Primary Cultures Of Vascular Smooth Muscle Cells Research Articles

Protein Kinase G Regulates Potassium Chloride Cotransporter-3 Expression in Primary Cultures of Rat Vascular Smooth Muscle Cells

K-Cl cotransport (KCC) is activated by nitric oxide donors and appears to be regulated by the cGMP signaling pathway. Expression of KCC mRNAs (KCC1-KCC4) in rat vascular smooth muscle cells (VSMCs) is unknown. We have reported the presence of KCC1 and KCC3 mRNAs in primary cultures of VSMCs by specific reverse transcription-polymerase chain reaction. KCC2 mRNA appeared at extremely low levels. KCC4 mRNA was undetectable. Semiquantitative reverse transcription-polymerase chain reaction revealed a 2:1 KCC1/KCC3 mRNA ratio in VSMCs. Depletion of protein kinase G (PKG)-1 from VSMCs did not change KCC3 mRNA expression. Analogous results were obtained with PKG-1-catalytic domain- and vector only-transfected VSMCs lacking endogenous PKG, suggesting no involvement of PKG-1 in the maintenance of basal KCC3 mRNA expression. However, 8-bromo-cGMP, a PKG stimulator, acutely increased KCC3 mRNA expression in a concentration- and time-dependent fashion; this effect was blocked by the PKG inhibitor KT5823 but not by actinomycin D. These findings show that VSMCs express mainly two mRNA isoforms, KCC1 and KCC3, and suggest that PKG participates post-transcriptionally in the acute KCC3 mRNA regulation. The role of KCC3 on cell volume and electrolyte homeostasis in response to PKG modulators remains to be determined.

Signalling Pathways Activated by 5-HT1B/5-HT1D Receptors in Native Smooth Muscle and Primary Cultures of Rabbit Renal Artery Smooth Muscle Cells

The potential of primary cultures of rabbit renal artery vascular smooth muscle cells (VSMCs) was assessed as a means to investigate the signalling pathways linked to 5-hydroxytryptamine (5-HT) 5-HT_1B/5-HT_1D receptors in native arteries. In renal artery segments denuded of endothelium, incubated with ketanserin and prazosin (each 1 µM), and prestimulated with 20 mM K⁺ Krebs buffer, 5-HT and CP 93,129, a 5-HT_1B receptor agonist, evoked concentration-dependent contractions. GR 127935, a 5-HT_1B/5-HT_1D receptor antagonist, significantly antagonised 5-HT-evoked contractions at nanomolar concentrations. Reverse transcription polymerase chain reaction (RT-PCR) of mRNA from smooth muscle cells from the isolated renal artery and from primary cultures of VSMCs from the same artery expressed mRNA transcripts for the 5-HT_1B receptor and the 5-HT_1D receptor in both preparations. The sequence of the PCR fragments corresponded to the known sequence for these receptors. Application of 5-HT evoked a concentration-dependent, pertussis toxin (PTx)-sensitive reduction in cyclic AMP in both cultured cells and intact artery (cyclic AMP concentration reduced by 65.53 ± 3.33 and 52.65 ± 5.34% from basal with 10 µM 5-HT, respectively). The effect of 10 µM 5-HT on cAMP was increased in the presence of 20 mM K⁺ (reduced by 82.50 ± 2.50 and 87.54 ± 3.97%, respectively). In intact arteries, contraction through 5-HT_1B/5-HT_1D receptors was significantly attenuated by inhibitors of phosphatidylinositol 3-kinase (wortmannin) and activated mitogen-activated protein kinase (MAPK), MEK (U01296). In the cultured VSMCs, activated MAPK was identified by immunocytochemistry and immunoblotting after stimulation with 5-HT, but only if 20 mM K⁺ was present at the onset of stimulation. These data provide the first direct evidence that 5-HT_1B/5-HT_1B receptors are linked to the activation of MAPK and indicate that primary cultures of renal VSMCs could provide a model system to study further the signalling pathways linked to these receptors.

