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.