OBJECTIVETo determine the autocrine effect of vascular endothelial growth factor (VEGF) on epidermal keratinocytes HaCaT cells.

METHODSCultured HaCaT cells were treated with various concentrations of VEGF(165) (0,1,5,10,25,50,100 ng/ml) or Avastin (0,0.063,0.125,0.25,0.50,1.0,2.0 mg/ml) in vitro. HaCaT cell proliferation was determined by MTT assay and the cell migration was measured by migration assay. The effect of VEGF(165) (10 ng/ml) on phosphorylation of ERK1/2 was detected in HaCaT cells pretreated or not pretreated with Avastin (0.5 mg/ml).

RESULTSVEGF enhanced the proliferation and migration of HaCaT cells in a dose-dependent manner, while Avastin inhibited the effects of VEGF also in a dose-dependent manner. VEGF(165) (10 ng/ml) induced the phosphorylation of ERK1/2 in HaCaT cells,but which was blocked by Avastin (0.5 mg/ml).

CONCLUSIONVEGF enhanced the proliferation and migration of HaCaT cells in a dose-dependent manner, while Avastin inhibited the effects of VEGF also in a dose-dependent manner. VEGF(165) (10 ng/ml) induced the phosphorylation of ERK1/2 in HaCaT cells,but which was blocked by Avastin (0.5 mg/ml).

Autocrine Communication ; Cell Line ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Epidermis ; cytology ; Humans ; Keratinocytes ; cytology ; Skin ; cytology ; Vascular Endothelial Growth Factor A ; pharmacology

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta(1-42)) induced ATP release, which in turn activated NADPH oxidase via the P2X(7) receptor (P2X(7)R). Reactive oxygen species (ROS) production in fAbeta(1-42)-treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X(7)R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta(1-42)-induced Ca2+ influx was mediated through P2X(7)R activation. In serial experiments, we found that microglial pretreatment with the P2X(7)R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 micrometer) or oxidized ATP (100 micrometer) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta(1-42)-stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.
Adenosine Triphosphate/*metabolism ; Amyloid beta-Protein/*pharmacology ; Animals ; Autocrine Communication/*drug effects/physiology ; Cells, Cultured ; Microglia/*drug effects ; Peptide Fragments/*pharmacology ; Pyridoxal Phosphate/analogs & derivatives/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; Receptors, Purinergic P2/physiology

To isolate bone marrow mesenchymal stem cells (BM-MSCs) and establish the model of chronic kidney disease (CKD) of Wuzhishan (WZS) mini-pig, and to study the repairment effect of BM-MSCs on CKD-induced renal fibrosis in vitro.
 Methods: Density gradient method was used to isolate and culture BM-MSCs. The cells were verified by morphology, phenotype, differentiation and so on. The left partial ureteral obstruction (LPUUO) was used to establish the CKD model, which was evaluated by B-ultrasound, single-photon emission computed tomography (SPECT), HE and Masson staining. The cells were divided into 3 groups, the tissue plus BM-MSCs group, the tissue group, and the BM-MSCs group, respectively. Seven days later, the supernatants were collected to observe the changes of hepatocyte growth factor (HGF) cumulative release. HE and Masson staining was used to observe the changes of renal tissue.
 Results: The isolated BM-MSCs possessed the features as follow: fibroblast-like adherent growth; positive in CD29 and CD90 expression while negative in CD45 expression; osteogenic induction and alizarin red staining were positive; alcian blue staining were positive after chondrogenic induction. Twelve weeks after the operation of LPUUO, B-ultrasound showed the thin renal cortical with pelvis effusion; SPETCT showed the left kidney delayed filling and renal impairment. The accumulation of HGF in the tissue plus BM-MSCs group was significantly higher than that in the tissue alone group at the 1st, 5th, 6th, 7th day, respectively (P<0.05). HE staining showed the different degree of renal lesions between the tissue plus BM-MSCs+CKD group and the tissue alone group, which was aggravated with the time going. Masson staining showed that the cumulative optical density of blue-stained collagen fibers in tissue plus BM-MSCs group was significantly lower than that in the tissue group at the 5th to 7th day (P<0.05).
 Conclusion: BM-MSCs from WZS mini-pig can inhibit or delay the progress of CKD-induced renal fibrosis through autocrine HGF in vitro.
Animals ; Autocrine Communication ; physiology ; Bone Marrow Cells ; Cells, Cultured ; Fibrosis ; physiopathology ; prevention & control ; Hepatocyte Growth Factor ; metabolism ; Kidney ; drug effects ; pathology ; physiopathology ; Mesenchymal Stem Cells ; drug effects ; Renal Insufficiency, Chronic ; complications ; physiopathology ; Swine ; Swine, Miniature ; Ureteral Obstruction ; complications

