Rabbit Aortic Smooth Muscle Cells Research Articles

Inhibitory Effect of Curcumin on Artery Restenosis Following Carotid Endarterectomy and Its Associated Mechanism in vitro and in vivo.

ObjectiveThe present study aimed to assess the effect of curcumin (Cur) on carotid artery restenosis following carotid endarterectomy (CEA) and its associated mechanism in vivo and in vitro.MethodsAng II was used to induce excessive proliferation of rabbit aortic smooth muscle cells (CCC-SMC-1) in order to establish a hemadostenosis cell model. Similarly, the animal model of carotid artery restenosis was established by carotid artery gas drying injury combined with high-fat feed prior to CEA. CCC-SMC-1 cells and animals were treated by Cur and its effects on neointimal hyperplasia, inflammation and oxidative stress were detected and observed. The proteins that were associated with the Raf/MEK/ERK pathway were detected in cells and rabbit carotid artery tissues.ResultsCur inhibited the proliferation of smooth muscle cells and neointimal formation and reduced the inflammation and oxidative stress indices. Concomitantly, Cur reduced the phosphorylation of the Raf/MEK/ERK pathway proteins.ConclusionCur could inhibit carotid restenosis following CEA by inhibiting the activation of the Raf/MEK/ERK pathway.

Bisphenol A exposure enhances atherosclerosis in WHHL rabbits.

Bisphenol A (BPA) is an environmental endocrine disrupter. Excess exposure to BPA may increase susceptibility to many metabolic disorders, but it is unclear whether BPA exposure has any adverse effects on the development of atherosclerosis. To determine whether there are such effects, we investigated the response of Watanabe heritable hyperlipidemic (WHHL) rabbits to 400-µg/kg BPA per day, administered orally by gavage, over the course of 12 weeks and compared aortic and coronary atherosclerosis in these rabbits to the vehicle group using histological and morphometric methods. In addition, serum BPA, cytokines levels and plasma lipids as well as pathologic changes in liver, adipose and heart were analyzed. Moreover, we treated human umbilical cord vein endothelial cells (HUVECs) and rabbit aortic smooth muscle cells (SMCs) with different doses of BPA to investigate the underlying molecular mechanisms involved in BPA action(s). BPA treatment did not change the plasma lipids and body weights of the WHHL rabbits; however, the gross atherosclerotic lesion area in the aortic arch was increased by 57% compared to the vehicle group. Histological and immunohistochemical analyses revealed marked increases in advanced lesions (37%) accompanied by smooth muscle cells (60%) but no significant changes in the numbers of macrophages. With regard to coronary atherosclerosis, incidents of coronary stenosis increased by 11% and smooth muscle cells increased by 73% compared to the vehicle group. Furthermore, BPA-treated WHHL rabbits showed increased adipose accumulation and hepatic and myocardial injuries accompanied by up-regulation of endoplasmic reticulum (ER) stress and inflammatory and lipid metabolism markers in livers. Treatment with BPA also induced the expression of ER stress and inflammation related genes in cultured HUVECs. These results demonstrate for the first time that BPA exposure may increase susceptibility to atherosclerosis in WHHL rabbits.

The NF-κB pathway: regulation of the instability of atherosclerotic plaques activated by Fg, Fb, and FDPs

Recently, the molecular mechanism responsible for the instability of atherosclerotic plaques has gradually become a hot topic among researchers and clinicians. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play an important role in the processes of formation and development of atherosclerosis. In this study, we established and employed the transwell co-culture system of rabbit aortic endothelial cells and smooth muscle cells to explore the relationship between fibrin (Fb), fibrinogen (Fg), and/or their degradation products (FDPs) in relation to the instability of atherosclerotic plaques; meanwhile, we observed the effects of Fg, Fb, and FDPs on the mRNA levels of MMPs and VEGF as well as on the activation of nuclear factor-kappa B (NF-κB). We concluded that Fb, Fg, and FDPs are involved in the progression of the instability of atherosclerotic plaques via increasing the expression of MMPs and VEGF. This effect might be mediated by the NF-кB pathway.

Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na+-K+ pump’s β1-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na+-K+ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na+-K+ pump activity is an important determinant of vascular tone through effects on [Ca2+]i, we have examined the role of oxidative regulation of the Na+-K+ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β1-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na+-K+ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K+-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na+-K+ ATPase and decrease in K+-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β1-subunit. Knock-out of FXYD1 dramatically decreased K+-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β1-subunit glutathionylation, and enhanced K+-induced vasorelaxation. Ang II inhibits the Na+-K+ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β1-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na+-K+ pump and may have an important protective role in the vasculature under conditions of oxidative stress.

The 2‐nitrate‐1,3‐dibuthoxypropan, a nitric oxide donor, alters autonomic function in spontaneously hypertensive rats

The 2‐nitrate‐1,3‐dibuthoxypropan (NDBP), an organic nitrate synthesized from glycerin, increases NO levels in rabbit aortic smooth muscle cells and causes a biphasic response: hypotension and bradycardia followed by hypertension and tachycardia in Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. However, its effect on autonomic control has not been investigated. This study evaluated the action of NDBP on autonomic function in SHR and WKY rats. Atropine (2 mg/kg) blunted the bradycardia induced by NDBP (15 mg/kg) in WKY and SHR (−265±25 vs −48±7 bpm, n=6 and −255±9 vs −25±10 bpm, n=6; p<0.05). Furthermore, the pressor response to the compound was potentiated (35±7 vs 65±4 mmHg in WKY; 33±10 vs 82±6 mmHg in SHR; p<0.05). Vagotomy also reduced the bradycardia in both WKY and SHR (−136±8 vs −17±2, n=4 and −171±36 vs −8±2, n=6, p<0.05). Moreover, hexamethonium (30 mg/kg) reduced both bradycardia (−261±10 vs 1±3 in WKY; −282±21 vs −26±25 in SHR, n=4; p<0.05) and pressor response (41±4 vs 0±1‐in WKY; 44±9 vs −12±7 in SHR, n=4; p<0.05) elicited by NDBP. In conclusion, NDBP, in addition to its effects as NO donor, induces cardiovascular effects by altering autonomic function.

Adventitial gene transfer of VEGFR-2 specific VEGF-E chimera induces MCP-1 expression in vascular smooth muscle cells and enhances neointimal formation

BackgroundThe role of vascular endothelial growth factors (VEGFs) in neointimal formation has been controversial. VEGF receptor (R)-2 signaling pathway is crucial in bringing about the effects of VEGFs including vasodilatation, endothelial cell migration and proliferation. In this study we have used an established adventitial gene transfer technique, in vitro studies and a novel VEGF-E/PlGF chimera that binds specifically to VEGFR-2, to investigate the role of VEGFR-2 in neointimal formation. MethodsIntimal hyperplasia was induced in the carotid arteries of cholesterol fed male New Zealand White rabbits using a silastic collar. Adenoviral vectors encoding VEGF-E chimera (1×109pfu/ml) were transferred to the adventitia of the carotid arteries either alone or together with adenoviruses encoding soluble VEGFR-2 (sVEGFR-2). Adenoviruses encoding LacZ were used as controls. All animals were sacrificed 7 days after the gene transfer. ResultsSignificant increases in neointimal formation, proliferating cells, inflammatory responses and adventitial angiogenesis were observed in the VEGF-E chimera transduced arteries. The number of medial smooth muscle cells expressing VEGFR-2 was significantly (p<0.001) higher. MCP-1 mRNA levels were significantly (p<0.01) increased in the VEGF-E chimera transduced arteries and transduced rabbit aortic smooth muscle cells (p<0.05). Soluble VEGFR-2 (sVEGFR-2) significantly inhibited VEGF-E chimera induced neointimal formation (p<0.01), cellular proliferation (p<0.01), inflammatory responses (p<0.01) and adventitial angiogenesis (p<0.01). ConclusionsThe results indicate that VEGFR-2 mediated signaling could aggravate neointimal formation and suggest a potential therapeutic role of sVEGFR-2 in inhibiting neointimal formation and adventitial angiogenesis.

