Primary Rat Aortic Smooth Muscle Research Articles

Hypoglycemic effects of dracorhodin and dragon blood crude extract from Daemonorops draco

BackgroundDragon blood is a red fruit resin from the palm tree Daemonorops draco and is a herbal ingredient used in the traditional Chinese medicine, “Jinchuang Ointment,” which is used to treat non-healing diabetic wounds. According to the Taiwan Herbal Pharmacopeia, the dracorhodin content in dragon blood should exceed 1.0%.ResultsOur findings indicate that dracorhodin and dragon blood crude extracts can stimulate glucose uptake in mouse muscle cells (C2C12) and primary rat aortic smooth muscle cells (RSMC). Dracorhodin is not the only active compound in dragon blood crude extracts from D. draco. Next, we orally administered crude dragon blood extracts to male B6 mice. The experimental group displayed a decreasing trend in fasting blood glucose levels from the second to tenth week. In summary, crude extracts of dragon blood from D. draco demonstrated in vivo hypoglycemic effects in B6 male mice.ConclusionsWe provide a scientific basis “Jinchuang ointment” in treating non-healing wounds in patients with diabetes.

Potential Actions of Baicalein for Preventing Vascular Calcification of Smooth Muscle Cells In Vitro and In Vivo.

Vascular calcification (VC) is associated with cardiovascular disease. Baicalein, a natural flavonoid extract of Scutellaria baicalensis rhizome has several biological properties which may inhibit VC. We investigated whether baicalein suppresses Runt-related transcription factor 2 (Runx2) and bone morphogenetic protein 2 (BMP-2) and upregulates smooth muscle 22-alpha (SM22-α) and alpha-smooth muscle actin (α-SMA). In an in vitro experiment, primary rat aortic vascular smooth muscle cells (VSMCs) were pretreated with 0.1, 1, and 5 μM baicalein, followed by β-glycerophosphate (β-GP) to induce calcification. In an in vivo experiment, VC was generated by vitamin D3 plus nicotine (VDN) administration to male Sprague Dawley (SD) rats randomly assigned into a control group, a VC group, a VC group pretreated with baicalein, and a baicalein alone group. Each group comprised 10 rats. Left ventricular (LV) morphology, function and performance were assessed by echocardiography. Calcium content was measured by Alizarin red S staining and alkaline phosphatase (ALP) activity assays. Apoptotic VSMCs were detected by flow cytometry. Protein levels and superoxide changes were evaluated using Western blotting and immunofluorescence assays respectively. Plasma malondialdehyde (MDA) was assayed. Baicalein pretreatment significantly reduced calcium content in calcified VSMCs (p < 0.001) as well as in VC rat aortic smooth muscle (p < 0.001). Additionally, ALP activity was decreased in calcified VSMCs and VC rat aortic smooth muscle (p < 0.001). Apoptosis was significantly attenuated by 1 μM baicalein pretreatment in calcified VSMCs. Runx2 and BMP-2 expressions were downregulated by the baicalein in calcified VSMCs. Baicalein pretreatment increased typical VSMCs markers SM22-α and α-SMA in calcified VSMCs. Baicalein pretreatment was associated with adverse changes in LV morphometry. Markers of oxidative stress declined, and endogenous antioxidants increased in VC rats pretreated with baicalein. Baicalein mitigates VC through the inhibition of Runx2/BMP-2 signaling pathways, enhancement of vascular contractile phenotype and oxidative stress reduction. However, our study is of basic experimental design; more advanced investigations to identify other molecular regulators of VC and their mechanisms of action is required.

Icariside II Attenuates Vascular Remodeling Via Wnt7b/CCND1 Axis.

