A7r5 Vascular Smooth Muscle Cells Research Articles

Treatment of β-thujaplicin counteracts di(2-ethylhexyl)phthalate (DEHP)-exposed vascular smooth muscle activation, inflammation and atherosclerosis progression

The initiation of atherosclerosis involves up-regulation of molecules such as E-selectin, VCAM-1, and ICAM-1. The progression of atherosclerosis is linked to proliferation and migration of vascular smooth muscle cell via MMP-2 and MMP-9 activities. However, the etiology of atherosclerosis concerning plasticizers is unknown. We evaluated β-thujaplicin in preventing the development of atherosclerosis in a model induced by pro-inflammatory cytokines. Moreover, we established a new atherosclerosis model in vascular smooth muscle cells (VSMC) exposed to a common contact plasticizer, di(2-ethylhexyl)phthalate (DEHP). SEVC4-10 endothelial cells were treated with 50% RAW conditioned medium and A7r5 VSMC was treated with the plasticizer, with/without β-thujaplicin (4 or 12 μM). Production of E-selectin, ICAM-1, and VCAM-1 in SEVC4-10 cells as well as MMP-2/MMP-9 (both expression and activity) in VSMC were monitored. Results showed that the conditioned medium induced E-selectin and ICAM were significantly prevented by β-thujaplicin. However, inhibition on the production of VCAM by β-thujaplicin was only seen in a concentration of 12 μM. Both concentrations of β-thujaplicin also significantly prevented DEHP-induced MMP-2 and MMP-9 expression and activities. Evidence uncovers that β-thujaplicin has additional factors in amelioration of atherosclerosis and corroborates that β-thujaplicin is a strong candidate in preventing the initiation and progression of atherosclerosis.

Estrogen modulates vascular smooth muscle cell function through downregulation of SIRT1.

BackgroundThere are sex differences in the incidence and severity of cardiovascular disease. Although an estrogen-mediated vasculoprotective effect is widely accepted, clinical trial results have been conflicting and the detailed mechanisms are still unclear. Sirtuin 1 (SIRT1), a class III histone deacetylase, may protect against vascular aging and atherosclerosis; however, the effects of estrogen on SIRT1 expression and vascular smooth muscle cell (VSMC) behavior remain unknown.Materials and MethodsWe ovariectomized (OVX) female, wild-type, C57BL/6J mice, which were randomized into non-estrogen- and estrogen-supplemented groups. We also treated A7r5 VSMCs with 17-β-estradiol and resveratrol, a SIRT1 activator, in vitro, and measured the expression of SIRT1 and apoptotic markers, as well as proliferation, viability, and migration.ResultsAortic tissue from OVX mice exhibited marked VSMC hyperplasia and upregulation of SIRT1, which was reversed by 17-β-estradiol supplementation, as assessed by western blotting and immunohistochemical staining. In vitro, 17-β-estradiol downregulated SIRT1 expression in a dose- and time-dependent manner, increased apoptosis, and reduced proliferation, viability, and migration. Resveratrol reversed these effects through the activation of SIRT1. Estrogen appeared to mediate its effects through the Akt and ERK pathways.ConclusionsEstrogen may regulate cardiovascular health via the expression of SIRT1, possibly through the AKT and ERK signaling pathways.

TGFβ1 induces stress fiber formation through upregulation of TRPC6 in vascular smooth muscle cells

Aberrant transforming growth factor β1 (TGFβ1) signaling plays a crucial role in the pathogenesis of vascular fibrosis. On the other hand, deregulated transient receptor potential canonical 6 (TRPC6) channel expression shows impaired vascular physiology and wound healing. However, it has little been known about the functional association between TGFβ1 and TRPC6 in vascular smooth muscle cells (VSMCs). In this study, we analyzed the microarray data obtained from TGFβ1-treated A7r5 VSMCs. We found that TGFβ1 specifically elevates the expression level of TRPC6 mainly through Smad-dependent canonical pathway. The siRNA against TRPC6 abolished TGFβ1-induced molecular and cellular phenotype changes, including myosin light chain phosphorylation, actin stress fiber formation, and cell migration. These results demonstrate that TRPC6 is an important component of TGFβ1 signaling pathway in VSMCs. Therefore, our findings provide a basis for future investigation aimed at developing novel therapeutic strategies for treatment of vascular fibrosis.

