Cultured Rabbit Vascular Smooth Muscle Research Articles

RNAi Targeting Embryonic Myosin Heavy Chain Isoform Inhibited Bound Thrombin-Induced Migration of Vascular Smooth Muscle Cells

To investigate the effect of bound thrombin, a complex of α-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell proliferation was measured by WST-1 reagent and migration was evaluated by counting migrated cells through pores of cell culture insert (8 μm size) after 48-hour treatment with bound thrombin (10 U/ml). To examine the role of an embryonic myosin heavy chain isoform (SMemb) in these effects by bound thrombin, the cells were subsequently treated for 48 h with an siRNA expression vector (ORF-2/pSilencer) directed against the open reading frame of SMemb mRNA. SMemb and plasminogen activator inhibitor-1 mRNA expressions were measured by Northern blot analysis. Bound thrombin significantly increased SMemb mRNA expression by 1.4 ± 0.01-fold and significantly increased plasminogen activator inhibitor-1 mRNA expression by 2.65 ± 0.69-fold (p < 0.01 vs. PBS treatment for each), which were abolished by treatment with ORF-2/pSilencer. Although bound thrombin had no effect on cell proliferation, bound thrombin significantly increased migration by 1.93 ± 0.20-fold (p < 0.05). ORF-2/pSilencer treatment significantly reduced the bound thrombin-stimulated migration activity by 1.28 ± 0.15-fold (p < 0.05). Thus, SMemb plays an important role in bound thrombin-induced cell migration activity of cultured vascular smooth muscle cells.

RNA interference targeting embryonic myosin heavy chain isoform inhibited mRNA expressions of phenotype markers in rabbit cultured vascular smooth muscle cells

To investigate whether the knockdown of SMemb gene expression induces phenotypic modulation of vascular smooth muscle (VSM) cells toward a contractile type, we constructed a siRNA targeting the 3' untranslated region (UTR) of SMemb gene (SMemb-siRNA). The SMemb-siRNA was introduced into cultured rabbit VSM cells for 48 h at 37 degrees C by the lipofection method. The mRNA expressions were estimated by comparative reverse transcription-polymerase chain reaction (RT-PCR). SMemb-siRNA significantly decreased the ratio of SMemb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in a dose-dependent manner (P < 0.01): 0 nM, 0.90 +/- 0.08; 100 nM, 0.43 +/- 0.07. Immunofluorescence and immunoblot analyses demonstrated that SMemb-siRNA markedly decreased SMemb protein expression to 56% +/- 7.8% (P < 0.01). Other MHC isoform (SM1 and SM2) mRNA expressions were not changed. The relative mRNA expressions of other phenotype markers (plasminogen activator inhibitor (PAI)-1 and beta-actin) were significantly decreased by SMemb-siRNA to 71% +/- 7.5% and 61% +/- 7.5%, respectively (P < 0.01). Expression of smooth muscle (SM) alpha-actin protein and cell proliferation was not changed by SMemb-siRNA. Thus, SMemb gene might be involved in the transcription of PAI-1 and beta-actin, but not involved in SM alpha-actin and cell proliferation in cultured VSM.

SMemb-specific siRNA expression vector inhibited bound thrombin-induced upregulation of SMemb gene in cultured rabbit vascular smooth muscle cells

SMemb-specific siRNA expression vector inhibited bound thrombin-induced upregulation of SMemb gene in cultured rabbit vascular smooth muscle cells

SMemb-specific siRNAs inhibited SMemb mRNA expression in cultured rabbit vascular smooth muscle cells

SMemb-specific siRNAs inhibited SMemb mRNA expression in cultured rabbit vascular smooth muscle cells

Argatroban, specific thrombin inhibitor, induced phenotype change of cultured rabbit vascular smooth muscle cells