Angioplasty often fails due to the abnormal proliferation of vascular smooth muscle cells (VSMCs). Success rates of angioplasty may increase following the administration of an agent that effectively ameliorates aberrant vascular remodeling. Icariside II (ICS-II) is a natural flavonol glycoside extract from the Chinese herbal medicine Epimedii that possesses several medicinal qualities that are beneficial in humans. Nevertheless, the role of ICS-II in addressing aberrant vascular remodeling have yet to be clarified. The current investigation studies the molecular effects of ICS-Ⅱ on balloon-inflicted neointimal hyperplasia in rats in vivo and on platelet-derived growth factor-induced vascular proliferation in primary rat aortic smooth muscle cells (VSMCs) in vitro. ICS-II was found to be as effective as rapamycin, the positive control used in this study. ICS-II inhibited neointimal formation in injured rat carotid arteries and notably reduced the expression of Wnt7b. ICS-Ⅱ significantly counteracted platelet-derived growth factor-induced VSMCs proliferation. Cell cycle analysis showed that ICS-II triggered cell cycle arrest during the G1/S transition. Western blot analysis further indicated that this cell cycle arrest was likely through Wnt7b suppression that led to CCND1 inhibition. In conclusion, our findings demonstrate that ICS-II possesses significant antiproliferative qualities that counteracts aberrant vascular neointimal hyperplasia. This phenomenon most likely occurs due to the suppression of the Wnt7b/CCND1 axis.

Galangin inhibits neointima formation induced by vascular injury via regulating the PI3K/AKT/mTOR pathway.

Aims: The proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in the pathological process of neointima formation after vascular injury. Galangin, an extract of the ginger plant galangal, is involved in numerous biological activities, including inhibiting the proliferation and migration of tumor cells, but its effect on VSMCs is unknown. This study focused on the role and mechanism of galangin in the neointima formation induced by vascular injury. Methods and results: In this study, we found that galangin restrained the PDGF-BB-induced proliferation, migration and phenotypic switching of VSMCs in a concentration-dependent manner. In vivo, we established a model of carotid artery balloon injury in rats, followed by intragastric administration of galangin (40 mg kg-1 day-1 or 80 mg kg-1 day-1) for 14 or 28 consecutive days. Then, the degree of neointima hyperplasia was evaluated by H&E staining, and the level of relevant protein expression was assessed by immunofluorescence and western blotting. In vitro, we isolated and grew primary rat aortic smooth muscle cells, which were treated with PDGF-BB and different doses of galangin, and then CCK-8 assay, wound healing assay, transwell assay, western blotting and immunofluorescence assays were performed. We found that galangin significantly inhibited PDGF-BB-induced proliferation, migration, and phenotypic switching of VSMCs and promoted autophagy in VSMCs in vitro, and galangin significantly inhibited neointimal hyperplasia after the common carotid artery balloon injury in rats. In terms of mechanisms, galangin inhibited the PI3K/AKT/mTOR pathway, thereby suppressing VSMC's switch from a contractile to a synthetic phenotype, inhibiting VSMC proliferation, migration and phenotypic switching and upregulating the Beclin1 protein expression levels and the ratio of LC3BII/I, promoting VSMC autophagy, and thereby inhibiting neointimal hyperplasia after vascular injury. Conclusion: Our study suggests that galangin inhibits neointimal hyperplasia after vascular injury by inhibiting smooth muscle cell proliferation, migration and phenotypic switching and by promoting autophagy, and that galangin may be a promising drug for the prevention and treatment of vascular restenosis after PCI.

Kahweol, a Diterpenoid Molecule, Inhibits CTGF-Dependent Synthetic Phenotype Switching and Migration in Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) phenotype switching from contractile to synthetic is essential for proliferation and migration in vascular pathophysiology. Connective tissue growth factor (CTGF) is a matricellular protein involved in cell adhesion, migration, and proliferation. Kahweol, a diterpene molecule in arabica coffee beans, has been reported to have anti-inflammatory, antiproliferative, and apoptotic effects in many cells. However, in VSMCs, the effects of kahweol on CTGF activities have not been investigated. Thus, in this study, the effects and associated mechanisms of kahweol in CTGF-dependent phenotype switching and migration in VSMCs were examined. Experiments were performed on primary rat aortic smooth muscle cells and a rat VSMC line, A7r5. Western blot analysis was used to determine the protein levels. The mRNA levels of synthetic markers were measured by qRT-PCR. Migration of VSMCs was evaluated by wound healing and transwell assays. Kahweol reduced the angiotensin II (Ang II)-induced CTGF expression. Further, kahweol inhibited expressions of synthetic phenotype markers of VSMC. The kahweol-reduced synthetic marker protein levels were reversed by the administration of rCTGF. However, expressions of contractile phenotype markers of VSMC were not affected. Kahweol suppressed Ang II-stimulated VSMC migration. Moreover, kahweol downregulated Ang II-induced p-FAK, p-Erk, and Yes-associated protein (YAP) protein expressions. Taken together, in Ang II-stimulated VSMCs, kahweol inhibited CTGF-dependent synthetic phenotype switching and migration, with focal adhesion kinase (FAK), Erk, and YAP involved in the underlying mechanisms of the kahweol effects. These results suggest that kahweol has a potential as a therapeutic agent to inhibit CTGF, which is a molecular target in sclerogenic vascular disease.

