PAR2 promotes M1 macrophage polarization and inflammation via FOXO1 pathway.

Macrophages polarization plays essential but different roles in most diseases such as atherosclerosis, adipose tissue inflammation, and insulin resistance. Our previous study revealed that protease-activated receptor 2 (PAR2), a G-protein coupled receptor influenced macrophage function, but little is known regarding the regulation of macrophage polarization process and its potential mechanisms. In the present study, bone marrow-derived macrophages (BMDM) isolated from C57/BL6 mice and cultured with L929-conditional medium and murine macrophage cell line RAW264.7 were used to study the function of PAR2 activation in vitro. BMDM was stimulated by the small molecular PAR2 agonist, 2-furoyl-LIGRLO-amide trifluoroacetate salt, followed by transcription factor microarray to screen the significantly activated signaling pathways under PAR2 activation. Western blot analysis, quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression of targeted genes and transcription factors. Immunofluorescence was used to observe the subcellular distribution of transcription factors. Our results demonstrated that M1-like polarization was presented by PAR2 agonist treatment with significant upregulation of interleukin-1ß, interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α in BMDM and RAW264.7. Microarray identified forkhead box protein O1 (FOXO1) was significantly increased under PAR2 agonist stimulation, which was confirmed by qPCR and Western blot analysis. Immunofluorescence demonstrated that increased FOXO1 accumulated in the nucleus, which is necessary to promote transcription for targeted genes. We further knocked down FOXO1 expression using small interfering RNA, which alleviated PAR2-induced proinflammatory gene expression. The PAR2/FOXO1 pathway mediated stimulation of proinflammatory genes was further confirmed by tryptase, an endogenous ligand of PAR2. In conclusion, this study demonstrated that PAR2 activation-induced M1 polarization and inflammation through the FOXO1-dependent pathway.

Trehalose Inhibits A53T Mutant α-Synuclein Overexpression and Neurotoxicity in Transduced PC12 Cells.

Fibrillar accumulation of A53T mutant α-synuclein (A53T-AS) in Lewy bodies is a symptom of Parkinsonism. Inhibitions of the overexpression and fibrillar aggregation of α-synuclein (AS) in vivo could be a promising strategy for treating Parkinson's disease (PD). In this study, at concentrations lower than 1 mM, trehalose decreased the A53T-AS expression level in transduced PC12 cells. Although H2O2 and aluminum ions increased the expression level and neurotoxicity of A53T-AS in cells, proper trehalose concentrations inhibited the event. These studies adequately prove that trehalose at an appropriate dose would be potentially useful for PD treatment.

Inhibitory effect of curcumin on the Al(III)-induced Aß42 aggregation and neurotoxicity in vitro.

The pathogenesis of Alzheimer's disease (AD) involves a key event which changes the morphology of amyloid-ß 42 (Aß)42 peptide from its soluble monomeric form into the fibrillated aggregates in the brain. Aluminum ion, Al(III), is known to act as a pathological chaperone of the Aß42 in this process; curcumin, a natural phenolic compound, is considered capable of binding Al(III) and Aß42; nevertheless, little is known about the combined action of curcumin and Al(III) on the Aß42 fibrillation and neurotoxicity. Here, combinations of circular dichroism spectroscopy, thioflavin T fluorescence, atomic force microscopy, Bradford and MTT assays, it is demonstrated that although Al(III) can promote the Aß42 fibrillation dose-dependently, leading to the high neurotoxicity to PC12 cells, curcumin can inhibit the events. Besides, we found that curcumin is able not only to inhibit the formation of Al(III)-induced Aß42 fibrillation, but also to form the Al(III)-curcumin complexes which in turn can remold the preformed, mature, ordered Aß42 fibrils into the low toxic amorphous aggregates. These findings suggest that curcumin could block the binding of Al(III) with Aß42 and form the Al(III)-curcumin complexes, so as to inhibit the Al(III)-induced Aß42 fibrillation and neurotoxicity. The Al(III)-curcumin complexes are worth potentially developing as a therapy agent against the neurodegenerative disorders in the future.

Effect of a novel proteoglycan PTP1B inhibitor from Ganoderma lucidum on the amelioration of hyperglycaemia and dyslipidaemia in db/db mice.

