Vascular endothelial growth factors（VEGFs） respectively bind to each of three receptor tyrosine kinases（RTKs）,known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases（VE...Vascular endothelial growth factors（VEGFs） respectively bind to each of three receptor tyrosine kinases（RTKs）,known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases（VEGFRs） are critical proteins which can regulate vascular development during angiogenesis,we decided to explore the inhibitory effects of soluble kinase insert domain-containing receptor（sKDR） on endothelial cells and angiogenesis.Total RNA was extracted from human umbilical vein endothelial cells（HUVEC）,and cDNA of extracellular domains 1―4 was amplified and recombined with pQE40 vector.After being expressed,affinity purified,renatured and analyzed by Western blot,the sKDR was assayed for its effects on endothelial cells by [3-（4,5-dimethylthiazol-2-yl）2,5-diphenyltetrazolium bromide]（MTT）,and on angiogenesis by chick chorioallantoic membrane（CAM） experiment.sKDR cDNA of 1150 bp was obtained via real-time polymerase chain reaction（RT-PCR）,and sKDR was expressed by pQE40 procaryotic expression system,purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） analysis with only one band and proved by Western blot.MTT assay demonstrateds that sKDR could inhibit the VEGF-stimulated HUVEC from proliferation,and CAM experiment showed sKDR could block the VEGF-induced angiogenesis.sKDR has the biological activity to bind with VEGF ligands and is a potential target for tumor anti-angiogenesis therapy.展开更多
Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of co...Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.展开更多
Exposure to fine ambient particulate matter(PM_(2.5)) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM_(2.5), we investigated alterat...Exposure to fine ambient particulate matter(PM_(2.5)) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM_(2.5), we investigated alterations in the protein profile of human umbilical vein endothelial cells(HUVECs) treated with PM_(2.5) using two-dimensional electrophoresis in conjunction with mass spectrometry(MS). A total of 31 protein spots were selected as differentially expressed proteins and identified by matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) MS. The results demonstrated that DNA damage and cell apoptosis are important factors contributing to PM_(2.5)-mediated toxicity in HUVECs. It is further proposed that PM_(2.5) can inhibit superoxide dismutase(SOD) activity and increase reactive oxygen species(ROS) and malonaldehyde(MDA) production in a concentration-dependent manner. Induction of apoptosis and DNA damage through oxidative stress pathways may be one of the key toxicological events occurring in HUVECs under PM_(2.5) stress. These results indicated that the toxic mechanisms of PM_(2.5) on cardiovascular disease are related to endothelial dysfunction.展开更多
This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide(H_(2)O_(2))-induced oxidative stress in human umbilical vein endothelial cells(HUVECs).We constructed a new func...This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide(H_(2)O_(2))-induced oxidative stress in human umbilical vein endothelial cells(HUVECs).We constructed a new functional L.plantarum(NC8-pSIP409-alr-angiotensin-converting enzyme inhibitory peptide(ACEIP))with a double-gene-labeled non-resistant screen as an expression vector.A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)colorimetric assay was carried out to determine the cell viability of HUVEC cells following pretreatment with NC8-pSIP409-alr-ACEIP.Flow cytometry(FCM)was used to determine the apoptosis rate of HUVEC cells.Cysteinyl aspartate specific proteinase(caspase)-3/8/9 activity was also assayed and western blotting was used to determine protein expression of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),inducible nitric oxide synthase(iNOS),nicotinamide adenine dinucleotide phosphate oxidase 2(gp91phox),angiotensin II(AngII),and angiotensin-converting enzyme 2(ACE2),as well as corresponding indicators of oxidative stress,such as reactive oxygen species(ROS),mitochondrial membrane potential(MMP),malondialdehyde(MDA),and superoxide dismutase(SOD).NC8-pSIP409-alr-ACEIP attenuated H_(2)O_(2)-induced cell death,as determined by the MTT assay.NC8-pSIP409-alr-ACEIP reduced apoptosis of HUVEC cells by FCM.In addition,compared to the positive control,the oxidative stress index of the H_(2)O_(2)-induced HUVEC(Hy-HUVEC),which was pretreated by NC8-pSIP409-alr-ACEIP,iNOS,gp91phox,MDA,and ROS,was decreased obviously;SOD expression level was increased;caspase-3 or-9 was decreased,but caspase-8 did not change;Bcl-2/Bax ratio was increased;permeability changes of mitochondria were inhibited;and loss of transmembrane potential was prevented.Expression of the hypertension-related protein(AngII protein)in HUVEC cells protected by NC8-pSIP409-alr-ACEIP decreased and expression of ACE2 protein increased.These plantarum results suggested that NC8-pSIP409-alr-ACEIP protects against H_(2)O_(展开更多
Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUV...Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.展开更多
文摘Vascular endothelial growth factors（VEGFs） respectively bind to each of three receptor tyrosine kinases（RTKs）,known as Flt-1,KDR and Flt-4.Since VEGFs and their respective families of receptor tyrosine kinases（VEGFRs） are critical proteins which can regulate vascular development during angiogenesis,we decided to explore the inhibitory effects of soluble kinase insert domain-containing receptor（sKDR） on endothelial cells and angiogenesis.Total RNA was extracted from human umbilical vein endothelial cells（HUVEC）,and cDNA of extracellular domains 1―4 was amplified and recombined with pQE40 vector.After being expressed,affinity purified,renatured and analyzed by Western blot,the sKDR was assayed for its effects on endothelial cells by [3-（4,5-dimethylthiazol-2-yl）2,5-diphenyltetrazolium bromide]（MTT）,and on angiogenesis by chick chorioallantoic membrane（CAM） experiment.sKDR cDNA of 1150 bp was obtained via real-time polymerase chain reaction（RT-PCR）,and sKDR was expressed by pQE40 procaryotic expression system,purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） analysis with only one band and proved by Western blot.MTT assay demonstrateds that sKDR could inhibit the VEGF-stimulated HUVEC from proliferation,and CAM experiment showed sKDR could block the VEGF-induced angiogenesis.sKDR has the biological activity to bind with VEGF ligands and is a potential target for tumor anti-angiogenesis therapy.