[Impact and related mechanisms of glucose fluctuations on aortic fibrosis in type 1 diabetic rats].

Objective: To investigate the impact and related mechanisms of glucose fluctuations on aortic fibrosis in rats with type 1 diabetes mellitus. Methods: After injection of streptozotocin (STZ), male Sprague Dawley (SD) (8-12 weeks) rats (n=24) were randomly divided into three groups in accordance with the random number table: controlled STZ-induced diabetes (C-STZ) group (n=8); uncontrolled STZ-induced diabetes (U-STZ) group (n=8); STZ-induced diabetes with glucose fluctuations (STZ-GF) group (n=8). After three weeks, rats were sacrificed and aorta was obtained, aortic fibrosis was detected by Masson trichrome staining. The expression of collagen type 1 (collagen Ⅰ) was tested by immunofluorescence. The expression of runt-related transcription factor 2 (Runx2) was tested by immunohistochemistry. The mRNA levels of collagen Ⅰ and Runx2 were detected by quantitative real-time PCR (qRT-PCR). The protein expressions of collagen Ⅰ, Runx2 and nuclear factor (NF)-κB were determined by Western blot. Primary rat aortic smooth muscle cells (VSMCs) were cultured in three conditions: normal glucose (NG), high glucose (HG) and glucose fluctuations (GF). Cells in GF group were incubated for 72 hours with glucose alternating between 5.5 and 25 mmol/L every 12 hours. TPCA-1, the inhibitor of NF-κB, the expression of collagenⅠin different groups of cells was tested by immunofluorescence. The protein expressions of collagen Ⅰ, Runx2 and NF-κB were also determined by Western blot. Results: (1) The quantitative ratios of the area of fibrosis in the C-STZ group, U-STZ group, STZ-GF group were (8.42±0.10)%, (21.30±0.74)% and (44.39±1.09)% (P<0.05), respectively. The means of integral optical density (IOD) of collagenⅠ in the three groups were 11.92±0.88, 50.04±3.56 and 77.52±2.69, respectively (P<0.05). The mRNA levels of collagenⅠ in the three groups were 1.00±0.10, 2.02±0.28 and 2.83±0.33, respectively (P<0.05). The protein expressions of collagenⅠ in the three groups were 1.05±0.03, 2.06±0.32 and 4.93±0.25, respectively (P<0.05). (2) The average IOD of Runx2 in the three groups were 150.00±7.35, 204.84±2.32 and 391.48±7.13, respectively (P<0.05). The mRNA levels of Runx2 in the three groups were 1.02±0.02, 1.27±0.04 and 2.18±0.12, respectively (P<0.05). The protein expressions of Runx2 in the three groups were 1.03±0.01, 2.34±0.36 and 4.52±0.75, respectively (P<0.05). (3) The protein expressions of NF-κB in the three groups were 1.02±0.01, 1.96±0.13 and 2.64±0.21, respectively (P<0.05). (4) In vitro, application of inhibitor of NF-κB reversed glucose fluctuations-induced upregulation of protein levels of Col Ⅰ and Runx2 (P<0.05). Conclusion: Glucose fluctuations could aggravate aortic fibrosis through activating Runx2 via NF-κB signaling pathways.

Angiotensin II-Regulated Autophagy Is Required for Vascular Smooth Muscle Cell Hypertrophy