TNAP stimulates vascular smooth muscle cell trans-differentiation into chondrocytes through calcium deposition and BMP-2 activation: Possible implication in atherosclerotic plaque stability

Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PPi), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PPi, and PPi provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications.

Effects of cell seeding density on real-time monitoring of anti-proliferative effects of transient gene silencing.

BackgroundReal-time cellular analysis systems enable impedance-based label-free and dynamic monitoring of various cellular events such as proliferation. In this study, we describe the effects of initial cell seeding density on the anti-proliferative effects of transient gene silencing monitored via real-time cellular analysis. We monitored the real-time changes in proliferation of Huh7 hepatocellular carcinoma and A7r5 vascular smooth muscle cells with different initial seeding densities following transient receptor potential canonical 1 (TRPC1) silencing using xCELLigence system. Huh7 and A7r5 cells were seeded on E-plate 96 at 10,000, 5000, 1250 and 5000, 2500 cells well−1, respectively, following silencing vector transfection. The inhibitory effects of transient silencing on cell proliferation monitored every 30 min for 72 h.ResultsTRPC1 silencing did not inhibit the proliferation rates of Huh7 cells at 10,000 cells well−1 seeding density. However, a significant anti-proliferative effect was observed at 1250 cells well−1 density at each time point throughout 72 h. Furthermore, significant inhibitory effects on A7r5 proliferation were observed at both 5000 and 2500 cells well−1 for 72 h.ConclusionsData suggest that the effects of transient silencing on cell proliferation differ depending on the initial cell seeding density. While high seeding densities mask the significant changes in proliferation, the inhibitory effects of silencing become apparent at lower seeding densities as the entry into log phase is delayed. Using the optimal initial seeding density is crucial when studying the effects of transient gene silencing. In addition, the results suggest that TRPC1 may contribute to proliferation and phenotypic switching of vascular smooth muscle cells.

Interaction of Gq‐ and Gs‐coupled Pathways in the Regulation of Ca2+ Spiking and Activity of Kv7.5 Potassium Channels in A7r5 Vascular Smooth Muscle Cells

The vasoconstrictor hormone arginine vasopressin (AVP), at picomolar concentrations, activates Gq‐coupled Va1 receptors and suppresses the activity of vascular Kv7.5 potassium (K+) channels in a protein kinase C (PKC) ‐dependent manner. The PKC‐dependent inhibition of Kv7.5 K+ current in vascular smooth muscle cells causes membrane depolarization and activation of L‐type voltage dependent Ca2+ channels, leading to an increase in cytosolic Ca2+ concentration, contraction of the vascular smooth muscle cells, and vasoconstriction. Conversely, we found that activity of vascular Kv7.5 channels is enhanced upon activation of β‐adrenergic Gs‐ coupled receptors, in a protein kinase A (PKA)‐ dependent manner. In the present study we investigated physiological consequences of the sequential activation of the PKC‐ and PKA‐dependent pathways. The amplitude of endogenous Kv7.5 currents in A7r5 vascular smooth muscle cells was measured using whole‐cell perforated‐patch voltage clamp, and the frequency of Ca2+ spiking was measured using the Ca2+ indicator fura‐2. We found that the Kv7.5 current amplitude was reduced by 100 pM AVP by 79 ± 5%, but addition of the β‐adrenergic agonist isoproterenol (1 μM) restored the amplitude to the original level. Applying the treatments in the opposite order, isoproterenol (1 μM) enhanced the Kv7.5 current by 6.1 ± 1.5 ‐fold and this much larger current was suppressed by 40% by addition of 100 pM AVP. In contrast, Ca2+ spiking, induced by 50 pM AVP and dependent on activation of L‐type voltage dependent Ca2+ channels, was not effected by treatment with 1 μM isoproterenol, added either before or after application of AVP. The discrepancy between the effectiveness of the isoproterenol to restore Kv7.5 current amplitude and its inability to reduce the frequency of AVP‐induced Ca2+ spiking in A7r5 cells might be partially explained by an isoproterenol‐induced increase (54 ± 8%) in the amplitude of L‐type Ca2+ currents.Support or Funding InformationLoyola University Chicago

Endogenous Sulfur Dioxide Inhibits Vascular Calcification in Association with the TGF-β/Smad Signaling Pathway.

