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The increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of vascular diseases. The intermediate conductance calcium‐activated potassium (IKCa) channel plays a critical role in VSMC proliferation by raising the intracellular calcium concentration ([Ca2+]i), but the underlying mechanism is still not unclear. Here we investigated the cooperation between IKCa and transient receptor potential canonical 1 (TRPC1) channels in mediating extracellular Ca2+ entry, which in turn activates downstream Ca2+ signalling in the regulation of VSMC proliferation using serum‐induced cell proliferation model. Serum‐induced cell proliferation was accompanied with up‐regulation of IKCa expression and an increase in [Ca2+]i. Serum‐induced cell proliferation and increase in [Ca2+]i were suppressed by IKCa inhibition with TRAM‐34 or IKCa knockdown. Serum‐induced cell proliferation was strongly reduced by the removal of extracellular Ca2+ with EGTA or intracellular Ca2+ with BAPTA‐AM and, additionally, by TRPC1 knockdown. Moreover, the increase in [Ca2+]i induced by serum or by IKCa activation with 1‐EBIO was attenuated by TRPC1 knockdown. Finally, serum induced ERK1/2 activation, which was attenuated by treatment with TRAM‐34 or BAPTA‐AM, as well as TRPC1 knockdown. Consistently, serum‐induced cell proliferation was suppressed by ERK1/2 inhibition with PD98059. Taken together, these results suggest that the IKCa and TRPC1 channels cooperate in mediating Ca2+ influx that activates the ERK1/2 pathway to promote cell proliferation, thus providing new mechanistic insights into VSMC proliferation. [ABSTRACT FROM AUTHOR]

Vascular stiffening, an early and common characteristic of cardiovascular diseases (CVDs), stimulates vascular smooth muscle cell (VSMC) proliferation which reciprocally accelerates the progression of CVDs. However, the mechanisms by which extracellular matrix stiffness accompanying vascular stiffening regulates VSMC proliferation remain largely unknown. In the present study, we examined the role of the intermediate‐conductance Ca2+‐activated K+ (IKCa) channel in the matrix stiffness regulation of VSMC proliferation by growing A7r5 cells on soft and stiff polydimethylsiloxane substrates with stiffness close to these of arteries under physiological and pathological conditions, respectively. Stiff substrates stimulated cell proliferation and upregulated the expression of the IKCa channel. Stiff substrate‐induced cell proliferation was suppressed by pharmacological inhibition using TRAM34, an IKCa channel blocker, or genetic depletion of the IKCa channel. In addition, stiff substrate‐induced cell proliferation was also suppressed by reducing extracellular Ca2+ concentration using EGTA or intracellular Ca2+ concentration using BAPTA‐AM. Moreover, stiff substrate induced activation of extracellular signal‐regulated kinases (ERKs), which was inhibited by treatment with TRAM34 or BAPTA‐AM. Stiff substrate‐induced cell proliferation was suppressed by treatment with PD98059, an ERK inhibitor. Taken together, these results show that substrates with pathologically relevant stiffness upregulate the IKCa channel expression to enhance intracellular Ca2+ signaling and subsequent activation of the ERK signal pathway to drive cell proliferation. These findings provide a novel mechanism by which vascular stiffening regulates VSMC function. [ABSTRACT FROM AUTHOR]

Background: Bromophenol-thiosemicarbazone hybrid was a novel synthetic brominated anticancer compound with two bromine atoms. Bromophenol-thiosemicarbazone hybrid showed considerable selective inhibitory activity against PARP1 (IC50 = 29.5 nmol/L). UHPLC-TOF-MS was used to establish a new method to quantify bromophenol-thiosemicarbazone hybrid bio-samples for indispensable quantitation analysis in further extensive pre-clinical studies. Methods: Chromatographic and mass spectrometry parameters were optimized for quantitative method establishment. Improved protein-precipitated method was applied to the extraction of bromophenol-thiosemicarbazone hybrid in rat plasma samples. Furthermore, this proposed method was applied to an intravenous bolus dose to male rats. Results: Mobile phase was consisted of water for A and acetonitrile for B with 25 mmol/L formic acid in both A and B. The flow rate was 0.30 mL/min, and the run time of bromophenol-thiosemicarbazone hybrid was 4.0 min. A Thermo Fisher Accucore 2.6 μm C18 column (50 × 2.1 mm i.d.; San Jose, USA) was used for chromatographic separation. High resolution mass spectrometry was used to quantify samples by exact mass number of compound which was operated on negative ionization mode. Linear dynamic range of the established method was widely with 13.7–10000 nmol/L. Pharmacokinetics properties of bromophenol-thiosemicarbazone hybrid were shown in the results. Conclusion: This method was reliable and reproducible from sample preparation to analysis and storage stability under the investigated conditions. It may be useful for analysis of halogenated compounds and brominated compounds in ultra-performance liquid chromatography-mass spectrometry. [ABSTRACT FROM AUTHOR]

Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca2+‐activated K+ channel, KCa3.1, is critically engaged in MSC proliferation but its role in mechanical regulation of MSC proliferation remains unknown. Here, we examined the KCa3.1 channel expression and its role in rat bone marrow‐derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up‐regulated the KCa3.1 channel expression and pharmacological or genetic inhibition of the KCa3.1 channel strongly suppressed stretch‐induced increase in cell proliferation and cell cycle progression. These results support that the KCa3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation. [ABSTRACT FROM AUTHOR]

A critical limitation for tissue engineering and autologous therapeutic applications of bone marrow derived EPCs is their low frequency, which is even lower in number and activity level in patients with cardiovascular risk factors and other diseases. New strategies for obtaining and reserving sufficient ready-to-use EPCs for clinical use have hit major obstacles, because effects of serial passage and cryopreservation on EPC phenotype and functions are still needed to be explored. The present study aims at investigating effects of a limited number of culture passages as well as cryopreservation on EPC phenotype and functions. We isolated EPCs from rat bone marrow and cultured them up to passage 12 (totaling achievements of 40 population doublings). The phenotype and functions of fresh cultured and post-cryopreserved EPCs at passages 7 and 12, respectively, were evaluated. EPCs at passage 12 maintained the morphological characteristics, marker phenotype, Dil-ac-LDL uptake and FITC-UEA-1 binding functions, enhanced EPCs proliferation, tube formation and migration, but decreased CD133 expression compared with EPCs at passage 7. Cryopreservation caused limited impairment in EPC phenotype and functions. In brief, our results demonstrated that a limited number of culture passages and cryopreservation did not change EPC phenotype and functions, and can be used for the development of robust strategies and quality control criterion for obtaining sufficient and high-quality ready-to-use EPCs for tissue engineering and therapeutic applications. [ABSTRACT FROM AUTHOR]

Background and Purpose: Protein tyrosine phosphatase 1B (PTP1B) negatively regulates insulin signalling by tyrosine dephosphorylation of the insulin receptor. It is a highly validated target for type 2 diabetes therapeutics. Here, the anti-diabetic effects of HPN were evaluated in the diabetic BKS db mice.Experimental Approach: The mode of inhibition of PTP1B by HPN was determined according to the Lineweaver-Burk plot. A surface plasmon resonance assay and molecular docking were used to study the interaction between HPN and PTP1B. C2C12 skeletal muscle cells were used to investigate the cell permeability of HPN and the effect of HPN on insulin signalling pathways. Long-term effects of HPN on glycaemic control were investigated in diabetic BKS db mice. Glycogen contents in liver and muscle were determined. Furthermore, changes in the number of beta cells were evaluated by Gomori staining.Key Results: HPN was identified as a specific PTP1B inhibitor. HPN directly interacted with PTP1B by binding to the catalytic domain through hydrogen bonds in a competitive mode. Approximately 56.98% of HPN entered into the cultured C2C12 myotubes. HPN ameliorated the impaired insulin signalling in palmitate-treated C2C12 myocytes. Notably, oral administration of HPN significantly protected mice from hyperglycaemia, dyslipidemia and hyperinsulinaemia. HPN also enhanced the storage of glycogen in liver and muscle. Moreover, HPN obviously improved the beta cell numbers of the pancreatic islets.Conclusion and Implications: Our results indicate that HPN is a specific PTP1B inhibitor, with anti-diabetic properties and good cell permeability and oral availability. [ABSTRACT FROM AUTHOR]

One of the major challenges in tissue engineering of small-diameter vascular grafts is to inhibit intimal hyperplasia and keep long-term patency after implantation. Rapid endothelialization of the grafts could be an effective approach. In this study, QK, a peptide mimicking vascular endothelial growth factor, was selected as the bioactive substrate and loaded in electrospun membranes for enhancement of vascular endothelial cell growth. In detail, QK peptide was firstly introduced with poly(ethylene glycol) diacrylate into a thiolated chitosan solution that could transfer into hydrogel. Then, suspensions or emulsions of poly(ethylene glycol)- b-poly( l-lactide- co- ε-caprolactone) (PELCL) containing QK peptide (with or without chitosan hydrogel) were electrospun into fibrous membranes. For comparison, the electrospun PELCL membrane without QK was also fabricated. Results of release behaviors showed that the electrospun membranes, especially that contained chitosan hydrogel prepared by suspension electrospinning, could successfully encapsulate QK peptide and maintain its secondary structure after released. In vitro cell culture studies exhibited that the release of QK peptide could accelerate the proliferation of vascular endothelial cells in the 9 days. It was suggested that the electrospun PELCL membranes loaded with QK peptide might have potential applications in vascular tissue engineering. [ABSTRACT FROM AUTHOR]