Cholesterol-lowering strategies reduce vascular LRP1 overexpression induced by hypercholesterolaemia

Low density lipoprotein receptor-related protein (LRP1) plays a key role on vascular functionality and is upregulated by hypercholesterolemia and hypertension. To investigate the effect of cholesterol-lowering interventions on vascular LRP1 over expression and whether simvastatin influences LRP1 expression. Male New Zealand rabbits were recruited into various groups, one group was fed a normal chow diet for 28 days (control group, n = 6), other group (n = 24) was fed a hypercholesterolemic diet (HC), six rabbits were euthanized at day 28 to test the capacity of HC diet to induce early atherosclerosis and the rest at day 60 (n = 18) after receiving either HC diet (HC group, n = 6), HC diet with simvastatin (2·5 mg/kg.day) (HC+simv group, n = 6), or a normal chow diet (NC group, n = 6) for the last 32 days. High-cholesterol diet raised vascular LRP1 concomitantly with increased lipid, VSMC and macrophage content in the arterial intima. Simvastatin and return to normocholesterolemic diet significantly reduced systemic cholesterol levels and vascular lipid content. Interestingly, these interventions also downregulate LRP1 overexpression in the vascular wall although to a different extent (HC+simv: 75 ± 3·6%vs NC: 50 ± 3·5% versus, P = 0·002). Immunohistochemistry studies showed that LRP1 diminushion was associated to a reduction in the number of intimal VSMC in HC+simv.group. Simvastatin per se did not exert any significant effect on LRP1 expression in rabbit aortic smooth muscle cells (rSMC). Our results demonstrate that cholesterol-lowering interventions exerted down regulatory effects on vascular LRP1 over expression induced by hypercholesterolemia and that simvastatin did not influence LRP1 expression beyond its cholesterol-lowering effects.

Effect of Statins on the Viability of Macrophages and Smooth Muscle Cells

Because macrophages play an important role in the destabilization of atherosclerotic plaques, and smooth muscle cells (SMCs) contribute to plaque stability, selective clearance of macrophages in atherosclerotic plaques is a promising strategy for plaque stabilization. In the present study, we investigated the effects of fluvastatin, simvastatin, lovastatin, and pravastatin on the viability of macrophages and SMCs. All statins, except pravastatin, induced cell death of J774A.1 macrophages after 24 hours, albeit with different sensitivity. The viability of rabbit aortic SMCs was hardly affected. Fluvastatin-induced macrophage cell death was characterized as apoptosis and could be reversed by isoprenoid intermediates of the mevalonate pathway. Peritoneal macrophages from male or female mice were much more resistant to statin-induced cell death. The high sensitivity of J774A.1 macrophages to statin-induced cell death was related to the strong 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in these cells. Macrophage and SMC content of rabbit atherosclerotic plaques was not affected after in vitro treatment with fluvastatin or lovastatin for 3 days. In conclusion, fluvastatin, simvastatin, and lovastatin, but not pravastatin, can selectively induce apoptosis in J774A.1 macrophages, but not in SMCs, primary macrophages or rabbit atherosclerotic plaques. This effect was related to the degree of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the different cell types.

Cellular prion protein (PrP C) and superoxide dismutase (SOD) in vascular cells under oxidative stress

The PrP C is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP C with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP C expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrP C expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome- c reduction, was 13.9±1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP C expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP C only in cells stimulated for 8 hours with the different stressors. The PrP C mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP C protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP C.

Blockade of Renin-Angiotensin System Attenuates Advanced Glycation End Products-Mediated Signaling Pathways

Advanced glycation end products (AGE) and a receptor for AGE (RAGE) play a key role in diabetic vascular complications. Matrix metalloproteinases (MMPs) and apoptosis contribute to plaque instability. The renin-angiotensin system (RAS) is crucial for NADPH oxidase-dependent redox signaling pathways in the vascular wall. We investigated the effects of RAS blockade on AGE-triggered signaling pathways and its downstream events, including MMP-9 and apoptosis. We used cultured rabbit aortic smooth muscle cells (SMCs), which were stimulated with AGE in the presence or absence of temocaprilat or olmesartan. Angiotensin converting enzyme (ACE) mRNA levels were increased 4 to 6 hours after adding AGE. AGE induced Rac1 and p47(phox) membrane translocation, reactive oxygen species (ROS) generation and NF-kappaB phosphorylation within 15 minutes, and various molecular expressions after 18 hours, which were attenuated by RAS blockade by temocaprilat or olmesartan. AGE-induced RAGE expression, as well as other molecules, including membrane type 1-MMP (MT1-MMP), monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1), was NADPH oxidase signaling-dependent and blunted by temocaprilat and olmesartan. The parameters of plaque instability, including MMP-9 expression and activity, and apoptosis were up-regulated by AGE, which was markedly attenuated by temocaprilat or olmesartan. Using isolated human monocyte culture, AGE-induced ROS generation and molecular expression were also attenuated by RAS blockade. The present study shows that AGE-triggered NADPH oxidase signaling pathways, including MMP-9 and apoptosis, were attenuated by RAS blockade, which may be an attractive strategy for treating plaque instability in diabetic vascular complications.

