TGF-beta1 phage model peptides isolated from a phage display 7-mer peptide library can inhibit the activity of keloid fibroblasts

Background Transforming growth factor-beta1 (TGF-beta1) is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-beta1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-beta1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-beta1. Enzyme-linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of Nuclear factor kappa B (NF-kappa B) mRNA, connective tissue growth factor (CTGF) mRNA and TGF-beta receptor II (TβRII) mRNA in keloid fibroblasts. Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-beta1 and one phage model peptide (No.4) could promote keloid fibroblasts proliferation, however, three phage model peptides (No.1-3) could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-Kappa B mRNA and CTGF mRNA in the three phage model peptides groups decreased, while the expression of TβRII mRNA slightly increased. Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-beta1 binding to its receptor and then regulating the expressions of NF-kappa B, CTGF and TβRII.     

Screening of specific binding peptide targeting blood vessel of human esophageal cancer in vivo in mice

Background  Cancer of the esophagus and gastroesophageal junction remains a virulent malignancy with poor prognosis. Rapid progresses were made in chemotherapeutic agents and the development of molecular markers allowed better identification of candidates for targeted therapy. This study aimed to identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer.

Methods  The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues (lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and VIII-related antigen in tumor xenograft by immunohistochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test.

Results  The number of phage recovered from tumor tissue of each round increased gradually in tumor group while decreased in control groups (P <0.01 in tumor and spleen, P <0.05 in lung). Immunohistochemical staining showed similar staining pattern with M13 antibody or VIII-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues. Two motifs, YSXNXW and PXNXXN, were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker factor VIII-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1.0×10¹¹ pfu/ml was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues (lung and spleen).

Conclusion  Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer. 

     

Keratinocyte growth factor phage model peptides can promote epidermal cell proliferation without tumorigenic effect

Background Keratinocyte growth factor （KGF） significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-（4,5-dimethylthiazol-2-yl） -2,5-diphenyl tetrazolium bromide （MTT） assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight （56.9%） of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor （EGF）. MTT assay data showed that four （No. 1-4） of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor （KGFR） mRNA in the KGF control group and the two phage model peptide groups （No. 1 and No. 2） increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups （No.1-4）. Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.     

Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

Background  Remodeling of the anterior cruciate ligament (ACL) graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site (IRES) sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 (TGFβ1) and vascular endothelial growth factor 165 (VEGF165) genes (named Ad-VEGF165-IRES-TGFβ1). We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts.
Methods  Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers were assessed by real-time PCR.
Results  The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type I, collagen type III, and fibronectin mRNA among matrix markers.
Conclusion  Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

     

Screening and Identification of a Novel Hepatocellular Carcinoma Cell Binding Peptide by Using a Phage Display Library

The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma(hHCC) cells using phage display of random peptide library in order to de-velope a peptide-based carrier for the diagnosis or therapy of hHCC.A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target.After panning,the phages that specifically bound to and internalized in hHCC cells were selected.The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis.57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM,and 4 amino acid residues,FLEP were extremely conservative.Based on the sequencing results,a 16-mer peptide(WH-16) was synthesized.The competitive ELISA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16.Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.     

Lipopolysaccharide induced activin A-follistatin imbalance affects cardiac fibrosis

Background  Inflammation plays a pivotal role in cardiac remodeling, especially in myocardial fibrosis. Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Previous study has demonstrated that many inflammation stimulating factors trigger transforming growth factor-β (TGF-β) induction and reactive myocardial fibrosis. Activin A (ACT A) is a member of TGF-β superfamily, and follistatin (FS) is an activin-binding protein, i.e. an antagonist of ACT A. Our previous studies have shown that ACT A-FS imbalance occurs in rats with heart failure, and overexpression of ACT A can lead to ventricular remodeling, and resultant heart failure. Low expression of FS after myocardial infarction further exacerbated heart failure. The pathogenic change resulting from overexpression of ACT A is consistent with that of overexpression of angiotensin II (Ang-II). Ventricular remodeling includes cardiocyte remodeling and myocardial interstitial collagen deposition and fibrosis. Therefore, the present study was designed to investigate the effects of inflammatory factors on the ACT A-FS and the secretions of cardiac fibroblasts in order to explore in depth the mechanism of myocardial fibrosis.