To investigate whether argatroban ((2 R,4 R)-4-methyl-1-[ N 2-((RS)-3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)- l-arginyl]-2-piperidinecarboxylic acid hydrate, a selective thrombin inhibitor, exerts a direct action on phenotype conversion of vascular smooth muscle cells, cultured rabbit aortic vascular smooth muscle cells were employed. Myosin heavy chain isoforms (SM1, SM2, and SMemb) mRNA expressions were evaluated by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). After the cells were incubated in serum-free medium containing argatroban (10 and 50 μg/ml) and platelet-derived growth factor (PDGF)-BB (10 and 50 ng/ml) for 3 h, total RNA was extracted. In situ hybridization demonstrated that myosin heavy-chain isoform mRNAs were homogenously expressed in argatroban- and PDGF-BB-treated cells. RT-PCR revealed that SM1/SM2 mRNA expressions were not changed with argatroban, while SMemb mRNA expression was increased to 1.6-fold with a statistical significance ( P<0.05). Treatment with argatroban (10 and 50 μg/ml) at 24 h did not change SM1/SM2 mRNA expressions. Although SMemb mRNA expression was slightly increased, there was no statistical significance. Other phenotype markers including plasminogen activator inhibitor-1 (PAI-1) and β-actin mRNAs were also significantly increased by argatroban. In conclusion, argatroban can directly induce phenotype conversion of vascular smooth muscle cells with the resultant up-regulation of SMemb, PAI-1, and β-actin mRNAs.

Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis.

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.

Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model

The ability of resveratrol to inhibit vascular intimal thickening was tested in an experimental model in which endothelial denudation was performed in the normal rabbit iliac artery. Resveratrol (2∼4mg/kg/d) was administered intragastrically for 5 weeks beginning 1 week before denudation. At the higher concentration of resveratrol, the intimal hyperplasia of injured vascular wall was effectively inhibited; the intimal proliferation index also was significantly less than that in the untreated control group (0.28±0.07 vs 0.41±0.13, respectively, p<0.01); the relative luminal area increased from 0.38±0.06 in the untreated control group to 0.53±0.10 in the resveratrol treatment group (p<0.001); and the count of smooth muscle cells in the thickened intima was statistically significantly reduced in the high dose resveratrol treatment group than that in the untreated group (1115±510 vs 1796±963, respectively, p<0.05). Resveratrol added to the culture media of cultured rabbit vascular smooth muscle cells inhibited DNA synthesis in a dose-dependent manner. These results showing that resveratrol is capable of inhibiting intimal hyperplasia of injured artery raise the possibility that this polyphenol might have clinical potential in prevention and treatment of restenosis after angioplasty.

Advanced Glycation Endproducts Stimulate Mitogen-Activated Protein Kinase and Proliferation in Rabbit Vascular Smooth Muscle Cells

Advanced glycation end products of bovine serum albumin (AGEs-BSA) exhibited biphasic effects on the proliferation of cultured rabbit vascular smooth muscle cells (VSMCs) in terms of [3H]thymidine incorporation and cell number count; a stimulatory effect was observed at 1-10 μg/ml and an inhibitory effect at more than 20 μg/ml, while it inhibited [3H]thymidine incorporation even at 1-10 μg/ml in cultured bovine vascular endothelial cells (VECs). Transient activation of p42 mitogen-activated protein kinase (MAPK) with a peak at around 5 min and a subsequent sustained phase was induced by AGEs-BSA in VSMCs, but not in VECs. The dependence of MAPK activation on AGEs-BSA dose was correlated with that of VSMCs proliferation.

Antiproliferative effect of baicalein, a flavonoid from a Chinese herb, on vascular smooth muscle cell

The effects of baicalein, baicalin and wogonin, the flavonoids from Scutellaria baicalensis, on the proliferative responses of cultured rabbit vascular smooth muscle cells were studied. The proliferative response was determined from the uptake of tritiated thymidine. In rabbit vascular smooth muscle cells, all three flavonoids dose dependently inhibited the proliferative response induced by 5% fetal calf serum at the dose range of 10 −6 to 10 −4 M. Baicalin and wogonin were less effective than baicalein as inhibitors of the serum-induced smooth muscle cell proliferation, indicating that the three hydroxyl groups on positions 5, 6 and 7 seem to be necessary and sufficient for full inhibitory activity against the proliferative response of smooth muscle cells. Baicalein had a greater inhibitory effect on the proliferative response stimulated by platelet-derived growth factor than on serum-stimulated proliferation. Baicalein, a flavonoid with antiproliferative and lipoxygenase-inhibitory activities, may be useful as another template for the development of better drugs to prevent the pathological changes of atherosclerosis and restenosis.