Hypertension is a disease associated to increased plasma levels of angiotensin II (Ang II). Ang II can regulate proliferation, migration, ROS production and hypertrophy of vascular smooth muscle cells (VSMCs). However, the mechanisms by which Ang II can affect VSMCs remain to be fully elucidated. In this context, autophagy, a process involved in self-digestion of proteins and organelles, has been described to regulate vascular remodeling. Therefore, we sought to investigate if Ang II regulates VSMC hypertrophy through an autophagy-dependent mechanism. To test this, we stimulated A7r5 cell line and primary rat aortic smooth muscle cells with Ang II 100 nM and measured autophagic markers at 24 h by Western blot. Autophagosomes were quantified by visualizing fluorescently labeled LC3 using confocal microscopy. The results showed that treatment with Ang II increases Beclin-1, Vps34, Atg-12–Atg5, Atg4 and Atg7 protein levels, Beclin-1 phosphorylation, as well as the number of autophagic vesicles, suggesting that this peptide induces autophagy by activating phagophore initiation and elongation. These findings were confirmed by the assessment of autophagic flux by co-administering Ang II together with chloroquine (30 μM). Pharmacological antagonism of the angiotensin type 1 receptor (AT1R) with losartan and RhoA/Rho Kinase inhibition prevented Ang II-induced autophagy. Moreover, Ang II-induced A7r5 hypertrophy, evaluated by α-SMA expression and cell size, was prevented upon autophagy inhibition. Taking together, our results suggest that the induction of autophagy by an AT1R/RhoA/Rho Kinase-dependent mechanism contributes to Ang II-induced hypertrophy in VSMC.

Tannic Acid Downregulates Angiotensin Type 1 Receptor in Aortic Smooth Muscle Cells Through EGFR Dependent Phosphoinositide 3‐kinase Pathway

Tannic acid (TA), a hydrolysable plant polyphenol commonly found in food and beverages, possesses several cardioprotective effects through its anti‐inflammatory and anti‐oxidant activities. In this study, we aimed to investigate the role of TA on the angiotensin type 1 receptor (AT1R) a major hypertension target and its associated signaling pathways in primary rat aortic smooth muscle cells (ASMCs). In the presence of TA (10 μg/ml for 20 h), cells exhibited down‐regulation of AT1R specific [3H] angiotensin II (AngII) binding compared to untreated control (3628.32±289.42 vs. 7244.66 ± 40.26 DPM/mg protein, P &lt; 0.001). TA mediated AT1R receptor downregulation in a dose dependent (0–40 μg/ml) and a time‐dependent (0–24 hours) manner, and was reversible. The reduction in AngII binding in TA‐treated cells was due to decreased receptor density rather than a change in receptor affinity as demonstrated by competition binding assay (Kd 1.302±0.152nM for TA treated vs. Kd 1.391±0.048 nM for untreated). Consistent with downregulation of AngII binding, AT1R mRNA was inhibited upon TA treatment (49.63% compared to control). TA mediated AT1R downregulation was associated with phosphorylation of extracellular signal regulated kinases (ERKs) p42/44. Pretreatment with MEK inhibitor PD98059 (20 mM) blocked TA induced ERKs p42/44 phosphorylation and AT1R downregulation. Moreover, AG1478 (5μm), an EGFR specific inhibitor, blocked TA mediated ERKs activation and down‐regulation of AT1R. Under similar conditions, treating cells with EGF (0.5 mg/ml) alone mediated ERKs activation and AT1R downregulation independent of TA (EGF; 3788.04±206.51 vs. Control; 6549.22±115.48 DPM/mg protein, P &lt; 0.001). LY294002 (10 mM), an inhibitor of phosphoinositide 3‐kinase (PI3K), inhibited TA and EGF mediated EGFR induced ERKs phosphorylation and reversed AT1R downregulation suggesting that TA mediated AT1R downregulation is through EGFR induced PI3K dependent ERK p42/44 pathway. Consistent with the receptor downregulation, quantitative calcium analysis revealed significant inhibition of AngII induced intracellular calcium release in TA treated cells, which was reversed when cells were pretreated with either MEK or EGFR inhibitor. Collectively, our study demonstrates for the first time that TA induced EGFR, PI3K and ERK p42/44 signaling axis is essential for downregulation of AT1R expression in ASMCs. Furthermore, the study highlights the significance of TA rich diet for the prevention of hypertension and associated cardiovascular disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Magnetoelastic galfenol as a stent material for wirelessly controlled degradation rates.