The effects of angiotensin II, endothelin-1, and protein kinase C inhibitor on DNA synthesis and intracellular calcium mobilization in vascular smooth muscle cells from young normotensive and spontaneously hypertensive rats

Angiotensin II (Ang-II) and endothelin 1 (ET-1) are important peptides that induce a prolonged vasoconstriction and enhance proliferation of vascular smooth muscle cells (VSMC). These substances may have an important role in the development of hypertension and atherosclerosis. Our objectives were to determine whether there are inborn differences in the proliferation patterns of VSMC obtained from spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY) by studying the effects of Ang-II and ET-1 on VSMC from those strains before the onset of hypertension, and to evaluate the roles of protein kinase C (PKC) and intracellular Ca 2+ in the mechanism of action of ET-1 and Ang-II. VSMC from aortas of young (1- to 2-week–old) SHR and WKY rats were grown as primary cultures in plates for 48 h. The cells were incubated with Ang-II (0.1 to 1000 nmol/L) or ET-1 (0.1 to 100 nmol/L). VSMC were also incubated in the presence of various concentrations of a PKC inhibitor, chelerythrine (0.1–10 nmol/L). Thymidine incorporation into DNA was measured as an indicator of DNA synthesis. Intracellular free Ca 2+ was determined by using FURA-2AM. ET-1 and Ang-II caused a marked dose-dependent enhancement of thymidine incorporation into DNA. The responses of VSMC from WKY and SHR to Ang-II and ET-1 were similar. In both strains, chelerythrine caused a dose-dependent suppression in the activity of ET-1 and Ang-II. However, VSMC from SHR incubated in the presence of ET-1 were more susceptible to the inhibitory effect of chelerythrine. Both Ang-II and ET-1 induced an increase of intracellular free Ca 2+. ET-1 induced a larger increase than Ang-II (190% and 100% greater than baseline free Ca 2+ levels, respectively), in spite of a lower concentration of ET-1 (ET-1 = 30 nmol/L; Ang-II = 100 nmol/L). Ang-II and ET-1 exerted a similar mitogenic effect on primary cultures of VSMC obtained from young SHR before the development of hypertension, compared with WKY. The mitogenic activity of Ang-II and ET-1 was accompanied by an increase of intracellular free Ca 2+. The effect of ET-1 upon intracellular Ca 2+ was stronger than that of Ang-II. VSMC cultures of SHR stimulated with ET-1 were more susceptible to PKC inhibition than those of WKY. The similarity of the effects of Ang- II and ET-1 on SHR and WKY does not exclude their role in the pathogenesis of hypertension and atherosclerosis, and further studies should be carried out to determine their role.

ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells.

Guanosine 3',5'-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32(Pi) to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [gamma-32P]ATP resulted in 32(Pi) incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.

Localization of 2 11beta-OH steroid dehydrogenase isoforms in aortic endothelial cells.

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) is expressed in vascular smooth muscle cells (VSMC) but has not been reported to be present in vascular endothelial cells. This enzyme assists in regulating the cellular concentration of active endogenous glucocorticoids (GCs). We have observed that endothelium intact rat aortic rings express message for both Type 1 and Type 2 11beta-HSD whereas primary cultures of VSMC express only mRNA for the Type I isoform. Since GCs diminish prostacyclin synthesis in endothelial cells, we hypothesized that 11beta-HSD is present in vascular endothelial cells. In primary cultures of rat aortic endothelial (RAE) cells, mRNA from both isoforms of 11beta-HSD could be detected by RT-PCR with higher levels of the Type 1 isoform. The oxo-reductase reaction "activating" 11-dehydro metabolites back to the parent steroid is the preferred enzyme direction (12:1 after a 120 minutes steroid incubation) in intact RAE cells. When RAE cells are grown in the presence of antisense oligonucleotides specific for Type 1 11beta-HSD, oxo-reductase activity is decreased by approximately 50% but the dehydrogenase reaction, which inactivates endogenous GCs and is characteristic of the Type 2 isoform, is unaffected. Thus endothelial cells appear to express both isoforms of 11beta-HSD; the Type 1 isoform dominates functioning in the oxo-reductase mode. Inhibition of the oxo-reductase reaction may lower the local concentrations of GC and indirectly allow for increased production of prostacyclin in endothelial cells.

Differentiation of Vascular Smooth Muscle Cells and the Regulation of Protein Kinase C-α

Dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) are important features of atherosclerosis. The molecular mechanisms are largely unclear; however, protein kinase C (PKC) is a key enzyme in the intracellular signaling pathways that mediate this process. We studied the activity and immunoreactivity of PKC-alpha in primary cultures of VSMCs from rat aortas under different conditions of growth and differentiation. PKC-alpha was determined under the following conditions: (1) during the growth phase and after confluence of cultured (passages 1 through 3) VSMCs, (2) before and after induction of differentiation in VSMCs by retinoic acid, and (3) in primary cultures of VSMCs from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats during early passages. PKC activity was measured by in vitro substrate phosphorylation. PKC-alpha immunoreactivity was assessed by Western blot using specific polyclonal antibodies and by immunostaining with confocal microscopy. Cell proliferation was measured by direct count. The cell phenotype was characterized by immunostaining and Western blot for alpha-actin and desmin. PKC-alpha expression and PKC activity during VSMC growth showed a decrease during rapid growth and an increase in confluent cells. This pattern was associated with the respective changes in cell differentiation. Retinoic acid induced an increase in PKC-alpha expression together with a more differentiated phenotype. Subcultured, rapidly growing VSMCs from SHR showed a decreased PKC-alpha expression compared with cells from WKY rats. To establish cause and effect, we next microinjected either PKC-alpha or inactivated material directly into dedifferentiated cells. We found that cells injected with active PKC-alpha expressed increased amounts of actin compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure-activity relationship between synthetic butylidenephthalide derivatives regarding the competence and progression of inhibition in primary cultures proliferation of mouse aorta smooth muscle cells.