[Objectives] AgHSFB2 of celery (Apium graveolens L.) belongs to HSF gene family, which plays key roles in protecting plants from heat stress. Studying the related genes and proteins of the HSF family is important to exploring the heat resistant mechanism of celery. [ Methods ] AgHSFB2 gene was isolated from three celery cultivars, 4 Liuhehuangxinqin' 'Jinnanshiqin' and 'Ventura', respectively. Sequence analysis and quantitative real-time PCR were used to detect the expression profdes under different temperature treatments(4,38 and 42 °C ). [Results]Sequence analysis indicated that the ORF length of AgHSFB2 was 918 bp,encoding 305 amino acids. The predicted molecular weight of AgHSFB2 was 34 048 and pi value was 5.09. Phylogenetic analysis between AgHSFB2 of celery and HSF family factors in Arabidopsis showed that AgHSFB2 was close to HSFB2a and HSFB2b in the HSFB2 subgroup. Phylogenetic analysis among differnt plants illuminated that the HSFB2 of celery was close to Solarium tuberosum and Solarium lycopersicum from Solanaceae. Structure analysis implied that AgHSFB2 was composed of 3 a helixes and 4 p sheets. Quantitative real-time PCR analysis demonstrated that AgHSFB2 was tissue-specific and cultivars-specific. AgHSFB2 was mainly expressed in leaves of the three celery cultivars. Under different temperature treatments (4,38 and 42 °C ) , the expression profiles of AgHSFB2 had similar response trend, but the response time and intensity of AgHSFB2 had significant differences. AgHSFB2 of 'Ventura' was greatly upregulated after 2 h in 4 °C and 38 °C treatments. AgHSFB2 in 'Jinnanshiqin' and 'Ventura' both were upregulated obviously after 1 h in 42 °C treatment. [ Conclusions] The excessive expression of heat shock proteins can be induced by the overexpression of AgHSFB2 gene, helping the plants resist damages from the high temperature stress. [ABSTRACT FROM AUTHOR]

Cress('Nanjingbaguazhoushuiqin')and celery('Liuhehuangxinqin')were selected to compare their structure of nutritional organ and photosynthetic characters under different light intensity· The results showed that root vascular bundles of cress were more sophisticated than those of celery· The structure of petiole in cress was hollow with big cavum medullare, while the structure was compact in celery without cavum medullare· There were more xylem cells, better developed phloem cells and vascular bundles in celery than in cress· The net photosynthetic rate(Pn ), stomatal conductance(Gs ), intercellular CO2 concentration(Ci ) and transpiration rate (Tr)of celery were 1·45, 1·81, 1·49 and 1·69 times more than those of cress at 90 μmol·m-2·s-1 light intensity, and were 1·77, 1· 61, 1·71 and 1·46 times more than those of cress at 270 μmol·m-2·s-1 light intensity, respectively· Under certain light intensity, Pn of cress and celery was positively correlated with Gs and Tr, and negatively correlated with Ci· Pn of celery was higher than that of cress. The results proved that photosynthetic characters of the cress and celery were related with vascular bundles, phloem and xylem of blade. [ABSTRACT FROM AUTHOR]

The neuropeptide galanin and its receptors (GalR) are found to be up-regulated in brains suffering from nerve injury, but the specific role played by galanin remains unclear. This study aimed to explore the neuroprotective role of galanin after shear stress induced nerve injury in the primary cultured cortical neurons of rats. Our results demonstrated that no significant changes in cell death and viability were found after galanin treatment when subjected to a shear stress of 5 dyn/cm2 for 12 h, after increasing magnitude of shear stress to 10 dyn/cm2 for 12 h, cell death was significantly increased, while galanin can inhibit the nerve injury induced by shear stress with 10 dyn/cm2 for 12 h. Moreover, Gal2-11 (an agonist of GalR2/3) could also effectively inhibit shear stress-induced nerve injury of primary cultured cortical neurons in rats. Although GalR2 is involved in the galanin protection mechanism, there was no GalR3 expression in this system. Moreover, galanin increased the excitatory postsynaptic currents (EPSCs), which can effectively inhibit the physiological effects of shear stress. Galanin was also found to inhibit the activation of p53 and Bax, and further reversed the down regulation of Bcl-2 induced by shear stress. Our results strongly demonstrated that galanin plays a neuroprotective role in injured cortical neurons of rats. [ABSTRACT FROM AUTHOR]