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50 = 15.8 microg/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.
Animals ; Autocrine Communication/*drug effects ; Catechin/*analogs & derivatives/metabolism/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects ; Female ; *Hepatocyte Growth Factor ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental/*metabolism/pathology ; Paracrine Communication/*drug effects ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-met/antagonists & inhibitors/metabolism ; Receptors, Growth Factor/antagonists & inhibitors/metabolism ; Signal Transduction

Normal human epidermal keratinocytes (NHEK) respond to the autocrine activated extracellular signal-regulated kinase (ERK) signaling pathway, which contributes to the survival of keratinocytes. However, during the condition of calcium-induced differentiation, how the autocrine ERK signaling is regulated and affected is poorly understood. The purpose of this study was to understand and to obtain clues to the possible function of the autocrine ERK activation during the calcium-induced differentiation of NHEK. We demonstrated that the autocrine activated ERK was not interrupted by calcium triggering and that it was sustained for at least one day after changing the medium. We also found that the autocrine ERK activation was associated with the expression of cyclin D1 and the cell cycle regulation at the early stage of calcium triggering by treating the cells with the mitogen-activated protein kinase inhibitor PD98059. However, the PD98059 treatment did not have a significant influence on the expression of involucrin and loricrin. In addition, we demonstrated that autocrine ERK activation was associated with protein kinase C and p38MAPK signaling. We suggest that the activation of autocrine ERK is not interrupted by calcium triggering and it might participate in cell growth during the early stage of calcium-induced differentiation in NHEK.
Keratinocytes/*cytology/drug effects/*physiology ; Humans ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Enzyme Activation/drug effects ; Dose-Response Relationship, Drug ; Cells, Cultured ; Cell Differentiation/drug effects ; Cell Cycle/drug effects/*physiology ; Calcium Signaling/drug effects/*physiology ; Calcium/*administration & dosage ; Autocrine Communication/drug effects/*physiology

OBJECTIVETo explore the regulatory effect and mechanism of Ningxin Hongqi Capsule on local ovarian autocrine and paracrine factors in peri-menopausal rats.

METHODSSD female rats aged 4 months were allocated in a normal control group (A) and those aged 14 months with vagino-cytologic figure of oestrus elongation were allocated in a senile female rat model group (B). Rats in Group B were subdivided into 5 groups randomly as the B1, B2 and B3 subgroups treated respectively with high, moderate and low dose Ningxin Hongqi Capsule, the B4 subgroup treated with estradiol and the B5 subgroup untreated for control. Rats' ovaries were obtained at the end of the experiment for observing the conditions of ovarian growing follicles and corpus luteum by HE staining, determining expressions of ovarian estradiol receptor (ER), progesterone receptor (PR), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin alpha (INHalpha), activin (ACT) alpha-beta, follistatin (FS), and insulin-like growth factor (IGF-1).

RESULTSAs compared with Group B5, the ovary index, number of growing follicle were higher and levels of FSH and LH were lower in Group B2 and B3, expression of ER was higher in Group B1 and B4, IGF-1 and INHalpha was higher in Group B2 and B3, and ACTalpha-beta and FS were lower (all P < 0.05).

CONCLUSIONNirigxin Hongqi Capsule could adjust and balance the local ovarian autocrine and paracrine factors to improve the ovarian function.

Animals ; Autocrine Communication ; drug effects ; physiology ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; Female ; Humans ; Models, Animal ; Ovary ; drug effects ; metabolism ; physiology ; Paracrine Communication ; drug effects ; physiology ; Perimenopause ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Estradiol ; biosynthesis ; Receptors, FSH ; biosynthesis ; Receptors, Progesterone ; biosynthesis

OBJECTIVETo evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma.

RESULTSThe proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased.

CONCLUSIONBlockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.

Antineoplastic Agents ; pharmacology ; Antisense Elements (Genetics) ; biosynthesis ; genetics ; Autocrine Communication ; drug effects ; Bone Neoplasms ; metabolism ; pathology ; Cell Division ; drug effects ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Humans ; Osteosarcoma ; metabolism ; pathology ; RNA, Messenger ; genetics ; Transfection ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1