The study was designed to investigate whether endogenous sulfur dioxide (SO2) plays a role in vascular calcification (VC) in rats and its possible mechanisms. In vivo medial vascular calcification was induced in rats by vitamin D3 and nicotine for four weeks. In vitro calcification of cultured A7r5 vascular smooth muscle cells (VSMCs) was induced by calcifying media containing 5 mmol/L CaCl2. Aortic smooth muscle (SM) α-actin, runt-related transcription factor 2 (Runx2), transforming growth factor-β (TGF-β) and Smad expression was measured. VC rats showed dispersed calcified nodules among the elastic fibers in calcified aorta with increased aortic calcium content and alkaline phosphatase (ALP) activity. SM α-actin was markedly decreased, but the osteochondrogenic marker Runx2 concomitantly increased and TGF-β/Smad signaling was activated, in association with the downregulated SO2/aspartate aminotransferase (AAT) pathway. However, SO2 supplementation successfully ameliorated vascular calcification, and increased SM α-actin expression, but inhibited Runx2 and TGF-β/Smad expression. In calcified A7r5 VSMCs, the endogenous SO2/AAT pathway was significantly downregulated. SO2 treatment reduced the calcium deposits, calcium content, ALP activity and Runx2 expression and downregulated the TGF-β/Smad pathway in A7r5 cells but increased SM α-actin expression. In brief, SO2 significantly ameliorated vascular calcification in association with downregulation of the TGF-β/Smad pathway.

Receptor for Advanced Glycation End-Products Signaling Interferes with the Vascular Smooth Muscle Cell Contractile Phenotype and Function.

Increased blood glucose concentrations promote reactions between glucose and proteins to form advanced glycation end-products (AGE). Circulating AGE in the blood plasma can activate the receptor for advanced end-products (RAGE), which is present on both endothelial and vascular smooth muscle cells (VSMC). RAGE exhibits a complex signaling that involves small G-proteins and mitogen activated protein kinases (MAPK), which lead to increased nuclear factor kappa B (NF-κB) activity. While RAGE signaling has been previously addressed in endothelial cells, little is known regarding its impact on the function of VSMC. Therefore, we hypothesized that RAGE signaling leads to alterations in the mechanical and functional properties of VSMC, which could contribute to complications associated with diabetes. We demonstrated that RAGE is expressed and functional in the A7r5 VSMC model, and its activation by AGE significantly increased NF-κB activity, which is known to interfere with the contractile phenotype of VSMC. The protein levels of the contraction-related transcription factor myocardin were also decreased by RAGE activation with a concomitant decrease in the mRNA and protein levels of transgelin (SM-22α), a regulator of VSMC contraction. Interestingly, we demonstrated that RAGE activation increased the overall cell rigidity, an effect that can be related to an increase in myosin activity. Finally, although RAGE stimulation amplified calcium signaling and slightly myosin activity in VSMC challenged with vasopressin, their contractile capacity was negatively affected. Overall, RAGE activation in VSMC could represent a keystone in the development of vascular diseases associated with diabetes by interfering with the contractile phenotype of VSMC through the modification of their mechanical and functional properties.

Mulberry water extracts inhibit atherosclerosis through suppression of the integrin-β₃/focal adhesion kinase complex and downregulation of nuclear factor κB signaling in vivo and in vitro.

Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in vivo and in vitro through stimulating apoptosis of vascular smooth muscle cells (VSMCs). Histological analysis was performed on atherosclerotic lesions from high-cholesterol diet (HCD)-fed rabbits after treatment with 0.5-1% MWEs for 10 weeks. Immunohistochemistry showed that the expressions of SMA, Ras, and matrix metalloproteinase-2 in the VSMCs were dose-dependently inhibited after MWE treatment. The antimigratory effects of MWEs on A7r5 VSMCs were assessed by western blot analysis of migration-related proteins, visualization of F-actin cytoskeleton, and reverse transcription polymerase chain reaction. The results showed that MWEs inhibited VSMC migration through reducing interactions of the integrin-β3/focal adhesion kinase complex, alterations of the cytoskeleton, and downregulation of glycogen synthase kinase 3β/nuclear factor κB signaling. Taken together, MWEs inhibited HCD-induced rabbit atherogenesis through blocking VSMC migration via reducing interactions of integrin-β3 and focal adhesion kinase and downregulating migration-related proteins.