Protein tyrosine phosphatase 1B (PTP1B) is implicated in the negative regulation of the insulin signalling pathway by dephosphorylating the insulin receptor (IR) and IR substrates. Ganoderma lucidum has traditionally been used for the treatment of diabetes in Chinese medicine; however, its anti-diabetic potency and mechanism in vivo is still unclear. Our previously published study reported a novel proteoglycan PTP1B inhibitor, named Fudan-Yueyang-Ganoderma lucidum (FYGL) from G. lucidum, with a half-maximal inhibitory concentration (IC50) value of 5·12 (sem 0·05) µg/ml, a protein:polyglycan ratio of 17:77 and 78 % glucose in polysaccharide, and dominant amino acid residues of aspartic acid, glycine, glutamic acid, alanine, serine and threonine in protein. FYGL is capable of decreasing plasma glucose in streptozotocin-induced diabetic mice with a high safety of median lethal dose (LD50) of 6 g/kg. In the present study, C57BL/6 db/db diabetic mice were trialed further using FYGL as well as metformin for comparison. Oral treatment with FYGL in db/db diabetic mice for 4 weeks significantly (P < 0·01 or 0·05) decreased the fasting plasma glucose level, serum insulin concentration and the homeostasis model assessment of insulin resistance. FYGL also controlled the biochemistry indices relative to type 2 diabetes-accompanied lipidaemic disorders. Pharmacology research suggests that FYGL decreases the plasma glucose level by the mechanism of inhibiting PTP1B expression and activity, consequently, regulating the tyrosine phosphorylation level of the IR ß-subunit and the level of hepatic glycogen, thus resulting in the improvement of insulin sensitivity. Therefore, FYGL is promising as an insulin sensitiser for the therapy of type 2 diabetes and accompanied dyslipidaemia.

Enhanced cell affinity of the silk fibroin- modified PHBHHx material.

Cell affinity is one of the important issues required for developing tissue engineering materials. Although the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has been attractive for its controllable mechanical properties recent years, its cell affinity is still necessary to be improved for the requirements. For this purpose, the regenerated silk fibroin (SF) was coated on the PHBHHx films and its porous scaffolds. The mechanical test showed that SF-modified PHBHHx (SF/PHBHHx) film has a maximum tensile strength of 11.5 +/- 0.5 MPa and elongation at break of 175 +/- 5%. ATR-FTIR spectroscopy demonstrated that SF firmly attached on the scaffold by the hydrogen bonding interaction between SF and PHBHHx even flushed for 21 days in the phosphate-buffer saline (PBS) solution (pH = 7.4). In order to characterize the cell affinity of the SF-modified material, endothelial-like cell line ECV304 cells were seeded on the SF/PHBHHx films and its porous scaffolds. The histochemical analyses of cells stained by the hematoxylin and eosin (HE) as well as cell nuclei stained by the 4',6-diamindine-2'-phenylindole (DAPI) demonstrated that cell attached and reached nearly 100% confluence on the SF/PHBHHx films when cultured for 4 days, which was much faster than that on the pure PHBHHx film. Moreover, the assay of cell activity by the 3-(4, 5-dimethyl thiazol -2-yl)-2, 5-diphenyl terazolium bromide (MTT) showed quantitatively that the number of cells on the SF/PHBHHx porous scaffolds was significant more than that on the unmodified ones after 4, 8, and 14 days culture, respectively. Scanning electron microscopy (SEM) revealed the similar results. Therefore, the SF-modified PHBHHx material is maybe a potential material applicable in the cardiovascular tissue engineering.

Differentiation of smooth muscle progenitor cells in peripheral blood and its application in tissue engineered blood vessels.

BACKGROUND: A major shortcoming in tissue engineered blood vessels (TEBVs) is the lack of healthy and easily attainable smooth muscle cells (SMCs). Smooth muscle progenitor cells (SPCs), especially from peripheral blood, may offer an alternative cell source for tissue engineering involving a less invasive harvesting technique. METHODS: SPCs were isolated from 5-ml fresh rat peripheral blood by density-gradient centrifugation and cultured for 3 weeks in endothelial growth medium-2-MV (EGM-2-MV) medium containing platelet-derived growth factor-BB (PDGF BB). Before seeded on the synthesized scaffold, SPC-derived smooth muscle outgrowth cell (SOC) phenotypes were assessed by immuno-fluorescent staining, Western blot analysis, and reverse transcription polymerase chain reaction (RT-PCR). The cells were seeded onto the silk fibroin-modified poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (SF-PHBHHx) scaffolds by 6x10(4) cells/cm2 and cultured under the static condition for 3 weeks. The growth and proliferation of the seeded cells on the scaffold were analyzed by 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT) assay, scanning electron microscope (SEM), and 4,6-diamidino-2-phenylindole (DAPI) staining. RESULTS: SOCs displayed specific "hill and valley" morphology, expressed the specific markers of the SMC lineage: smooth muscle (SM) alpha-actin, calponin and smooth muscle myosin heavy chain (SM MHC) at protein and messenger ribonucleic acid (mRNA) levels. RT-PCR results demonstrate that SOCs also expressed smooth muscle protein 22alpha (SM22alpha), a contractile protein, and extracellular matrix components elastin and matrix Gla protein (MGP), as well as vascular endothelial growth factor (VEGF). After seeded on the SF-PHBHHx scaffold, the cells showed excellent metabolic activity and proliferation. CONCLUSION: SPCs isolated from peripheral blood can be differentiated into the SMCs in vitro and have an impressive growth potential in the biodegradable synthesized scaffold. Thus, SPCs may be a promising cell source for constructing TEBVs.