The gold standard of care for coronary artery disease, a leading cause of death for in the world, is balloon angioplasty in conjunction with stent deployment. However, implantation injuries and long-term presence of foreign material often promotes significant luminal tissue growth, leading to a narrowing of the artery and severely restricted blood flow. A promising method to mitigate this process is the use of biodegradable metallic stents, but thus far they have either degraded too slowly (iron) or disappeared prematurely (magnesium). The present work investigates the use of a unique type of magnetic material, galfenol (iron-gallium), for postoperative wireless control of stent degradation rates. Due to its magnetoelastic property, galfenol experiences longitudinal micron-level elongations when exposed to applied magnetic fields, allowing generation of a microstirring effect that affect its degradation behavior. In vitro indirect cytotoxicity tests on primary rat aortic smooth muscle cells indicated that galfenol byproducts must be concentrated approximately seven times from collected 60 day degradation medium to cause ∼15% of death from all cells. Surface and cross-sectional characterization of the material indicate that galfenol (Fe80 Ga20 ) degradation rates (∼0.55% per month) are insufficient for stenting applications. While this material may not be ideal for comprising the entire stent, there is potential for use in combination with other materials. Furthermore, the ability to control degradation rates postimplantation opens new possibilities for biodegradable stents; additional magnetoelastic materials should be investigated for use in stenting applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 232-241, 2019.

2-Methoxyestradiol causes matrix metalloproteinase 9-mediated transactivation of epidermal growth factor receptor and angiotensin type 1 receptor downregulation in rat aortic smooth muscle cells.

Studies have demonstrated the therapeutic potential of estrogen metabolite 2-methoxyestradiol (2ME2) in several cardiovascular disorders, including hypertension. However, the exact mechanism(s) remains unknown. In this study, primary rat aortic smooth muscle cells (RASMCs) were exposed to 2ME2, and angiotensin type 1 receptor (AT1R) expression, function, and associated signaling pathways were evaluated. In RASMCs, 2ME2 downregulated AT1R expression in a concentration- and time-dependent manner, which was correlated with reduced mRNA expression. The 2ME2 effect was through G protein-coupled receptor 30 (GPR30) that inhibits second messenger cAMP. Moreover, 2ME2 exposure phosphorylated ERK1/2 that was sensitive to MEK inhibitor PD98059. Selective epidermal growth factor receptor (EGFR) inhibitor AG1478 blocked 2ME2-induced EGFR transactivation and attenuated subsequent phosphorylation of ERK1/2 preventing AT1R downregulation. The transactivation was dependent on 2ME2-induced release of matrix metalloproteinase 9 (MMP9) and epidermal growth factor demonstrated by ELISA. Furthermore, transfection with small interfering (si) RNA targeting MMP9 impeded ERK1/2 activation and AT1R downregulation in response to 2ME2 and G1 stimulation. Interestingly, under similar conditions, stimulation of GPR30 with the selective agonist G1 elicited similar signaling pathways and downregulated the AT1R expression that was reversed by GPR30 antagonist G15. Furthermore, 2ME2 and G1 inhibited angiotensin II (ANG II) induced Ca2+ release, a response consistent with AT1R downregulation. Collectively, our study demonstrates for the first time that 2ME2 binding to GPR30 induces MMP9 specific transactivation of EGFR that mediates ERK1/2-dependent downregulation of AT1R in RASMCs. The study provides critical insights into the newly discovered role and signaling pathways of 2ME2 in the regulation of AT1R in vascular cells and its potential to be developed as a therapeutic agent that ameliorates hypertension.

Wedelolactone, a plant coumarin, prevents vascular smooth muscle cell proliferation and injury-induced neointimal hyperplasia through Akt and AMPK signaling

Wedelolactone (WDL) is a natural compound derived from Chinese herbal medicine Eclipta prostrate L, and has been reported to exhibit various effects potentially beneficial for human health. However, the possible preventive effects of WDL toward vascular remodeling and mechanisms involved have not been investigated to date. In this study, we investigated the effects of WDL on proliferation induced by platelet-derived growth factor (PDGF) in primary rat aortic smooth muscle cells (VSMCs) and on neointimal hyperplasia resulted from balloon injury in rats. WDL exhibited strong inhibitory effects against PDGF-induced VSMC proliferation. Cell cycle analysis revealed that WDL induced G0/G1 arrest and prevented cell cycle from entering S phase. Immunoblot analysis suggested that the cell cycle arrest induced by WDL was through Akt suppression and adenosine 5′-monophosphate-activated protein kinase (AMPK) activation, with a subsequent cyclin-dependent kinase inhibitor p21 induction and cyclin D1 inhibition. We also observed that WDL notably reduced neointima-to-media area ratio of balloon-injured rat common carotid arteries (CCAs) in comparison with those untreated balloon-injured CCAs. The regulation of WDL on protein expressions of Akt, AMPK and cyclin D1 in vivo were also consistent with that in vitro. Taken together, our results suggest WDL exhibits potential preventive effects toward vascular remodeling and neointimal hyperplasia through the reduction of VSMC proliferation via inhibition of Akt and activation of AMPK.