Effects of passage number on proliferation and store-operated calcium entry in A7r5 vascular smooth muscle cells

IntroductionEmbryonic rat aortic smooth muscle cells, A7r5, have been used extensively as an in vitro vascular smooth muscle cell model. They are usually provided at 11th passage by supplier and generally used before 25th passage. However, the exact passage number (P#) used is not reported in general. MethodsIn this study, A7r5 cells (P#<18 and P#>23) were used in quantitative gene (5-HT2A and SERCA2b) expression as well as in functional analyses including measurements of real-time changes in cell proliferation and in 5-HT- and CPA-induced intracellular Ca2+ levels. ResultsCPA-induced SR Ca2+ release and store-operated Ca2+ entry significantly increased (p<.05) while cell proliferation decreased in P#>23 cells (p<.01). Furthermore, 5-HT-induced Ca2+ elevations and 5-HT2A mRNA levels did not change whereas SERCA2b mRNA levels and CPA-induced [Ca2+]i levels were significantly elevated in P#>23 cells (p<.05, p<.01, respectively). DiscussionChanges in SERCA2b expression and SOCE may contribute to suppression of cell proliferation during A7r5 subculturing. Therefore, passage numbers and subculturing procedure should be reported and taken into account during expressional and functional analyses for an accurate comparison of published data.

Traumatic brain injury in vivo and in vitro contributes to cerebral vascular dysfunction through impaired gap junction communication between vascular smooth muscle cells.

Gap junctions (GJs) contribute to cerebral vasodilation, vasoconstriction, and, perhaps, to vascular compensatory mechanisms, such as autoregulation. To explore the effects of traumatic brain injury (TBI) on vascular GJ communication, we assessed GJ coupling in A7r5 vascular smooth muscle (VSM) cells subjected to rapid stretch injury (RSI) in vitro and VSM in middle cerebral arteries (MCAs) harvested from rats subjected to fluid percussion TBI in vivo. Intercellular communication was evaluated by measuring fluorescence recovery after photobleaching (FRAP). In VSM cells in vitro, FRAP increased significantly (p<0.05 vs. sham RSI) after mild RSI, but decreased significantly (p<0.05 vs. sham RSI) after moderate or severe RSI. FRAP decreased significantly (p<0.05 vs. sham RSI) 30 min and 2 h, but increased significantly (p<0.05 vs. sham RSI) 24 h after RSI. In MCAs harvested from rats 30 min after moderate TBI in vivo, FRAP was reduced significantly (p<0.05), compared to MCAs from rats after sham TBI. In VSM cells in vitro, pretreatment with the peroxynitrite (ONOO(-)) scavenger, 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron[III], prevented RSI-induced reductions in FRAP. In isolated MCAs from rats treated with the ONOO(-) scavenger, penicillamine, GJ coupling was not impaired by fluid percussion TBI. In addition, penicillamine treatment improved vasodilatory responses to reduced intravascular pressure in MCAs harvested from rats subjected to moderate fluid percussion TBI. These results indicate that TBI reduced GJ coupling in VSM cells in vitro and in vivo through mechanisms related to generation of the potent oxidant, ONOO(-).

Schisandrin B suppresses TGFβ1-induced stress fiber formation by inhibiting myosin light chain phosphorylation

Ethnopharmacological relevanceSchisandra chinensis fruit extract (SCE) has been used as a traditional oriental medicine for treating vascular diseases. However, the pharmacologic effects and mechanisms of SCE on vascular fibrosis are still largely unknown. Transforming growth factor β1 (TGFβ1)-mediated cellular changes are closely associated with the pathogenesis of vascular fibrotic diseases. Particularly, TGFβ1 induces actin stress fiber formation that is a crucial mechanism underlying vascular smooth muscle cell (VSMC) migration in response to vascular injury. In this study, we investigated the effect of SCE and its active ingredients on TGFβ1-induced stress fiber assembly in A7r5 VSMCs. Materials and methodsTo investigate pharmacological actions of SCE and its ingredients on TGFβ1-treated VSMCs, we have employed molecular and cell biological technologies, such as confocal microscopy, fluorescence resonance energy transfer, western blotting, and radiometric enzyme analyses. ResultsWe found that SCE inhibited TGFβ1-induced stress fiber formation and cell migration. Schisandrin B (SchB) showed the most prominent effect among the active ingredients of SCE tested. SchB reduced TGFβ1-mediated phosphorylation of myosin light chain, and this effect was independent of RhoA/Rho-associated kinase pathway. Fluorescence resonance energy transfer and radiometric enzyme assays confirmed that SchB inhibited myosin light chain kinase activity. We also showed that SchB decreased TGFβ1-mediated induction of α-smooth muscle actin by inhibiting Smad signaling. ConclusionsThe present study demonstrates that SCE and its active ingredient SchB suppressed TGFβ1-induced stress fiber formation at the molecular level. Therefore, our findings may help future investigations to develop multi-targeted therapeutic strategies that attenuate VSMC migration and vascular fibrosis.

