·AIM: To determine the effects of laser photocoagulation on serum levels of angiopoietin-1(Ang-1),angiopoietin-2(Ang-2), soluble angiopoietin receptor Tie-2(Tie-2), Ang-1/Ang-2 ratio and vascular endothelial grow...·AIM: To determine the effects of laser photocoagulation on serum levels of angiopoietin-1(Ang-1),angiopoietin-2(Ang-2), soluble angiopoietin receptor Tie-2(Tie-2), Ang-1/Ang-2 ratio and vascular endothelial growth factor(VEGF) in patients with type 2diabetes mellitus(T2DM) and proliferative diabetic retinopathy(PDR). We also explored the role of the Ang/Tie system in PDR.·METHODS:Totally 160patientswithT2 DM, including50 patients with non-diabetic retinopathy(NDR), 58 patients with non-proliferative diabetic retinopathy(NPDR), and52 patients with PDR were enrolled in this study. Serum Ang-1, Ang-2, Tie-2 receptor and VEGF levels were measured using enzyme-linked immunosorbent assays for all patients and were repeated in 26 patients who underwent laser photocoagulation two months after the procedure.·RESULTS:ThemedianlevelsofAng-2andVEGFinserum were significantly higher in the NPDR group(4.23 ng/mL and 303.2 pg/mL, respectively) compared to the NDR group(2.67 ng/mL and 159.8 pg/mL, respectively, P 【0.01), with the highest level in the PDR group(6.26 ng/mL and531.2 pg/mL, respectively, P 【0.01). The median level of Ang-1 was significantly higher in the NPDR group(10.77ng/mL) compared to the NDR group(9.31 ng/mL) and the PDR groups(9.54 ng/mL)(P 【0.05), while no difference was observed between the PDR and NDR groups. Ang-1/Ang-2 ratio of PDR group was lowest in three groups(1.49 vs 2.69 and 2.90, both P 【0.01). The median level of Tie-2was not significantly different among three groups(P 】0.05).Ang-2 was positively correlated with VEGF and Tie-2 in the PDR and NPDR groups(both P 【0.05). Among the 26 patients who underwent laser photocoagulation, serum Ang-2 and VEGF levels significantly decreased(both P 【0.05), whereas serum Ang-1 level and Ang-1/Ang-2ratio were weakly increased(P 】0.05). The median levels of Ang-2 and VEGF in serum were highest in PDR group,however, Ang-1/Ang-2 ratio of PDR group was lowest in three groups.·CONCLUSION: Laser photocoagulation can reduce serum Ang-2 and VEGF levels. The Ang/Tie system and VEGF play an important role in the development and progression of T2 DM patients with PDR.展开更多
BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR typ...BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.展开更多
This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purifie...This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.展开更多
文摘·AIM: To determine the effects of laser photocoagulation on serum levels of angiopoietin-1(Ang-1),angiopoietin-2(Ang-2), soluble angiopoietin receptor Tie-2(Tie-2), Ang-1/Ang-2 ratio and vascular endothelial growth factor(VEGF) in patients with type 2diabetes mellitus(T2DM) and proliferative diabetic retinopathy(PDR). We also explored the role of the Ang/Tie system in PDR.·METHODS:Totally 160patientswithT2 DM, including50 patients with non-diabetic retinopathy(NDR), 58 patients with non-proliferative diabetic retinopathy(NPDR), and52 patients with PDR were enrolled in this study. Serum Ang-1, Ang-2, Tie-2 receptor and VEGF levels were measured using enzyme-linked immunosorbent assays for all patients and were repeated in 26 patients who underwent laser photocoagulation two months after the procedure.·RESULTS:ThemedianlevelsofAng-2andVEGFinserum were significantly higher in the NPDR group(4.23 ng/mL and 303.2 pg/mL, respectively) compared to the NDR group(2.67 ng/mL and 159.8 pg/mL, respectively, P 【0.01), with the highest level in the PDR group(6.26 ng/mL and531.2 pg/mL, respectively, P 【0.01). The median level of Ang-1 was significantly higher in the NPDR group(10.77ng/mL) compared to the NDR group(9.31 ng/mL) and the PDR groups(9.54 ng/mL)(P 【0.05), while no difference was observed between the PDR and NDR groups. Ang-1/Ang-2 ratio of PDR group was lowest in three groups(1.49 vs 2.69 and 2.90, both P 【0.01). The median level of Tie-2was not significantly different among three groups(P 】0.05).Ang-2 was positively correlated with VEGF and Tie-2 in the PDR and NPDR groups(both P 【0.05). Among the 26 patients who underwent laser photocoagulation, serum Ang-2 and VEGF levels significantly decreased(both P 【0.05), whereas serum Ang-1 level and Ang-1/Ang-2ratio were weakly increased(P 】0.05). The median levels of Ang-2 and VEGF in serum were highest in PDR group,however, Ang-1/Ang-2 ratio of PDR group was lowest in three groups.·CONCLUSION: Laser photocoagulation can reduce serum Ang-2 and VEGF levels. The Ang/Tie system and VEGF play an important role in the development and progression of T2 DM patients with PDR.
基金Supported by the Agencia Nacional de Promoción Científica y TecnológicaNo. PICT-2013-1441+8 种基金Consejo Nacional de Investigaciones Científicas y TécnicasNo. PIP11220150100350Instituto Nacional del Cáncer Asistencia Financiera ⅡRESOL 493/14, No. 2002-4395-14-1Instituto Nacional del Cáncer Asistencia Financiera Ⅲ-2016-2017, RESOL-2016-1006-E-APN-MSNo. 2002-3862-16-1 CANCERUniversidad Nacional del SurNo. PGI:24/B230 and No. PGI:24/B303Fundación Alberto J. Roemmers of Argentina
文摘BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.
基金supported by the Rhone Alps Region of France, MIRA grants for the collaboration,the Agence National de la Recherche (ANR) RIB,the CNRS (International program for Scientific Cooperation, "PICS")
文摘This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.