S1P in HDL promotes interaction between SR-BI and S1PR1 and activates S1PR1-mediated biological functions: calcium flux and S1PR1 internalization

HDL normally transports about 50-70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.

Iron Oxide Nanoparticles Stimulates Extra-Cellular Matrix Production in Cellular Spheroids.

Nanotechnologies have been integrated into drug delivery, and non-invasive imaging applications, into nanostructured scaffolds for the manipulation of cells. The objective of this work was to determine how the physico-chemical properties of magnetic nanoparticles (MNPs) and their spatial distribution into cellular spheroids stimulated cells to produce an extracellular matrix (ECM). The MNP concentration (0.03 mg/mL, 0.1 mg/mL and 0.3 mg/mL), type (magnetoferritin), shape (nanorod—85 nm × 425 nm) and incorporation method were studied to determine each of their effects on the specific stimulation of four ECM proteins (collagen I, collagen IV, elastin and fibronectin) in primary rat aortic smooth muscle cell. Results demonstrated that as MNP concentration increased there was up to a 6.32-fold increase in collagen production over no MNP samples. Semi-quantitative Immunohistochemistry (IHC) results demonstrated that MNP type had the greatest influence on elastin production with a 56.28% positive area stain compared to controls and MNP shape favored elastin stimulation with a 50.19% positive area stain. Finally, there are no adverse effects of MNPs on cellular contractile ability. This study provides insight on the stimulation of ECM production in cells and tissues, which is important because it plays a critical role in regulating cellular functions.

86 - Are S-Nitrosothiols Predominantly Stable End-Effectors of Protein Function or Transient Intermediates Leading to Disulfide Formation?

S-nitrosation is considered to be a ubiquitous, stable post-translational modification that directly targets and modulates a vast number of proteins. This widespread role appears incompatible with the inherent lability of the nitroso bond and its propensity to rapidly react with thiols to generate disulfide bonds. We present evidence that S-nitrosothiols primarily exist as transient intermediates leading to disulfides. Exposure of primary rat aortic smooth muscle cells (SMCs) to the nitrosating reagent S-nitrosocysteine (CysNO) generated robust and widespread protein S-nitrosation. However, CysNO also induced disulfides in multiple candidate proteins which preceded a significant increase in widespread protein S-nitrosation. Quantitative analysis of SMCs exposed to CysNO demonstrated that disulfides, not S-nitrosothiols, were the dominant modification that accumulated. S-glutathiolation and S-cysteinylation were significantly increased after exposure of SMCs to biotinylated forms of CysNO or S-nitrosoglutathione respectively. 10 μM CysNO depleted non-protein thiols, predominantly reduced glutathione, 7-fold as measured by the Ellman's assay. Furthermore, this intervention also reduced the activity of NAPDH-dependent enzymes (MTS assay) 2-fold, which together indicates CysNO compromises the cells reducing system (p

Abstract 378: Cytochrome b5 Reductase 3 Regulates Vascular Smooth Muscle Phenotypic Switching