Pharmacological Properties of Homomeric Kv7.4, Kv7.5 and Heteromeric Kv7.4/7.5 Channels: Effects of ICA069673 and Ml213

Kv7.4 and Kv7.5 voltage-activated potassium channels are proposed to contribute to the maintenance of resting membrane voltage in smooth muscle cells. We had previously provided evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels in vascular smooth muscle cells. Among new activators of Kv7 channels, ICA069673 and ML213 were reported to discriminate between Kv7.4 and Kv7.5 isoforms. Here we compare effects of ICA069673 and ML213 on homomeric human Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that in contradiction to previously published reports, ML213 does not discriminate among Kv7.4 and Kv7.5 isoforms. ML213 significantly increased the maximum conductance of Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels. In each case, the increase in maximal conductance was accompanied by a negative shift of the activation curves by 23 mV, 47 mV and 32 mV; EC50: 1.5 μM, 3.4 μM and 3.2 μM for Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels, respectively. Current deactivation rates decreased in a voltage dependent manner in the presence of the ML213 (10 μM) for all three channel combinations. In contrast to ML213, ICA069673 was unable to enhance currents through Kv7.5 homomeric channels. However, both Kv7.4 and heteromeric Kv7.4/7.5 channels responded to ICA069672 with a negative shift of activation curves by 57 mV (EC50 = 21 μM) and 66 mV (EC50 = 50 μM) respectively without increase in maximal conductance. Current deactivation rates decreased in the presence of ICA069673 with an opposite voltage dependence in comparison with deactivation rates in the presence of ML213. Based on these results, neither ML213 nor ICA069673 are able to fully discriminate between homomeric Kv7.4, Kv7.5 and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells.

Association of matrix Gla protein gene functional polymorphisms with loss of bone mineral density and progression of aortic calcification

Two matrix Gla protein (MGP) polymorphisms were associated with progression of aortic calcification and femoral neck bone loss in men. All these findings were also functionally corroborated in two vascular and bone in vitro systems indicating that MGP genetic variations can be partly responsible of higher risk of bone loss and vascular calcification. MGP plays an important role in bone and vascular mineralization as confirmed by MGP-deficient murine model. We therefore aimed to find a genetic association among -138T>C, -7G>A, and Thr83Ala MGP single-nucleotide polymorphisms (SNPs), bone loss, and progression of aortic calcification in a randomly selected general population of 296 individuals who participated in the European Vertebral Osteoporosis Study. To evaluate the rate of change in bone mineral density (BMD) and the progression of aortic calcification, dual X-ray absorptiometry and lateral spine X-rays were performed at baseline and after 4 years of follow-up. Genotyping for the three polymorphisms was carried out using polymerase chain reaction and restriction fragment length analysis. In addition, functional studies of MGP-7G>A and Thr83Ala SNPs were performed on transiently transfected osteoblast-like UMR-106 and vascular smooth muscle A7r5 cells. The proportion of men who had lost BMD in the femoral neck was higher among homozygous -7AA and 83Ala-Ala (p = 0.039 and p = 0.009, respectively), and also featured a higher risk of progression of aortic calcifications (OR = 5.6, 95% CI = 1.2-27.8 and OR = 6.8, 95% CI = 1.4-32.3, respectively). No effect was observed in women. The MGP-7A allele produced a reduction in luciferase activity compared to MGP-7G: 47% less in vascular cells and 34% less in bone cells (p = 0.001 and 0.012, respectively). In vascular cells under calcifying conditions, the MGP 83Thr allele showed a slightly higher, although not significant, inhibition than the MGP 83 Ala allele in calcium content suggesting functional differences between both variants. These results suggest that MGP genetic variations could predict a higher risk of bone loss and progression of vascular calcification in men.

Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility

Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca(2+) signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca(2+) signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca(2+) release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca(2+) influx through L-type Ca(2+) channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca(2+) release, resulting in increased VSM contraction in response to agonist stimulation.