Methods  A rat model of heart failure was established, and the results showed that there was a greater degree of cardiac fibrosis in heart failure (HF) rats. In addition, we found that there was an imbalance of the ACT A/FS system in HF rats, which was characterized by increased levels of ACT A. Further, primary rat cardiac fibroblasts were cultured and the MTT assay was performed to determine the effect of the inflammatory factor-bacterial endotoxin lipopolysaccharide (LPS) on cardiac fibroblast proliferation.

Results  The results showed that LPS can stimulate the cardiac fibroblasts to proliferate in a dose dependent manner. Cellular immunohistochemical staining showed that the rat cardiac fibroblasts themselves could express ACT A and FS proteins, and stimulation by LPS could apparently promote the cultured primary rat cardiac fibroblasts to secrete ACT A, but inhibit the secretion of FS. The results also showed that ACT A promoted, in a dose dependent manner, the proliferation of the cultured primary rat cardiac fibroblasts, and the expression of collagen types I and III. Moreover, ACT A promoted, in a dose dependent manner, the cardiac fibroblasts to secrete nitric oxide (NO), and unregulated the expression of inducible nitric oxide synthase (iNOS) mRNA.

Conclusions  These results suggest that the inflammatory mediator LPS can promote ACT A-FS imbalance in cardiac fibroblasts, mainly overexpression of ACT A. Overexpression of ACT A promotes the proliferation and the secretion of collagens in cardiac fibroblasts through autocrine/paracrine stimulation of NO, and is involved in the pathological process of myocardial fibrosis.

     

Lipopolysaccharide induced activin A-follistatin imbalance affects cardiac fibrosis

Background  Inflammation plays a pivotal role in cardiac remodeling, especially in myocardial fibrosis. Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Previous study has demonstrated that many inflammation stimulating factors trigger transforming growth factor-β (TGF-β) induction and reactive myocardial fibrosis. Activin A (ACT A) is a member of TGF-β superfamily, and follistatin (FS) is an activin-binding protein, i.e. an antagonist of ACT A. Our previous studies have shown that ACT A-FS imbalance occurs in rats with heart failure (HF), and overexpression of ACT A can lead to ventricular remodeling, and resultant HF. Low expression of FS after myocardial infarction further exacerbated HF. The pathogenic change resulting from overexpression of ACT A is consistent with that of overexpression of angiotensin II (AngII). Ventricular remodeling includes cardiocyte remodeling and myocardial interstitial collagen deposition and fibrosis. Therefore, the present study was designed to investigate the effects of inflammatory factors on the ACT A-FS and the secretions of cardiac fibroblasts in order to explore in depth the mechanism of myocardial fibrosis.

Methods  A rat model with HF was established, and the results showed that there was a greater degree of cardiac fibrosis in HF rats. In addition, we found that there was an imbalance of the ACT A/FS system in HF rats, which was characterized by increased levels of ACT A. Further, primary rat cardiac fibroblasts were cultured and the MTT assay was performed to determine the effect of the inflammatory factor-bacterial endotoxin lipopolysaccharide (LPS) on cardiac fibroblast proliferation.

Results  The results showed that LPS can stimulate the cardiac fibroblasts to proliferate in a dose-dependent manner. Cellular immunohistochemical staining showed that the rat cardiac fibroblasts themselves could express ACT A and FS proteins, and stimulation by LPS could apparently promote the cultured primary rat cardiac fibroblasts to secrete ACT A, but inhibit the secretion of FS. The results also showed that ACT A promoted, in a dose-dependent manner, the proliferation of the cultured primary rat cardiac fibroblasts, and the expression of collagen types I and III. Moreover, ACT A promoted, in a dose dependent manner, the cardiac fibroblasts to secrete nitric oxide (NO), and unregulated the expression of inducible nitric oxide synthase (iNOS) mRNA.