Antiproliferative effect of esculetin on vascular smooth muscle cells: possible roles of signal transduction pathways

The effect of esculetin, a coumarin derivative with lipoxygenase inhibitor activity, on the proliferation response of cultured rabbit vascular smooth muscle cells was studied. Proliferation response was determined by the uptake of tritiated thymidine. Esculetin (10 −5−10 −4 M) dose dependently inhibited the enhanced proliferation stimulated by 5% fetal calf serum. The structure-activity relationship of esculetin and eight other coumarin derivatives indicates that two adjacent phenolic hydroxyl groups at the C-6 and C-7 positions in the coumarin skeleton are necessary for the potent antiproliferative effect. The antiproliferative effects of other lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid (ETYA) and ketoconazole, were comparable to the effect of esculetin. However, esculetin exhibited the greatest maximal suppression. The enhanced releases of 12-hydroxyeicosatetraenoic acid (12-HETE), prostaglandin E 2 and 6-keto-prostaglandin F 1α in the culture medium of smooth muscle cells stimulated by 5% fetal calf serum were significantly reduced by esculetin. Furthermore, the fetal calf serum-stimulated protein tyrosine kinase activity was reduced by esculetin (10 −5−10 −4 M) in a dose-dependent manner. In contrast, the protein kinase C activity stimulated by phorbol-12-myristale-13-acetate was not affected by esculetin (10 −6−10 −4 M). These results suggest that the antiproliferative effect of esculetin on vascular smooth muscle cells may be partly mediated through inhibition of protein tyrosine kinase and modulated by inhibition of lipoxygenase.

Regulation of vascular smooth muscle cell integrin expression by transforming growth factor beta1 and by platelet-derived growth factor-BB.

We have examined the ability of transforming growth factor-beta 1 (TGF-beta 1) and platelet-derived growth factor-BB (PDGF-BB) to regulate the expression of various integrins in cultured rabbit vascular smooth muscle cells (SMC). We found that expression of the alpha v beta 3 integrin complex was induced by both growth factors, although TGF-beta 1 appeared to be the more potent inducer. mRNA level of the beta 3 integrin subunit was undetectable in quiescent cells and enhanced by both growth factors, while the alpha v integrin subunit mRNA level did not change with growth factor addition. Therefore, appearance of the alpha v beta 3 integrin protein complex after growth factor stimulation was due to increased expression of the beta 3 integrin subunit mRNA. The TGF-beta 1 induced increase in beta 3 integrin mRNA was delayed, but did not require prior protein synthesis, since cycloheximide was unable to block the increase in beta 3 mRNA level. By contrast, PDGF-BB induced a more rapid increase in beta 3 integrin mRNA level that peaked by 6 h after growth factor addition and no detectable beta 3 integrin mRNA remained after 24 h. Interestingly, the PDGF-BB induced elevation of beta 3 integrin, although more rapid, was completely inhibited by cycloheximide. Expression of the alpha 5 integrin subunit in response to growth factors was very similar to beta 3. However, in contrast to beta 3 and alpha 5, neither TGF-beta 1 nor PDGF-BB were able to alter the expression of the beta 1 integrin subunit in vascular SMC. However, in TGF-beta 1 treated cells, there was a large increase in expression of a 190 kDa polypeptide that was associated with the beta 1 integrin subunit. This 190 kDa polypeptide was not detected in PDGF treated SMC or in TGF-beta 1 treated fibroblasts. The alpha 1 integrin subunit has a MW of approximately 190 kDa and is capable of complexing with beta 1. Analysis of the alpha 1 integrin subunit mRNA level indicated that it was indeed induced by TGF-beta 1, but not by PDGF-BB, suggesting that the 190 kDa polypeptide may be the alpha 1 integrin subunit. These results indicate that TGF-beta 1 and PDGF-BB are potent but distinct activators of integrin expression in vascular SMC.