Inhibitory effect of dimeric beta peptide on the recurrence and metastasis of hepatocellular carcinoma in vitro and in mice.

AIM: To block the adhesion of tumor cells to the extracellular matrix, and prevent tumor metastasis and recurrence, the dimer of the beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta2) was designed and synthesized and its anti-adhesion and anti-invasion effects on hepatocellular carcinoma cells were assessed. Additionally, its influence on the metastasis and recurrence of mouse hepatocellular carcinoma was measured. METHODS: The anti-adhesion effect of beta2 on the highly metastatic hepatocellular carcinoma cell line HCCLM6 cells and fibronectin (FN) was assayed by the MTT assay. The inhibition of invasion of HCCLM6 cells by beta2 was observed using a Transwell (modified Boyden chamber) and matrigel. Using the hepatocellular carcinoma metastasis model and LCI-D20 nude mice, the influence of beta2 on the metastasis and recurrence of hepatocellular carcinoma after early resection was investigated. RESULTS: HCCLM6 cells co-incubated with 100 mumol/L, 50 micromol/L, 20 micromol/L or 10 micromol/L beta2 for 3 h showed an obvious decrease in adhesion to FN. The adhesion inhibition ratios were 11.8%, 21.7%, 29.6% and 48.7%, respectively. Additionally, HCCLM6 cells cultured with 100 mumol/L beta2 had a dramatic decrease in cell invasion. beta2 was also observed to inhibit the incisal edge recurrence and the distant metastasis of nude mice hepatocellular carcinoma after early resection (P < 0.05). CONCLUSION: The beta2 peptide can specifically block the adhesion and invasion of HCCLM6 cells, and can inhibit HCC recurrence and metastasis of LCI-D20 model posthepatectomy in vivo. Thus, beta2 should be further studied as a new anti-tumor drug.

Construction of tissue-engineered heart valves by using decellularized scaffolds and endothelial progenitor cells.

BACKGROUND: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study was designed to develop a tissue engineering heart valve by using human umbilical cord blood-derived endothelial progenitor cells (EPCs) and decellularized valve scaffolds. METHODS: Decellularized valve scaffolds were prepared from fresh porcine heart valves. EPCs were isolated from fresh human umbilical cord blood by density gradient centrifugation, cultured for 3 weeks in EGM-2-MV medium, by which time the resultant cell population became endothelial in nature, as assessed by immunofluorescent staining. EPC-derived endothelial cells were seeded onto the decellularized scaffold at 3 x 10(6) cells/cm(2) and cultured under static conditions for 7 days. Proliferation of the seeded cells on the scaffolds was detected using the MTT assay. Tissue-engineered heart valves were analyzed by HE staining, immunofluorescent staining and scanning electron microscopy. The anti-thrombogenic function of the endothelium on the engineered heart valves was evaluated by platelet adhesion experiments and reverse transcription-polymerase chain reaction (RT-PCR) analysis for the expression of endothelial nitric oxide synthase (eNOS) and tissue-type plasminogen activator (t-PA). RESULTS: EPC-derived endothelial cells showed a histolytic cobblestone morphology, expressed specific markers of the endothelial cell lineage including von Willebrand factor (vWF) and CD31, bound a human endothelial cell-specific lectin, Ulex Europaeus agglutinin-1 (UEA-1), and took up Dil-labeled low density lipoprotein (Dil-Ac-LDL). After seeding on the decellularized scaffold, the cells showed excellent metabolic activity and proliferation. The cells formed confluent endothelial monolayers atop the decellularized matrix, as assessed by HE staining and immunostaining for vWF and CD31. Scanning electron microscopy demonstrated the occurrence of tight junctions between cells forming the confluent monolayer. Platelets adhesion experiments suggested that the neo-endothelium was non-thrombogenic. The expression levels of eNOS and t-PA genes in the neo-endothelium were quite similar to those in human umbilical vein endothelial cells. CONCLUSIONS: EPCs isolated from the human umbilical cord blood can differentiate into endothelial cells in vitro and form a functional endothelium atop decellularized heart valve scaffolds. Thus, EPCs may be a promising cell source for constructing tissue-engineered heart valves.