The inhibitory effects of synthetic butylidenephthalide (BP) derivatives on 10% fetal bovine serum-stimulated proliferation were assayed by measuring the proliferative cell number at an interval of 12 h in primary cultures of mouse aorta smooth muscle cells (SMC). Their potencies for the anti-proliferation effect were in the order BP-42 (4,5-dihydroxy BP) > BP-92 (4,5-dihydroxy butylphthalide) > BP-97 (6,7-dihydroxy-3-(3-bromo-1-octenyl)-phthalide) > BP-82 (6,7-dihydroxy BP) > BP-86 (5,6-dihydroxy BP) > BP-87 (4,5,6-trihydroxy BP) > BP-85 (4,7-dihydroxy BP) > BP-84 (5,7-dihydroxy BP) > BP-4C3 (4-methoxy propylphthalide) > BP-7 (4-hydroxy BP) > BP-40 (4,5-dimethoxy butylphthalide) > BP-5C3 (4-hydroxy propylphthalide). We divided these anti-proliferative effects into anti-competence and anti-progression effects by using a convenient assay. BP-42 had the greatest potency in used phthalides for competence inhibition of the SMC proliferation. BP-92 had small potency for competence inhibition. BP-97 had greater potency for competence inhibition than BP-82. These results demonstrated that the anti-proliferative effect of BP-42 was greatest in used phthalides in primary cultures of vascular SMC. The 4,5-dihydroxy group and 3-butylidene or 3-(3-bromo-1-octenyl) group in these synthetic BP derivatives contributed to the anti-competence effect on SMC. BP-42 may become a prototype of an anti-atherosclerotic drug.

Transforming growth factor β decreases the rate of proliferation of rat vascular smooth muscle cells by extending the G2 phase of the cell cycle and delays the rise in cyclic AMP before entry into M phase

Transforming growth factor beta 1 (TGF-beta 1) decreased the rate of proliferation of rat aortic vascular smooth muscle cells (VSMCs) stimulated with serum showing a maximal effect at > 5 ng/ml (200 pM). However, it did not reduce the proportion of cells which passed through S phase (> 90%) and entry into S phase was delayed by less than 3 h. The proportion of cells passing through M phase (> 90%) was also unaffected, but entry into mitosis was delayed by approx. 24 h. This increase in cell cycle time was therefore due mainly to an increase in the G2 to mitotic metaphase period. Addition of TGF-beta 1 late in G1 or late in S phase failed to delay the onset of mitosis, but the presence of TGF-beta 1 between 0 and 12 h after the addition of serum to quiescent cells was sufficient to cause the maximal delay in mitosis of approx. 24 h. The role of cyclic AMP in the mechanism of the TGF-beta 1 effects on the cell cycle was examined. Entry into mitosis was preceded by a transient 2-fold increase in cyclic AMP concentration and TGF-beta 1 delayed both this increase in cyclic AMP and entry into mitosis to the same extent. Addition of forskolin or 8-(4-chlorophenylthio)-cyclic AMP to cells 30 h after stimulation with serum completely reversed the increase in duration of G2 in the presence of TGF-beta 1, suggesting that the rise in cyclic AMP levels which precedes mitosis might trigger entry of the VSMCs into M phase. Addition of forskolin late in S phase (26 h after stimulation with serum) advanced the entry of the cells into M phase and they divided prematurely. This effect was unaffected by the addition of cycloheximide with the forskolin; however, the effect of forskolin on cell division was completely inhibited when cycloheximide was added late in G1. TGF-beta 1 prevented the loss of smooth-muscle-specific myosin heavy chain (SM-MHC), which occurs in primary VSMC cultures in the presence or absence of serum, and the cells proliferated while maintaining a differentiated phenotype. However, TGF-beta 1 did not cause re-differentiation of subcultured VSMCs which contained very low amounts of SM-MHC and the effect of TGF-beta 1 in extending the G2 phase of the cell cycle is exerted independently of its effect on differentiation.