APD791, 3-Methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a Novel 5-Hydroxytryptamine 2A Receptor Antagonist: Pharmacological Profile, Pharmacokinetics, Platelet Activity and Vascular Biology

We have evaluated the receptor pharmacology, antiplatelet activity, and vascular pharmacology of APD791 [3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide] a novel 5-hydroxytryptamine 2A (5-HT(2A)) receptor antagonist. APD791 displayed high-affinity binding to membranes (K(i) = 4.9 nM) and functional inverse agonism of inositol phosphate accumulation (IC(50) = 5.2 nM) in human embryonic kidney cells stably expressing the human 5-HT(2A) receptor. In competition binding assays, APD791 was greater than 2000-fold selective for the 5-HT(2A) receptor versus 5-HT(2C) and 5-HT(2B) receptors, and was inactive when tested against a wide panel of other G-protein-coupled receptors. APD791 inhibited 5-HT-mediated amplification of ADP-stimulated human and dog platelet aggregation (IC(50) = 8.7 and 23.1 nM, respectively). Similar potency was observed for inhibition of 5-HT-stimulated DNA synthesis in rabbit aortic smooth muscle cells (IC(50) = 13 nM) and 5-HT-mediated vasoconstriction in rabbit aortic rings. Oral administration of APD791 to dogs resulted in acute (1-h) and subchronic (10-day) inhibition of 5-HT-mediated amplification of collagen-stimulated platelet aggregation in whole blood. Two active metabolites, APD791-M1 and APD791-M2, were generated upon incubation of APD791 with human liver microsomes and were also indentified in dogs after oral administration of APD791. The affinity and selectivity profiles of both metabolites were similar to APD791. These results demonstrate that APD791 is an orally available, high-affinity 5-HT(2A) receptor antagonist with potent activity on platelets and vascular smooth muscle.

Receptor tyrosine kinases as mediators of injury-induced bradykinin B 1 receptor expression in rabbit aortic smooth muscle

Prolonged in vitro incubation of rabbit aortic rings allows recording contractile responses mediated by the inducible bradykinin B 1 receptors; addition of interleukin (IL)-1 or epidermal growth factor (EGF) to the bathing fluid increases the rate of sensitization, a process partially inhibited by the nonspecific Tyr-kinase inhibitor genistein. The recent development of specific inhibitors for receptor associated Tyr-kinase activities (tyrphostin AG 1478 for EGF receptor, sunitinib for VEGF receptor and others) allows assessing the role of such signaling molecules in this process. AG 1478 reduced the potentiating effects of exogenous EGF, and also the spontaneous sensitization to the agonist des-Arg 9-bradykinin. Sunitinib or GM 6001, a wide spectrum inhibitor of metalloproteinases, had no effect on these responses. In rabbit aortic smooth muscle cells, the cytokines IL-1β and EGF increased the density of binding sites for [ 3H]Lys-des-Arg 9-bradykinin in 4 h; AG 1478 reduced only the effect of exogenous EGF. IL-1 receptor antagonist decreased both the effect of IL-1β and of EGF in rabbit smooth muscle cells. EGF was weakly and slowly coupled to nuclear factor-κB nuclear translocation in these cells, as compared to the effect of IL-1β. EGF-induced EGF receptor autophosphorylation and ERK1/2 phosphorylation was selectively inhibited by AG 1478 in smooth muscle cells; Lys-des-Arg 9-bradykinin did not transactivate EGF receptor in these cells. While the Tyr-kinase activity sensitive to AG 1478 is recruited by tissue damage and exogenous EGF to upregulate bradykinin B 1 receptors in freshly isolated aortas, this signaling system interacts with others (e.g., IL-1) for the optimal expression of B 1 receptors.