Vitamin D 3 stimulates the production of prostacyclin by vascular smooth muscle cells

The effects of vitamin D 3 on the production of prostacyclin (PGI 2) by cultured rabbit vascular smooth muscle cells (VSMCs) were investigated. PGI 2 synthesis by VSMCs was significantly increased in the presence of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) and 1α hydroxyvitamin D 3 (1α(OH)D 3) at 48 hours [1,25(OH) 2D 3 > 1 α (OH)D 3]. Physiological concentration of 1,25(OH) 2D 3 (10 −10 M) significantly increased the synthesis of PGI 2. Further, we observed that treatment with 1,25(OH) 2D 3 significantly induced the activity of cyclooxygenase without changing the activity of phospholipase A 2. These findings suggest that the mechanism of action of 1,25(OH) 2D 3 on the synthesis of PGI 2 is mediated by the cyclooxygenase pathway. It seems possible that vitamin D 3 is a vasoactive agent and may play a protective role in the development of atherosclerosis.

Serotonin plays a major role in serum-induced phospholipase C-mediated hydrolysis of phosphoinositides and DNA synthesis in vascular smooth muscle cells

Whole blood serum (WBS) rapidly induced the phospholipase C-mediated hydrolysis of phosphoinositides and subsequently stimulated DNA synthesis in cultured rabbit vascular smooth muscle cells (VSMCs). Ketanserin, a serotonin (S2) receptor antagonist, markedly inhibited the WBS-induced phospholipase C reaction and DNA synthesis. Serotonin by itself had a weak mitogenic activity for VSMCs, but this vasoconstrictor markedly stimulated the platelet-derived growth factor- and epidermal growth factor-induced DNA synthesis. The stimulatory effect of serotonin on the growth factor-induced DNA synthesis was inhibited by ketanserin. The amount of serotonin contained in WBS was sufficient to induce the phospholipase C reaction and stimulate the growth factor-induced DNA synthesis. These results indicate that serotonin plays a major role in the WBS-induced phospholipase C-mediated hydrolysis of phosphoinositides and DNA synthesis in rabbit VSMCs and suggest that serotonin may act as an important growth regulator for VSMCs in addition to acting as a vasoconstrictor.

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 μM and was inhibited by the specific angiotensin II receptor antagonist, [Sar 1, Ile 8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c- fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c- fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vaso-constrictor.

Mass analysis of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells. Comparison of stimulation by angiotensin II and endothelin.

We compared the effects of angiotensin II and endothelin on mass levels of 1,2-diacylglycerol, and endogenous activator of protein kinase C, in cultured rabbit vascular smooth muscle cells with the effects of these vasoconstrictors on contractile responses of rabbit aortic strips. At a high concentration (1 microM), both angiotensin II and endothelin induced a biphasic formation of 1,2-diacylglycerol with an early transient phase and a late sustained phase. At this high concentration, angiotensin II caused a transient contraction followed by a gradual relaxation, whereas endothelin caused a slowly developing and sustained contraction. At a low concentration (EC50 for the early phase of 1,2-diacylglycerol formation), angiotensin II also induced a biphasic formation of 1,2-diacylglycerol and caused a transient contraction, but endothelin induced a monophasic formation of 1,2-diacyglycerol with only an early peak. Despite a rapid decrease of 1,2-diacylglycerol, endothelin at this low concentration still caused a sustained contraction. At both the high and low concentrations, the 1,2-diacylglycerol level was sustained higher for angiotensin II, whereas the tension during the late tonic phase of contraction was greater for endothelin. These results suggest that the unique persistent nature of endothelin-induced contraction is not attributed simply to the stimulatory effect of this peptide on the 1,2-diacylglycerol/protein kinase C pathway.

Endothelin-induced biphasic formation of 1,2-diacylglycerol in cultured rabbit vascular smooth muscle cells — Mass analysis with a radioenzymatic assay

Mass analysis of 1,2-diacylglycerol (DG) with a radioenzymatic assay revealed that endothelin induced a biphasic formation of DG with an early transient phase peaking at 30 sec and a late sustained phase peaking at 5 min in cultured rabbit vascular smooth muscle cells (VSMCs). The amounts of DG after the 30-sec and 5-min incubation with endothelin were 0.74 ± 0.08 (mean ± SE) nmol and 0.87 ± 0.10 nmol/100 nmol of lipid phosphorus, representing 2.6- and 3.1-fold increases of the resting level, respectively. The EC 50 values of endothelin for the early and late phases of DG formation were about 1 nM and 40 nM, respectively. In the { 3H}inositol-labeled VSMCs, endothelin induced a rapid transient formation of inositol tris- and bisphophates which peaked at 30 sec and a sustained formation of inositol monophosphate which peaked at 5 min. The EC 50 values for the formation of these inositol phosphates were the same and about 1 nM. These results suggest that the early transient phase of DG is derived from the hydrolysis of polyphosphoinositides, while a large part of the late sustained phase of DG is from the reaction(s) other than the hydrolysis of phosphoinositides but its sources remain to be clarified.

Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells

Incubation of the [ 3H]inositol-labeled cultured rabbit vascular smooth muscle cells (VSMCs) with either endothelin or angiotensin II caused a rapid formation of inositol mono-, bis- and trisphosphates (IP 1, IP 2 and IP 3, respectively). Time courses of the endothelin- and angiotensin II-induced formation of these inositol phosphates were similar. The maximal levels of IP 1, IP 2 and IP 3 formation induced by endothelin were about 50%, 25% and 40%, respectively, of those induced by angiotensin II. The doses of endothelin necessary for the half maximal and maximal extents of the formation of IP 1 were about 1 nM and 100 nM, respectively. Protein kinase C-activating 12- O -tetradecanoylphorbol-13-acetate (TPA) inhibited the endothelin-induced formation of IP 1 with the half maximal extent of inhibition seen at 3 nM. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate, but not by 4α-phorbol-12,13-didecanoate, known to be inactive for this enzyme. These results indicate that endothelin causes the phospholipase C-mediated hydrolysis of phosphoinositides, though to a lesser extent than angiotensin II, in cultured VSMCs and suggest that protein kinase C modulates the signaling mechanism of endothelin to the phospholipase C.

Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization

Cultured endothelial cells release a potent vasoconstrictor peptide, endothelin. Cumulative addition of synthetic endothelin to isolated rabbit aortic rings elicited a concentration-dependent increase in contractile tension which was endothelium-independent. In cultured rabbit vascular smooth muscle cells loaded with the fluorescent dye fura 2, endothelin induced a concentration-dependent increase in [Ca2+]i over the range of 0.01 to 100 nM. Moreover, in the absence of extracellular Ca2+, endothelin could still induce an increase in [Ca2+]i. In addition, endothelin stimulated 45Ca2+ efflux from preloaded vascular smooth muscle cells in the presence and absence of extracellular Ca2+, as well as stimulating 45Ca2+ influx in a concentration-dependent manner. Measurement of inositol phosphates in [3H]-myoinositol-labelled vascular vascular trisphosphate. Unlabelled endothelin inhibited (125I)-endothelin binding to cultured rabbit vascular smooth muscle cells in a concentration-dependent manner. Binding was not inhibited by other vasoactive hormones or calcium channel ligands, suggesting cell surface receptors specific for endothelin. We conclude that one of the initial membrane events in the action of endothelin is to induce phospholipase C-stimulated PIP2 hydrolysis and that this signalling mechanism is initiated by endothelin/receptor interaction at the plasma membrane.

Platelet-derived growth factor (PDGF)-induced phospholipase C-mediated hydrolysis of phosphoinositides in vascular smooth muscle cells — different sensitivity of PDGF- and angiotensin II-induced phospholipase C reactions to protein kinase C-activating phorbol esters

In cultured rabbit vascular smooth muscle cells (VSMC), platelet-derived growth factor (PDGF), a potent mitogen for VSMC, induced the dose- and time-dependent formation of inositol mono-, bis- and trisphosphates (IP1, IP2 and IP3, respectively). The doses of PDGF necessary for these reactions were similar to those for DNA synthesis. The maximal level of IP1 was comparable to, and those of IP2 and IP3 were about half of those induced by angiotensin II, a potent vasoconstrictor. However, the time courses of the PDGF-induced reactions were slower than those of the angiotensin II-induced ones. Moreover, protein kinase C-activating phorbol esters inhibited the angiotensin II-induced reactions, but did not the PDGF-induced ones. These results indicate that PDGF induces the phospholipase C reactions in VSMC but suggest that the signaling mechanism of PDGF to the phospholipase C is different from that of angiotensin II.