The effect of L-stepholidine, a novel extract of Chinese herb, on the acquisition, expression, maintenance, and re-acquisition of morphine conditioned place preference in rats.

The effect of L-stepholidine (SPD), a novel alkaloid extract of the Chinese herb Stephania with partial dopamine D1 receptor agonistic and D2 receptor antagonistic dual actions, on morphine conditioned place preference (CPP) was studied. Daily injection of morphine (10 mg/kg, i.p.) for 6 days induced CPP in rats, and daily treatment with SPD at 10 or 20 mg/kg before morphine injection dose-dependently attenuated morphine-induced CPP. On the day following acquisition of morphine CPP, a single administration of SPD at 10 or 20 mg/kg failed to block the expression of CPP. However, daily administration of SPD at 20 mg/kg for 7 days attenuated the maintenance of CPP. Morphine-induced CPP extinguished after a 21-day saline training and then a single injection of morphine (3 mg/kg, i.p.) induced re-acquisition of morphine CPP; however, pretreatment with SPD at 10 or 20 mg/kg 30 min before morphine injection dose-dependently blocked morphine (3 mg/kg, i.p.)-induced re-acquisition of morphine CPP. Furthermore, our data indicate that SPD had no effect on food-induced CPP or state-dependent learning, suggesting that the observed effect of SPD does not result from an inhibition of general learning ability. These results demonstrate that SPD can inhibit acquisition, maintenance, and re-acquisition of morphine conditioned place preference and suggest its potential for treatment of opioid addiction.

[Expression of IgG receptor on human umbilical vein endothelial cells stimulated with TNF-alpha and IFN-gamma].

AIM: To detect the expression of IgG receptors (FcgammaR) on cytokine-stimulated human umbilical vein endothelial cells (HUVECs). METHODS: By using ELISA, immunocytochemical staining, immunofluorescent staining and RT-PCR, the expression and subtypes of FcgammaR were detected. RESULTS: Non-stimulated HUVECs expressed very low level of FcgammaRIIa. FcgammaRIIa mRNA was dramatically up-regulated upon 24 hour stimulation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma(IFN-gamma). ELISA results indicated that the expression of FcgammaRIIa increased 16 folds after stimulation with TNF-alpha and IFN-gamma for 3 days (P<0.01). Immunofluorescent staining showed that FcgammaRIIa was expressed on the surface of the stimulated HUVECs. CONCLUSION: TNF-alpha and IFN-gamma could increase FcgammaRIIa expression on HUVECs. The enhanced expression of FcgammaRIIa may mediate the deposition of immune complexes to blood vessels under vasculitic conditions.

[Molecular cloning and expression of anti-tumor adhesion peptide (beta3)].

To block tumor cell adhesion, inhibit tumor metastasis and recurrence, the anti-adhesion peptide-trimeric beta peptide (DLYYLMDLSYSMKGGDLYYLMDLSYSMKGGDLYYLMDLSYSMK, beta3) was designed. The DNA fragment of beta3 was cloned into expression vector pET-His and the fusion protein His-beta3 was expressed in E. coli. BL21(DE3)plysS. After 1.5 hours' induction with IPTG, His-beta3 peptide was expressed significantly amounting to 10% of the insoluble proteins and 4% of the total proteins. 20mg of beta3 peptide was obtained from one litter culture medium after purification by using metal-chelating sepharose 6B FF. The purity of beta3 is 92.2% according to Gel-Pro analysis. The anti-adhesion effects of beta3 peptide, beta1 peptide (DLYYLMDLSYSMK) and GRGDS on the hepatocellular carcinoma cell line SMMC-7721 and the high metastasis hepatocellular carcinoma cell line HCCLM6 were studied. The result showed the beta3 blocked the adhesion of HCCLM6 cells and SMMC-7721 cells to fibronectin (FN) specifically. The inhibition effect was dose-dependent and time-dependent and the inhibition rate of beta3 was higher than three times concentration of beta1 and GRGDS. This suggested that pET-His-beta3/BL21(DE3)plysS was a suitable expression system for beta3, and the expressed beta3 specially inhibited the adhesion of cancer cells.

Biocompatibility of Poly(epsilon-caprolactone) scaffold modified by chitosan--the fibroblasts proliferation in vitro.