Conclusions  These results suggest that the inflammatory mediator LPS can promote ACT A-FS imbalance in cardiac fibroblasts, mainly overexpression of ACT A. Overexpression of ACT A promotes the proliferation and the secretion of collagens in cardiac fibroblasts through autocrine/paracrine stimulation of NO, and is involved in the pathological process of myocardial fibrosis.

     

Controlled release of transforming growth factor-beta receptor kinase inhibitor from thermosensitive Chitosan-based hydrogel: Application for prevention of capsular contracture

Background  Capsular contracture has become the most common complication associated with breast implant. Transforming growth factor-beta (TGF-β) is well known for a prominent role in fibrotic diseases. Due to the critical role of TGF-β in pathogenesis of capsular formation, we utilized thermosensitive C/GP hydrogel to controlled release of TGF-β receptor kinase inhibitor (SD208) and investigated their effects on capsular contracture.

Methods  In vitro degradation and drug release of C/GP hydrogel were performed. Twenty-four rabbits underwent subpanniculus implantation with 30 ml smooth silicone implants and were randomly divided into four groups as follows: Group 1 received saline solution; Group 2 received SD208; Group 3 received SD208-C/GP; Group 4 received C/GP. At 8 weeks, the samples of capsular tissues were analyzed by hematoxylin and eosin and immunohistological staining. The mRNA expression of collagen III and TGF-β1 was detected by RT-PCR assay.

Results  C/GP hydrogel could be applied as an ideal drug delivery vehicle which supported the controlled release of SD208. SD208-C/GP treatment showed a significant reduction in capsule thickness with fewer vessels. The histological findings confirmed that the lower amounts of inflammatory cells and fibroblasts infiltrate in SD208-C/GP group. In contrast, typical capsules with more vessel predominance were developed in control group. We did not observe the same inhibitory effect of SD208 or C/GP treatment on capsular contracture. Moreover, SD208-C/GP therapy yielded an evident down-regulation of collagen III and TGF-β1 mRNA expression.

Conclusions  This study demonstrated that controlled release of TGF-β receptor kinase inhibitor from thermosensitive C/GP hydrogel could significantly prevent capsule formation after mammary implants.

     

Effects of HepII domain peptides V of fibronectin on corneal permeability, endothelial cells, intraocular pressure and morphology of trabecular meshwork in rats

Background  Trabecular meshwork (TM) cell volume may be an important determinant of aqueous humor outflow in the eye. This study aimed to evaluate the role of HepII domain peptides V on corneal permeability, corneal endothelial cells, intraocular pressure (IOP) and morphology of trabecular meshwork in rats.

Methods  The IOP of rat eyes was measured before and 3, 5, 7 and 8 hours after topical delivery of HepII domain peptides V through intracameral injections. The peptide’s concentration in aqueous humor was assessed by high performance liquid chromatography (HPLC). The shape and density of endothelial cells were observed by laser confocal microscopy 8 hours, 3 and 14 days after intracameral injections of HepII domain peptides V. The morphological changes in TM of rat eyes were assessed by transmission electron microscopy (TEM).

Results  Intracameral injection of HepII domain peptides V significantly (P <0.001) decreased IOP by (5.71±2.10) mmHg in rats at 5 hours after injection. There were no obvious changes of the shape and the density of corneal endothelial cells. In addition, morphological changes in the TM of rats were observed including the expansion of intercellular spaces in the juxtacanalicular meshwork, removal of extracellular material, cellular relaxation, and cytoskeleton reorganization.