Function and role of voltage-gated sodium channel NaV1.7 expressed in aortic smooth muscle cells

Voltage-gated Na(+) channel currents (I(Na)) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of I(Na), its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, I(Na) was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular Na(+) with N-methyl-d-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding Na(V)1.7, as determined by quantitative RT-PCR. I(Na) was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type Ca(2+) channel current was recorded, and I(Na) was not observed in freshly isolated rASMCs, whereas TTX-sensitive I(Na) was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for Na(V)1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that I(Na) is expressed in cultured and diseased conditions but not in normal aorta. The Na(V)1.7 plays an important role in cell migration, endocytosis, and secretion. Na(V)1.7 is also expressed in aorta after balloon injury, suggesting a potential role for Na(V)1.7 in the progression of intimal hyperplasia.

Interactions between cell death induced by statins and 7‐ketocholesterol in rabbit aorta smooth muscle cells

7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects. Cultured rabbit aorta SMCs (passage 2-6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined. NR uptake declined upon 18 h exposure to 25 microM 7-ketocholesterol (-41+/-3%, n=13), 100 microM fluvastatin (-59%) or 30-100 microM simvastatin (-28 to -74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10-30 microM, simvastatin 1-10 microM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10-100 microM, 70-545% increase) and simvastatin (3-100 microM 43-322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 microM was always inactive. 7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings.

Complex regulation of store-operated Ca2+ entry pathway by PKC-ε in vascular SMCs

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

Guided growth of smooth muscle cell on poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) scaffolds with uniaxial microtubular structures

Scaffolds of biopolyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) with uniaxial microtubular structure were successfully prepared by directional freezing and phase-separation techniques. These special structures guided the rabbit aorta smooth muscle cells (RaSMCs) to grow along the tubular structure. The structure of the scaffolds including tubular size and architecture, as well as scaffold mechanical properties could be adjusted by changing the PHBHHx concentration, PHBHHx solvent, and phase-separation temperature. In addition, the scaffolds demonstrated anisotropic mechanical properties, with much more improved strength along the longitudinal direction of the microtubules compared with those along the transverse direction. Three-day and 7-day cell culture of RaSMCs seeded on the scaffolds with different microtubular sizes showed similar cellular metabolic activity but different cell distribution and morphology owing possibly to different scaffold tubular sizes. The scanning electron microscopy and H&E staining demonstrated that RaSMCs were guided to grow along the microtubular structures of scaffolds prepared from the 2.5% or 3.0% PHBHHx/benzene solution. This study demonstrated the possibility of using PHBHHx scaffolds to achieve guided cell growth.

Bio-protocols for directly forming active encapsulations containing living primary cells.

Electrosprays and electrospinning were recently pioneered for directly handling living cells. These recent discoveries are now widely referred to as "bio-electrosprays" and "cell electrospinning", which have been demonstrated as having tremendous applicability to the life sciences in regenerative and therapeutic medicine. In the current work, we report our developmental studies with these protocols as submerged entities with primary rabbit aorta smooth muscle cells for generating cell-bearing encapsulations, which demonstrate proof-of-concept for a plethora of biomedical applications. Cell viability of the post-treated cells was assessed in comparison with two controls by way of flow cytometry over a three week period, establishing a viable cellular population >70%. Hence these investigations demonstrate the ability to explore these electrified encapsulating approaches for directly forming biologically viable emulsions, which could potentially be exploited from mechanisms for cancer therapy, hormone, and diabetic treatment to applications with cosmetics. Therefore, these studies elucidate the strong implications these bio-protocols have to offer the life sciences.

Inhibition of Neointimal Hyperplasia Development by MCI-186 is Correlated With Downregulation of Nuclear Factor-.KAPPA.B Pathway