In this study, the surface of poly(epsilon-caprolactone) (PCL) scaffold was modified by chitosan (CS) in order to enhance its cell affinity and biocompatibility. It is demonstrated by scanning electronic microscopy (SEM) that when 0.5-2.0 wt% chitosan solutions are used to modify the PCL scaffold, the amount of adhesion of the fibroblasts on the chitosan-modified PCL scaffolds dramatically increase when compared to the control after 7 days cell culture. The results indicate that the chitosan-modified PCL scaffolds are more favorable for cell proliferation by improving the scaffold biocompatibility. The improvement may be helpful for the extensive applications of PCL scaffold in heart valve and blood vessel tissue engineering.

[Preparation of heart valve scaffold and cell seeding].

To prepare scaffolds for heart valve tissue engineering, porcine heart valves were treated with varied concentrations of trypsin for 32, 56, 80 and 104 h or followed with DNase. And then the structure of acellular valves was observed under light microscope, scanning and transmission electron microscope. Porcine endothelial cells, human endothelial cells, and canine myofibroblasts were reseeded onto the acellularized porcine heart valve scaffolds once a day for 3 days. The valves were analyzed by immunohistochemical staining and electron microscopy. Results show that all endothelial cells and the majority of interstitial cells were removed from the heart valves after digestion with trypsin for 104 h, and the collagen fiber structure remains intact, but the space between collagen fibers increased slightly. Incubation with trypsin for 80 h and then with DNase almost removed all cells, and the collagen fiber structure and the space between the fibers remain intact. After reseeding, human endothelial cells almost fully cover the valve scaffold surface as shown by H-E staining and platelet endothelial cell adhesion molecules (PECAM-1) staining. Xenogeneic porcine endothelial cells also adhered to and grew on the scaffolds. As shown by H-E staining and actin staining, canine myofibroblasts not only adhered to the surface of valve scaffold but also migrated to the inner part of matrix after one week culture. These results suggest that the digestion of porcine heart valves with trypsin combining with DNase is a suitable method to remove cells. The acellular porcine heart valve scaffolds have a quite favorable biocompatibility with human and porcine endothelial cells as well as canine myofibroblasts.

Silk fibroin modified porous poly(epsilon-caprolactone) scaffold for human fibroblast culture in vitro.

In order to develop scaffolds with improved biocompatibility for cell culture, hybrid scaffolds were fabricated by modifying poly(epsilon-caprolactone) (PCL) with silk fibroin (SF) in a porous structure. Scanning electronic microscopy revealed that the morphology of the PCL-SF hybrid scaffold was affected by the concentration of the SF solution. Availability of SF on the surface and the conformational transition induced by methanol treatment were proved by attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR), and wettability of the hybrid scaffold was greatly improved. To evaluate scaffold biocompatibility, human fibroblasts were cultured on the hybrid scaffold with the unmodified PCL scaffold as control. An MTT assay indicated that although fewer cells were initially held on the hybrid scaffold after one day of culture, comparable cell numbers were achieved after four days and significantly more cells proliferated on the hybrid after seven days. The cell morphology also indicated that the PCL-SF hybrid scaffold was favorable for cell culture. This study suggests that surface modification with SF would be an effective way to improve the biocompatibility of PCL, facilitating its application in practical tissue engineering.

Signaling transduction by IgG receptors.

OBJECTIVE: To review and summarize literature regarding stimulatory and inhibitory signaling pathways from different types of Fc gamma receptors (FcgammaRs). DATA SOURCE: Articles were obtained from Medline from January 1991 to April 2002. STUDY SELECTION: Over 100 English language papers and reviews published over the last 11 years were selected. RESULTS AND CONCLUSIONS: Stimulatory Fcgamma receptors include FcgammaRI, FcgammaRIIA, FcgammaRIIC, and FcgammaRIII A. They transduce signals through the immunoreceptor tyrosine-based activation motif (ITAM) in subunits or in the cytoplasmic domain. Inhibitory Fcgamma receptors, such as FcgammaRIIB, are single chain receptors, transducing signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM) in cytoplasmic domains. Stimulatory signals include protein phosphorylation, increase in intracellular free calcium, the production of 1,4,5-triphosphate inositol (IP(3)) and diacylglycerol (DAG) mainly through the Src-family kinases, phosphoinositide 3-kinase (PI3-K) and phospholipase C (PLC). Inhibitory signaling has been implicated in the repression of the above activities as well as inhibition of B cell responses through Src homology 2-containing inositol phosphatase (SHIP).