Contractile vascular smooth muscle cells (VSMC) play a key role in the regulation of arterial blood vessel tone and cardiovascular health. However, in many vascular diseases, VSMCs undergo a phenotypic switch from a contractile state to a synthetic phenotype, where loss of the contractile markers myosin heavy chain 11 (Myh11), smooth muscle alpha actin (ACTA2) and transgelin (SM22) are observed and proliferation is increased. Recent evidence from our lab demonstrates that cytochrome b5 reductase 3 (Cyb5R3) regulates the redox state of soluble guanylate cyclase to control cGMP levels in VSMCs. Because cGMP modulates protein kinase G activity, a critical kinase that maintains VSMCs in a contractile state, we tested the hypothesis that Cyb5R3 is critical for maintenance of the contractile phenotype. To test this hypothesis, we transduced primary rat aortic smooth muscle cells with non-targeting (NT) or Cyb5R3 shRNA followed by serum starvation for 24, 48, and 72 hours to induce phenotypic switching. After each time point, mRNA measurements of Cyb5R3, Myh11, ACTA2, and SM22 were conducted using RT-PCR. In NT shRNA transduced VSMCs, we observed a significant increase in Cyb5R3, Myh11, ACTA2, and SM22 mRNA, but not in Cyb5R3 knockdown VSMCs. Next, we conducted proliferation studies by serum starving NT shRNA and Cyb5R3 shRNA treated VSMCs for 24 hours followed by stimulation of platelet growth factor BB (PDGF-BB, 40 ng/mL). After 24 hours of PDGF-BB treatment, Cyb5R3 deficient cells showed augmented proliferation compared to control cells measured by 3 H-thymidine incorporation. Together, our data suggest that Cyb5R3 is essential for VSMC phenotypic switching and proliferation, which may unravel a new therapeutic target for treating individuals with cardiovascular disease.

Zinc Prevents Abdominal Aortic Aneurysm Formation by Induction of A20-Mediated Suppression of NF-κB Pathway.

Chronic inflammation and degradation of elastin are the main processes in the development of abdominal aortic aneurysm (AAA). Recent studies show that zinc has an anti-inflammatory effect. Based on these, zinc may render effective therapy for the treatment of the AAA. Currently, we want to investigate the effects of zinc on AAA progression and its related molecular mechanism. Rat AAA models were induced by periaortic application of CaCl2. AAA rats were treated by daily intraperitoneal injection of ZnSO4 or vehicle alone. The aorta segments were collected at 4 weeks after surgery. The primary rat aortic vascular smooth muscle cells (VSMCs) were stimulated with TNF-α alone or with ZnSO4 for 3 weeks. The results showed that zinc supplementation significantly suppressed the CaCl2-induced expansion of the abdominal aortic diameter, as well as a preservation of medial elastin fibers in the aortas. Zinc supplementation also obviously attenuated infiltration of the macrophages and lymphocytes in the aortas. In addition, zinc reduced MMP-2 and MMP-9 production in the aortas. Most importantly, zinc treatment significantly induced A20 expression, along with inhibition of the NF-κB canonical signaling pathway in vitro in VSMCs and in vivo in rat AAA. This study demonstrated, for the first time, that zinc supplementation could prevent the development of rat experimental AAA by induction of A20-mediated inhibition of the NF-κB canonical signaling pathway.

Fully automated software for quantitative measurements of mitochondrial morphology

Mitochondria undergo dynamic changes in morphology in order to adapt to changes in nutrient and oxygen availability, communicate with the nucleus, and modulate intracellular calcium dynamics. Many recent papers have been published assessing mitochondrial morphology endpoints. Although these studies have yielded valuable insights, contemporary assessment of mitochondrial morphology is typically subjective and qualitative, precluding direct comparison of outcomes between different studies and likely missing many subtle effects. In this paper, we describe a novel software technique for measuring the average length, average width, spatial density, and intracellular localization of mitochondria from a fluorescent microscope image. This method was applied to distinguish baseline characteristics of Human Umbilical Vein Endothelial Cells (HUVECs), primary Goto-Kakizaki rat aortic smooth muscle cells (GK SMCs), primary Wistar rat aortic smooth muscle cells (Wistar SMCs), and SH-SY5Ys (human neuroblastoma cell line). Consistent with direct observation, our algorithms found SH-SY5Ys to have the greatest mitochondrial density, while HUVECs were found to have the longest mitochondria. Mitochondrial morphology responses to temperature, nutrient, and oxidative stressors were characterized to test algorithm performance. Large morphology changes recorded by the software agreed with direct observation, and subtle but consistent morphology changes were found that would not otherwise have been detected. Endpoints were consistent between experimental repetitions (R=0.93 for length, R=0.93 for width, R=0.89 for spatial density, and R=0.74 for localization), and maintained reasonable agreement even when compared to images taken with compromised microscope resolution or in an alternate imaging plane. These results indicate that the automated software described herein allows quantitative and objective characterization of mitochondrial morphology from fluorescent microscope images.