Conclusions  HepII domain peptides V could not penetrate cornea and was safe to corneal endothelial cells. HepII domain peptides V could significantly decrease IOP in rat probably by disorganizing actin cytoskeleton and cell-junction in the TM.

     

Identification and Characterization of Peptide Mimics of Blood Group A Antigen

In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase （GST） fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.     

Pericyst may be a new pharmacological and therapeutic target for hydatid disease

Background  Most hydatid cysts with calcified walls are biologically and clinically silent and inactive. Transforming growth factor-beta 1 (TGF-β1) plays a critical role in the calcification process of cells. The aim of this study was to assess the effect of modulating TGF-β1 signaling on the calcification of hydatid cysts.

Methods  Pericyst cells isolated from hepatic hydatid cysts were cultured with osteogenic media. These cells were assessed for alkaline phosphatase activity and mineralization capacity using Alizarin Red staining. Cells were also treated with recombinant human TGF-β1 and TGF-β inhibitor, and the expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) were analyzed using Western blotting. The effects of inhibiting TGF-β1 signaling on calcification of pericyst walls were assessed using different doses of TGF-β inhibitor for 7 weeks in a preclinical disease model of liver cystic echinococcosis.

Results  Cells within the pericyst displayed high levels of alkaline phosphatase activity and mineralized nodule formation, as induced by osteogenic media. These activities, as well as expression profiles of osteoblast markers (RUNX2, osterix, and osteocalcin) could be inhibited by addition of recombinant human TGF-β1 (rhTGF-β1) and enhanced by TGF-β inhibitor. In the animal model of cystic echinococcosis, inhibition of TGF-β1 signaling increased calcification of the pericyst wall, which was associated with decreased cyst load index and lower viability of protoscoleces.

Conclusions  Cells within the pericysts adopt an osteoblast-like phenotype and have osteogenic potential. Inhibition of TGF-β1 signaling increases hydatid cyst calcification. Pharmacological modulation of calcification in pericysts may be a new therapeutic target in the treatment of hydatid disease.

     

Ultraviolet B light-induced apoptosis in human keratinocytes enriched with epidermal stem cells and normal keratinocytes

Background  The stem-cell compartment is the primary target for the accumulation of oncogenic mutations. Overexposure to solar ultraviolet radiation is responsible for the development and progression of >90% of skin cancers. Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.

Methods  Keratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen IV and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0–80 mJ/cm². The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 Western blotting.

Results  Keratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm² UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm² UVB.

Conclusions  Human epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.

     

Effects of ovarian cancer G protein coupled receptor 1 on the proliferation, migration, and adhesion of human ovarian cancer cells

Background  OGR1 was found as a G-protein coupled receptor (GPCR) and proton sensor. Our previous studies have found that OGR1 has inhibitory effect on the metastasis of prostate cancer. In order to investigate the roles of OGR1 gene in the biological activities of ovarian cancer, we studied the OGR1 effects on ovarian cancer cells, HEY cells.

Methods  OGR1 gene was transfected into HEY cell, in which endogenous expression is low. OGR1-overxepressed cells and vector-transfected cells were compared in different assays. Western blotting was employed to confirm the high expression level of OGR1. Cell proliferation was determined by MTT assay and cell doubling time assay. Cell migration assay (transwell assay) and cell adhesion assay were performed to determine the migration and adhesion potential of cells. Student’s t test was employed for statistical analysis.

Results  Proliferation of OGR1-overexpressed cells was significantly reduced (P <0.01); cell migration was significantly inhibited in the OGR1-transfected cells (P <0.01); cell adhesion to extracellular matrix including fibronectin, vitronectin, collagen I/ IV was significantly increased (P <0.01).

Conclusions  OGR1 expression in human ovarian cancer cells significantly inhibited the cell proliferation and migration, but significantly enhanced cell adhesion to the extracellular matrix. It indicated that OGR1 may be a tumor suppressor gene for ovarian cancer.