Evidence from ground-based animal studies using tail-suspended hindlimb unloaded rats model has clearly demonstrated that simulated microgravity-induced smooth muscle cell phenotype conversion, a characteristic vascular structural and functional remodeling, may be one of the key contributors to postspaceflight orthostatic intolerance. However, the rats model involves multiple collective effects of microgravity including cephalic fluid shift and postural muscle unloading on smooth muscle cells (SMCs). It cannot isolate a single factor from the collective ones and therefore is not ideal to study the effects of gravitational vector alteration alone on SMCs. To test the hypothesis that gravitational vector alteration per se might affect smooth muscle cell phenotype, a roller culture apparatus was employed to expose cultured rat aortic smooth muscle cells (RASMCs) to simulated microgravity. Cell proliferation, cell cycle distribution, apoptosis, migration, and nitric oxide production rates were measured and compared between the control and the simulated microgravity groups. Cell cytoskeleton reorganization induced by simulated microgravity was observed by confocal microscopy. Specific contractile and synthetic Gene expression at the mRNA level was quantified by reverse transcriptional polymerase chain reaction. It was observed that simulated microgravity suppressed RASMC proliferation and migration, enhanced cell apoptosis, stimulated NO release, and destroyed the original well-organized cytoskeleton. Moreover, at the mRNA level, long-time exposure (≥72 h) to simulated microgravity induced a contractile phenotype tendency by up-regulating smMHC expression. All these findings suggest that the phenotype modulation of vascular smooth muscle cells may be gravity dependent. [ABSTRACT FROM AUTHOR]

The large conductance Ca-activated K (BK) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BK channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BK channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BK protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BK in SS-induced inhibition of proliferation was examined by BK inhibition using a BK specific blocker, iberiotoxin (IBTX), and by BK transfection in BK non-expressing CHO cells. The changes in [Ca] were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC(3). We found that laminar SS inhibited VSMC proliferation and stimulated BK channel expression. Furthermore, laminar SS induced an increase in [Ca] and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BK channel activity on cell proliferation in response to SS was also confirmed in BK-transfected CHO cells showing a decline in proliferation. Blocking BK channel reversed its inhibitory effect, providing additional support for the involvement of BK in SS-induced proliferation reduction. Our results suggest, for the first time, that BK channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation. [ABSTRACT FROM AUTHOR]

VEGF-loaded core/shell fibrous membranes were prepared by coaxial electrospinning with dextran (DEX) as the core component and poly(lactide-co-glycolide) (PLGA) as the shell polymer, respectively. The electrospun DEX/PLGA fibers were observed by scanning electron microscopy, transmission electron microscopy and confocal microscopy to identify the core/shell fiber structure and the protein distribution. The results of tensile tests showed that the DEX/PLGA membranes possessed lower tensile strength and higher Young's modulus than PLGA one. The release profiles demonstrated that vascular endothelial growth factor (VEGF) release sustained for more than 28 days. Studies on cell viability and spreading demonstrated that the DEX(VEGF)/PLGA membranes positively promoted cell proliferation and cell-membrane interaction, which further testified that the processed VEGF remained bioactivities. Furthermore, the detections for the up-regulation of intercellular adhesion molecular-1 and the release of von Willebrand factor under pathological stimuli, which are related to inflammation process and thrombus formation, exhibited a normal immune response for the DEX(VEGF)/PLGA membrane. These data suggested that the VEGF-loaded fibers could be feasible in vascular tissue engineering. [ABSTRACT FROM AUTHOR]

RNA-binding proteins (RBPs) lie at the center of posttranscriptional regulation and the dysregulation of RBPs contributes to diabetes. Therefore, the modulation of RBPs is anticipated to become a potential therapeutic approach to diabetes. CYC27 is a synthetic derivative of marine bromophenol BDB, which is isolated from red alga Rhodomela confervoides. In this study, we found that CYC27 significantly lowered the blood glucose levels of diabetic BKS db mice. Moreover, CYC27 effectively ameliorated dyslipidemia in BKS db mice by reducing their total serum cholesterol (TC) and triglyceride (TG) levels. Furthermore, CYC27 was an insulin-sensitizing agent with increased insulin-stimulated phosphorylation of insulin receptors and relevant downstream factors. Finally, to systemically study the mechanisms of CYC27, label-free quantitative phosphoproteomic analysis was performed to investigate global changes in phosphorylation. Enriched GO annotation showed that most regulated phosphoproteins were related to RNA splicing and RNA processing. Enriched KEGG analysis showed that a spliceosome-associated pathway was the predominant pathway after CYC27 treatment. Protein-protein interaction (PPI) analysis showed that CYC27 modulated the process of mRNA splicing via phosphorylation of the relevant RBPs, including upregulated Cstf3 and Srrt. Our results suggested that CYC27 treatment exerted promising anti-diabetic effects by sensitizing the insulin signaling pathways and modulating RNA splicing-associated RBPs. [ABSTRACT FROM AUTHOR]