Mulberry 1-Deoxynojirimycin Pleiotropically Inhibits Glucose-Stimulated Vascular Smooth Muscle Cell Migration by Activation of AMPK/RhoB and Down-regulation of FAK

Mulberry 1-deoxynojirimycin (DNJ), an inhibitor of α-glucosidase, has been reported to help prevent diabetes mellitus and suppress lipid accumulation. The aim of this study was to determine whether mulberry DNJ has pleiotropic effects on the development of atherosclerosis. The mechanisms by which mulberry DNJ might inhibit migration of A7r5 vascular smooth muscle cells (VSMCs) under hyperglycemic conditions mimicking diabetes were investigated. The antimigratory effects of DNJ on VSMCs were assessed by Western blot analysis of migration-related proteins and by electric cell-substrate impedance sensing (ECIS) and visualization of F-actin cytoskeleton. Two pathways of DNJ-mediated inhibition of VSMC migration were identified. The first involved AMPK activation to inhibit fatty acid synthase (FASN) and Akt activity and then RhoB activation to inhibit nuclear factor-κB (NF-κB) and matrix metalloproteinase-2 (MMP) activity. The second involved inhibition of focal adhesion kinase (FAK), Ras, and RhoA activity leading to inhibition of F-actin activity.

The G Protein-Coupled Estrogen Receptor 1 (GPER1/GPR30) Agonist G-1 Regulates Vascular Smooth Muscle Cell Ca2+ Handling

The G protein-coupled estrogen receptor GPER1/GPR30 is implicated in blood pressure regulation but the mechanisms are not identified. Here, we hypothesize that GPER1 controls blood pressure by regulating vascular smooth muscle cell Ca²⁺ handling. Treatment with the GPER1 agonist G-1 (in the µ<smlcap>M</smlcap> concentration range) acutely reduced spontaneous and synchronous Ca²⁺ spike activity in A7r5 vascular smooth muscle cells expressing mRNA for GPER1. Furthermore, G-1 (1 µ<smlcap>M</smlcap>) attenuated the thromboxane A₂ analogue U46619-stimulated Ca²⁺ spike activity but had no effect on the U46619-induced increase in the basal level of Ca²⁺. The voltage-sensitive L-type Ca²⁺ channel blocker nifedipine (100 n<smlcap>M</smlcap>) reduced Ca²⁺ spike activity similar to G-1. Pharmacological, but not physiological, concentrations of the estrogen 17β-estradiol reduced Ca²⁺ spike activity. The GPER1 antagonist G-15 blocked G-1-induced downregulation of Ca²⁺ spike activity, supporting a GPER1-dependent mechanism. G-1 (1 µ<smlcap>M</smlcap>) and nifedipine (100 n<smlcap>M</smlcap>) attenuated the 30-m<smlcap>M</smlcap> KCl-evoked rise in intracellular Ca²⁺ concentration, suggesting that G-1 blocks inflow of Ca²⁺ via voltage-sensitive Ca²⁺ channels. In conclusion, we demonstrate that the GPER1 agonist G-1 regulates vascular smooth muscle cell Ca²⁺ handling by lowering Ca²⁺ spike activity, suggesting a role for this mechanism in GPER1-mediated control of blood pressure.

Zn deficiency promotes calcification in vascular smooth muscle cells independent of ALP action and in part by Pit1 up‐regulation

Zn deficiency more recently has been related to the development of cardiovascular disease, which involves aberrant calcification. ALP is a key regulator in osteogenic calcification to increase local inorganic phosphate (Pi) concentration. Even though vascular smooth muscle cells (VSMCs) generally do not express alkaline phosphatase (ALP), its presence has been reported on calcified VSMCs. However, under Zn deficiency, our initial findings showed undetectable level of ALP activity in VSMCs at calcifying conditions. We therefore hypothesized that Zn deficiency‐induced calcification may not be initiated by ALP in VSMCs. To test this hypothesis, we cultured rat aortic A7r5 VSMCs using β‐glycerophosphate (β‐GP, as ALP substrate, 0–15 mM) or Na phosphate (NaP, as non‐ALP substrate, 0–7 mM) as source of Pi under Zn deficiency (1 μM) for up to 15 days to induce VSMC calcification. Under Zn deficiency, P and Ca accumulation (calcification) increased with increasing NaP concentration (3–7 mM) but not with β‐GP treatment in which requires ALP activity to generate Pi. ALP activity was neither affected by β‐GP nor by NaP concentrations. In addition, Zn status did not affect ALP activity in VSMCs. However, Pit1 as a sodium‐dependent phosphate transporter into the cells, was upregulated under zinc deficiency. Our findings illustrate that Zn deficiency‐induced calcification in VSMCs is not initiated by ALP action but in part by Pit1 upregulation. Supported by the fund of National Research Foundation of Korea (No. NRF‐2008–220‐F00013).