Heparin decreases the rate of proliferation of rat vascular smooth muscle cells by releasing transforming growth factor beta-like activity from serum.

Various heparins have been reported to inhibit the proliferation of vascular smooth muscle cells (VSMCs). The effects of eight chemically distinct heparins on the cell cycle and differentiation of primary and passaged cultures of rat aortic VSMCs have been characterised and the mechanism of heparin action investigated. VSMCs from adult rat aorta were prepared by enzyme dispersion and stimulated to enter the cell cycle with 10% serum in the presence or absence of heparin. Progressions through S phase and M phase were measured by [3H]-thymidine incorporation and cell counting respectively. Flow cytometry was used to confirm the effects of heparin on VSMC cell cycle progression. The effect of heparin on VSMC differentiation was investigated by analysing smooth muscle specific myosin heavy chain content of the cells after heparin treatment. Eight heparins at concentrations between 5 micrograms.ml-1 and 100 micrograms.ml-1 partially inhibited VSMC proliferation (27% to 76% 96 hours after addition of heparin), but did not affect the entry of the cells into S phase. Flow cytometry confirmed that VSMC populations in the presence of heparin contained significantly (p < 0.005) more cells in the G2/M phase of the cell cycle than control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of heparin were completely reversed by the presence of a neutralising antiserum to transforming growth factor beta (TGF beta) and heparin attached to agarose beads was as effective as free heparin as a growth inhibitor of VSMCs. Heparins of varying molecular weight and anticoagulant properties all partially inhibited VSMC proliferation predominantly by extending the G2/M phase of the cell cycle. Heparin also inhibited dedifferentiation of primary cultures of VSMCs. Heparin (< 100 micrograms.ml-1) acted extracellularly to release TGF beta from serum, which accounted for the effects of heparin on proliferation and differentiation.

Evidence for involvement of phospholipase C-gamma 2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells.

In order to examine the mechanisms underlying smooth-muscle cell proliferation, we investigated effect of platelet-derived growth factor (PDGF) dimers on proliferation of rabbit vascular smooth-muscle cells (VSMCs) and also involvement of phospholipase C (PLC) isoforms in the signal transduction. PDGF-BB and -AB, but not -AA, stimulated cell proliferation and intracellular production of inositol trisphosphate. Northern and Western analyses demonstrated that VSMCs mainly expressed PLC-gamma 2 and PLC-delta 1 among four PLC isoforms tested. A number of cellular proteins, including PLC-gamma 2, but not PLC-delta 1, were phosphorylated on a tyrosine residue by the stimulation of either PDGF-BB or -AB. These results suggest a functional association of PDGF receptor and PLC-gamma 2 that might be responsible for PDGF-dependent VSMC growth. In addition, the expression of PLC-gamma 2 was extremely low in the primary VSMC cultures and was induced during further cultivation of the primary cultures, indicating that an acquisition of PDGF-signal-transducing components, including PLC-gamma 2, may be an important step for proliferation of smooth-muscle cells.

Receptor-mediated mitogenic effect of thromboxane A 2 in vascular smooth muscle cells

The effects of thromboxane A 2 (TXA 2) on the proliferation of vascular smooth muscle cells (VSMC) were examined using primary cultures of VSMC from rat aorta. U46619, a stable TXA 2 mimetic, stimulated DNA synthesis of VSMC only in the presence of insulin. The effect was concentration-dependent with a half-maximal effect obtained at approximately 1 × 10 −8M. The mitogenic effect of U46619 was larger than that of endothelin, another mitogen derived from endothelium. Among several TXA 2/PGH 2 analogs, the proliferative activity was detected only in the agonists, and not in the antagonists or in the metabolite of TXA 2. A series of TXA 2/PHG 2 receptor antagonists completely suppressed the U46619-stimulated DNA synthesis as well as the [ 3H]SQ29,548 binding to the TXA 2/ PGH 2 receptors in VSMC. The rank order of binding affinities to the receptors among the respective antagonists correlated well with the potencies for suppression of the proliferative effects of U46619. The mitogenic effects of U46619 were also attenuated by the presence of calcium antagonists. U46619 caused activation of phospholipase C with the production of inositol trisphosphate, leading to increases in the intracellular free Ca 2+ concentration as measured with the fluorescent indicator fura-2. These results suggest that TXA 2 induces mitogenic effects on VSMC through binding to its specific receptors. This effect of TXA 2 on the proliferation of VSMC may be related to the development of atherosclerosis