     

Granulocyte-macrophage colony-stimulating factor and interleukin 4 induce the malignant transformation of the bone marrow-derived human adult mesenchymal stem cells

Background  The purpose of the study was to examine the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) on the bone-marrow-derived human adult mesenchymal stem cells (hMSCs).

Methods  The hMSCs were isolated and cultured with GM-CSF and IL-4 for a period of one month. A single colony of transformed cells was then isoloated and their phenotype was characterized by morphology, surface marker expression, and in vivo tumorigenesis.

Results  After one month culture, the transformed mesenchymal cells exhibited the morphology and phenotype similar to those of tumor cells, and also caused multiple fast growing lung deposits when it was injected into immunodeficient mice.

Conclusion  Cytokines-driven malignant transformation of hMSCs may be a useful model for studying signaling pathways initiating malignant transformation of hMSC.

     

Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina

Background  Endothelial progenitor cells (EPCs) transplantation is a promising therapeutic strategy for ischemic retinopathy. The current study aimed to establish a simple, reliable and fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) in laser-injured mouse retina.

Methods  EPCs were isolated from human umbilical cord blood mononuclear cells, cultivated, and labeled with various concentrations of CFSE. Based on fluorescence intensity and cell morphology, a 15 minutes incubation with 5 μmol/L CFSE at 37°C was selected as the optimal labeling condition. The survival capability and the apoptosis rate of CFSE-labeled EPCs were measured by Trypan blue staining and Annexin V/PI staining assay respectively. Fluorescence microscopy was used to observe the label stability during the extended culture period. Labeled EPCs were transplanted into the vitreous cavity of pigmented mice injured by retinal laser photocoagulation. Evans Blue angiography and flat mounted retinas were examined to track the labeled cells.

Results  EPCs labeled with 5 μmol/L CFSE presented an intense green fluorescence and maintained normal morphology, with no significant changes in the survival capability or apoptosis rate after being labeled for 2 days, 1 and 4 weeks. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. Evans Blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks.

Conclusion  EPCs could be labeled by CFSE in vitro and monitored in vivo for at least 4 weeks, and participate in the repair of injured retinal vessels.

     

Induction of actin disruption and downregulation of P-glycoprotein expression by solamargine in multidrug-resistant K562/A02 cells

Background  Solamargine (SM), a steroidal glycoalkaloid isolated from the Chinese herb Solanum incanum, has been shown to inhibit the growth of some cancer cell lines and induce significant apoptosis. However, the effects of SM on multidrug-resistant (MDR) cells and the molecular mechanisms involved are poorly understood. The purpose of this study was to evaluate the anti-MDR effects of SM and the associated mechanisms in MDR K562/A02 cells.

Methods  The cytotoxicity of SM was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay. The 14′,6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry were used to detect SM-induced apoptosis. The mRNA expression of P-glycoprotein (P-gp) was investigated by real-time PCR (RT-PCR). Western blotting was used to determine the expression of Bcl-2, Bax, and actin. The changes in the morphology of actin were examined with immunofluorescence staining.

Results  MTT results showed that SM effectively killed the MDR sublines K562/A02, KB/VCR, and H460/paclitaxel (Taxol), and their parental cell lines K562, KB, and H460 to an equivalent or more sensitive degree. Based on the results by flow cytometry and immunostaining, the pro-apoptotic effects of SM were observed in MDR K562/A02 cells. Furthermore, the RT-PCR results showed that SM induced the downregulation of MDR1 mRNA. In addition, the expression of P-gp and actin was decreased in the SM-treated cells, as measured by western blotting and immunostaining.

Conclusions  These results demonstrate that SM effectively triggers apoptosis in MDR tumor cells, which is associated with actin disruption and downregulation of MDR1 expression. This compound may merit further investigation as a potential therapeutic agent that bypasses the MDR mechanism for the treatment of MDR tumors.