Long-term zinc deprivation accelerates rat vascular smooth muscle cell proliferation involving the down-regulation of JNK1/2 expression in MAPK signaling

BackgroundThe accelerated proliferation of vascular smooth muscle cells (VSMCs) is a contributor for atherosclerosis by thickening the vascular wall. Since zinc modulation of VSMC proliferation has not been clarified, this study investigated whether zinc affects VSMC proliferation. Methods and resultsBoth a rat aorta origin vascular smooth muscle cell line (A7r5 VSMCs) and primary VSMCs which were collected from rat aorta (pVSMCs) were cultured with zinc (0–50 μM Zn) for short- (≤12 d) and long-term (28 d) periods under normal non-calcifying (0 or 1 mM P) or calcifying (>2 mM P) P conditions. Mouse vascular endothelial cells (MS I cells) were also cultured (under 0–50 μM Zn and 10 mM P for 20 d) to compare with VSMC cultures. While during short-term culture of VSMCs, zinc deprivation decreased cell proliferation in a zinc-concentration manner both under non-calcifying and calcifying conditions in A7r5 and pVSMCs (P < 0.05), during long-term cultures (28 d), A7r5 VSMC proliferation was inversely related to medium zinc concentration under normal physiological P conditions (regression coefficient r2 = −0.563, P = 0.012). The anti-cell proliferative effect of zinc supplementation (>50 μM) was VSMC-specific. Long-term (35 d), low zinc treatment down-regulated JNK expression and activation, while not affecting ERK1/2 MAPK signaling in A7r5 VSMCs. ConclusionThe results showed that chronic zinc deprivation accelerated VSMC proliferation, perhaps due to down-regulation of MAPK-JNK signaling, and that the anti-cell proliferative role of zinc is VSMC-specific. The findings suggested that zinc may have anti-VSMC proliferative properties in atherosclerosis.

Optogenetic Control of Vascular Tone with High Temporal Resolution

Blood pressure is directly controlled by the contractile state of vascular smooth muscle cells (VSMCs) which is regulated by membrane potential-dependent Ca2+-entry through Ca2+-channels and Ca2+-release from intracellular stores. Modulation of membrane potential in VSMCs in intact vessels can be performed by electrical field stimulation or elevation of extracellular potassium concentration, but both methods are not cell-specific and have low spatio-temporal resolution. To overcome these limitations, we have used the light-gated cation channel channelrhodopsin2 for optogenetical control of VSMCs membrane potential. ChR2 was expressed in a7r5 VSMCs and patch clamp experiments showed light-induced inward currents. Brief light pulses with increasing light intensities led to graded depolarization and eventually initiation of action potentials. Prolonged illumination resulted in constant depolarization and elevated intracellular Ca2+-concentrations. Optogenetic stimulation of VSMCs in intact vessels was performed using transgenic mice that express ChR2 (Nat Methods. 2010;7:897-900) in VSMCs but not endothelial cells. Isometric force was analyzed in aortic rings with a wire-myograph. Illumination with blue light (475nm, 2.7mW/mm2) reliably induced contractions with very fast on- (< 800ms) and off-kinetics (< 2.5s) and contraction could be maintained up to 10 min. The amplitude of contraction could be graded by variation of light intensity and reached a maximum comparable to stimulation with noradrenalin (10μM). Control vessels from wild-type mice or transgenic mice expressing EGFP did not show light-induced contractions. Taken together ChR2 enables light-induced contraction of VSMCs with high temporal resolution and represents a powerful technique to analyze membrane-potential dependent mechanisms in VSMCs. Optogenetic stimulation allows cell-specific and localized stimulation down to single cell level and therefore can be used in the future to investigate the interplay of endothelial cells and VSMCs as well as the electrical coupling between cells within intact vessels.