BET Bromodomain Blockade Mitigates Intimal Hyperplasia in Rat Carotid Arteries

BackgroundIntimal hyperplasia is a common cause of many vasculopathies. There has been a recent surge of interest in the bromo and extra-terminal (BET) epigenetic “readers” including BRD4 since the serendipitous discovery of JQ1(+), an inhibitor specific to the seemingly undruggable BET bromodomains. The role of the BET family in the development of intimal hyperplasia is not known. MethodsWe investigated the effect of BET inhibition on intimal hyperplasia using a rat balloon angioplasty model. ResultsWhile BRD4 was dramatically up-regulated in the rat and human hyperplastic neointima, blocking BET bromodomains with JQ1(+) diminished neointima in rats. Knocking down BRD4 with siRNA, or treatment with JQ1(+) but not the inactive enantiomer JQ1(−), abrogated platelet-derived growth factor (PDGF-BB)-stimulated proliferation and migration of primary rat aortic smooth muscle cells. This inhibitory effect of JQ1(+) was reproducible in primary human aortic smooth muscle cells. In human aortic endothelial cells, JQ1(+) prevented cytokine-induced apoptosis and impairment of cell migration. Furthermore, either BRD4 siRNA or JQ1(+) but not JQ1(−), substantially down-regulated PDGF receptor-α which, in JQ1(+)-treated arteries versus vehicle control, was also reduced. ConclusionsBlocking BET bromodomains mitigates neointima formation, suggesting an epigenetic approach for effective prevention of intimal hyperplasia and associated vascular diseases.

Regulation of RGS5 GAP activity by GPSM3.

Heterotrimeric G protein signaling is limited by intracellular proteins that impede the binding of or accelerate the hydrolysis of the activating nucleotide GTP, exemplified respectively by the G protein-signaling modifier (GPSM) and regulator of G protein-signaling (RGS) families of proteins. Little is known about how members of these groups of proteins might influence the impact of the other on G protein activity. In the present study, we have identified novel binding and functional interactions between GPSM3 (also known as activator of G protein-signaling 4 (AGS4) or G18) and RGS5, both of which were found to be expressed in primary rat aortic smooth muscle cell cultures. The binding of GPSM3 to RGS5 appears to be selective as no interactions were detected with other RGS proteins tested. In solution-based experiments, the addition of GPSM3 was found to enhance the ability of RGS5 to accelerate GTP hydrolysis by Gαi1 but not that of RGS4. In membrane-based assays utilizing M2 muscarinic receptor-activated Gαi1, GPSM3 decreased the rate of GTP hydrolysis in the presence of RGS4 but not RGS5, suggesting that the enhancement of RGS5 activity by GPSM3 is maintained under these conditions and/or that the binding of RGS5 to GPSM3 impedes its inhibitory effect on GTP turnover. Overall these findings show that it is possible for GPSM and RGS proteins to bind to one another to produce distinct regulatory effects on heterotrimeric G protein activity.

Serotonin 5-HT2A Receptor Activation Blocks TNF-α Mediated Inflammation In Vivo

Tumor necrosis factor alpha (TNF-α) plays a key role in inflammation, and its production and signaling contribute to many inflammatory related diseases. Recently, we discovered that selective activation of serotonin 5-HT2A receptors with the agonist (R)-DOI produces a super-potent blockade of proinflammatory markers in primary rat aortic smooth muscle cells. Here, we demonstrate that systemic administration of (R)-DOI can block the systemic effects of TNF-α in whole animal, with potent anti-inflammatory effects in the aortic arch and small intestine. This includes blockade of TNF-α-induced expression of pro-inflammatory cell adhesion (Icam-1, Vcam-1), cytokine (Il-6, IL-1b), and chemokine (Mcp-1, Cx3cl1) genes, and expression of VCAM-1 protein in the intestine. Further, systemic (R)-DOI also prevents the TNF-α-induced increase of circulating IL-6. Importantly, utilizing receptor selective antagonists, we have demonstrated that the mechanism underlying the systemic anti-inflammatory effects of (R)-DOI is activation of serotonin 5-HT2A receptors. Our results highlight a powerful new role for the serotonin 5-HT2A receptor in inflammatory processes, and indicate that agonism of serotonin receptors may represent an effective and novel approach to develop powerful small molecule therapeutics for inflammatory diseases and conditions such as atherosclerosis and inflammatory bowel disease.