Atherosclerosis is a progressing inflammatory response mediated by various signaling molecules among which nuclear factor kappaB (NF-kappaB) is thought to have a pivotal role. This study demonstrated the efficacy of antioxidant MCI-186 in preventing the progression of atherosclerosis by inhibiting signaling molecules such as NF-kappaB. Balloon injury of intima was performed in the right common carotid artery of Japanese male white rabbits, which were then fed a 1% high cholesterol diet for 4 weeks, after assigning them to either the control (n=7) or MCI-186 (0.5 mg .kg(-1) . day(-1), n=7) group. Histological analysis revealed a reduction in neointimal thickness and lipid deposition in the subendothelial area of the MCI-186 group. Immunohistochemical analysis revealed attenuation of E-selectin expression, macrophage migration and proliferation of smooth muscle cells in the MCI-186 treated group. In in vitro studies, rabbit aorta smooth muscle cells were incubated with rIL-1betain either the presence or absence of MCI-186. MCI-186 significantly inhibited rIL-1beta-induced proliferation of smooth muscle cells from rabbit aorta, as well as the activation of NF-kappaB. Moreover, western blot analysis showed the inhibitory action of MCI-186 on the nuclear translocation of NF-kappaB in human umbilical vein endothelial cells under rIL-1betastimulation. MCI-186 could provide a novel therapeutic strategy for atherosclerosis by inhibiting the NF-kappaB pathway.

Cell Electrospinning Highly Concentrated Cellular Suspensions Containing Primary Living Organisms Into Cell-Bearing Threads and Scaffolds

We recently pioneered the cell electrospinning of living cells as viable biological threads and scaffolds. In that study, we demonstrated the process with an immortalized human brain astrocytoma (1321N1, European Collection of Cell Cultures) cell line at a cell concentration of 10(6) cells/ml. The next stage was to demonstrate the ability to cell electrospin primary living cells at cell concentrations of 10(7) cells/ml (the highest-ever cell concentration threaded by any threading methodology). Furthermore, the post-threaded cells needed their viability assessed over a long period of time by way of flow cytometry, which accurately assesses the viable cell populations. In this work, we employ primary porcine vascular and rabbit aorta smooth-muscle cells prepared as cellular suspensions at cell concentrations of 10(7) cells/ml. The cell electrospinning device employs a coaxial needle arrangement that enables the flow of either highly concentrated cellular suspension in the inner needle while the outer needle accommodates the flow of a viscoelasticity medical-grade polydimethylsiloxane medium. Cell viability was assessed over a long timeframe by way of flow cytometry in comparison with controls. The work reported here demonstrates the ability to cell electrospin primary living organisms as highly concentrated cellular suspensions. The viable population of cells post-cell electrospinning are significant and remain viable over both the short and long term, as assessed by flow cytometry. Our work elucidates the ability to cell electrospin primary cells as highly concentrated cellular suspensions. The post-cell electrospun organisms are viable over long periods of time, demonstrating a significant active cell population when compared with controls.

Trapping of adrenergic decongestant drugs into cellular endomembrane compartments: Toxicological and pharmacological consequences

Rhinitis of allergic and viral origin is often self-treated by patients with locally applied vasoconstrictor decongestant drugs. In turn, prolonged use of these agents produce an inflammatory condition termed rhinitis medicamentosa. Cationic drugs are sequestered into cells via various mechanisms, including mitochondrial concentration and V-ATPase-driven trapping in vacuoles that swell by an osmotic mechanism. We hypothesized that receptor-independent endomembrane sequestration of topically applied concentrated α-adrenoceptor agonists (decongestants, mydriatics) could contribute to their toxicity and prolonged duration of action. The morphological and functional effects of phenylephrine and xylometazoline on rabbit aortic smooth muscle cells were examined and their possible sequestration evaluated using the contractility of rabbit aorta rings. Synthetic agonists produced V-ATPase-dependent cell vacuolization (prevented by bafilomycin A1; xylometazoline 250 μM, phenylephrine 2.5 mM). V-ATPase-mediated cytotoxicity was slow (24 h; phenylephrine only, 5–10 mM); a rapid xylometazoline-induced cytotoxicity (≥ 500 μM, 4 h) correlated to mitochondrial functional alterations. Xylometazoline had slower contraction and relaxation kinetics than the other α-adrenoceptor agonists in the aorta; bafilomycin pre-treatment influenced its kinetics (accelerated contraction and relaxation) and concentration–effect relationship (potentiation). V-ATPase-driven sequestration contributed to a component of the tissue reservoir of both phenylephrine and xylometazoline as assessed by aortic rings contracted with the concentrated agonists and subsequently washed. Phenylephrine and xylometazoline caused the V-ATPase-dependent cytopathology at a fraction of the usual topical concentrations; this form of sequestration influenced the toxicity and pharmacology of individual agents.