文摘Objective: To investigate the effect of activated protein C (APC) on inflammatory responses in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS). Methods: The second passage of collagenase digested HUVEC was divided into the following groups: serum free medium control group (SFM control), phosphate buffer solution control group (PBS control), LPS group with final concentration of 1 μg/ml (LPS group), APC group with final concentration of 7 μg/ml, Pre-APC group (APC pretreatment for 30 min prior to LPS challenge), and Post-APC group (APC administration 30 min after LPS challenge). Supernatant was harvested at 0, 4, 8, 12 and 24 h after LPS challenge. Interleukin-6 (IL-6) and Interleukin-8 (IL-8) levels were analyzed with ELISA. Cells were harvested at 24 h after LPS challenge, and total RNA was extracted. Mes-senger RNA levels for IL-6 and IL-8 were semi-quantitatively determined by RT-PCR. Results: Compared with control group, IL-6 and IL-8 levels steadily increased 4 to 24 h after LPS stimulation. APC treatment could increase LPS-induced IL-6 and IL-8 production. The mRNA levels of IL-6 and IL-8 exhibited a similar change. Conclusion: APC can further increase the level of IL-6 and IL-8 induced by LPS. The effect of these elevated cytokines is still under investigation.
基金Project supported by the Medical and Health Science and Technology Fund of Zhejiang Province(No.2016KYB224)the Scientific Research Fund of Zhejiang Chinese Medicine University(No.2015ZG17),China
文摘Exposure to fine ambient particulate matter(PM_(2.5)) is known to be associated with cardiovascular disease. To uncover the molecular mechanisms involved in cardiovascular toxicity of PM_(2.5), we investigated alterations in the protein profile of human umbilical vein endothelial cells(HUVECs) treated with PM_(2.5) using two-dimensional electrophoresis in conjunction with mass spectrometry(MS). A total of 31 protein spots were selected as differentially expressed proteins and identified by matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) MS. The results demonstrated that DNA damage and cell apoptosis are important factors contributing to PM_(2.5)-mediated toxicity in HUVECs. It is further proposed that PM_(2.5) can inhibit superoxide dismutase(SOD) activity and increase reactive oxygen species(ROS) and malonaldehyde(MDA) production in a concentration-dependent manner. Induction of apoptosis and DNA damage through oxidative stress pathways may be one of the key toxicological events occurring in HUVECs under PM_(2.5) stress. These results indicated that the toxic mechanisms of PM_(2.5) on cardiovascular disease are related to endothelial dysfunction.
基金This work is supported the National Key Research and Development Program of China(Nos.2017YFD0501000 and 2017YFD0501200)the National Natural Science Foundation of China(Nos.31672528,31941018,and 32072888)+1 种基金the Science and Technology Project of Jilin Provincial Department of Education(No.JJKH20190942KJ)the Science and Technology Development Program of Jilin Province(Nos.20180201040NY and 20190301042NY),China.
文摘This study probed the protective effect of recombinant Lactobacillus plantarum against hydrogen peroxide(H_(2)O_(2))-induced oxidative stress in human umbilical vein endothelial cells(HUVECs).We constructed a new functional L.plantarum(NC8-pSIP409-alr-angiotensin-converting enzyme inhibitory peptide(ACEIP))with a double-gene-labeled non-resistant screen as an expression vector.A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide(MTT)colorimetric assay was carried out to determine the cell viability of HUVEC cells following pretreatment with NC8-pSIP409-alr-ACEIP.Flow cytometry(FCM)was used to determine the apoptosis rate of HUVEC cells.Cysteinyl aspartate specific proteinase(caspase)-3/8/9 activity was also assayed and western blotting was used to determine protein expression of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),inducible nitric oxide synthase(iNOS),nicotinamide adenine dinucleotide phosphate oxidase 2(gp91phox),angiotensin II(AngII),and angiotensin-converting enzyme 2(ACE2),as well as corresponding indicators of oxidative stress,such as reactive oxygen species(ROS),mitochondrial membrane potential(MMP),malondialdehyde(MDA),and superoxide dismutase(SOD).NC8-pSIP409-alr-ACEIP attenuated H_(2)O_(2)-induced cell death,as determined by the MTT assay.NC8-pSIP409-alr-ACEIP reduced apoptosis of HUVEC cells by FCM.In addition,compared to the positive control,the oxidative stress index of the H_(2)O_(2)-induced HUVEC(Hy-HUVEC),which was pretreated by NC8-pSIP409-alr-ACEIP,iNOS,gp91phox,MDA,and ROS,was decreased obviously;SOD expression level was increased;caspase-3 or-9 was decreased,but caspase-8 did not change;Bcl-2/Bax ratio was increased;permeability changes of mitochondria were inhibited;and loss of transmembrane potential was prevented.Expression of the hypertension-related protein(AngII protein)in HUVEC cells protected by NC8-pSIP409-alr-ACEIP decreased and expression of ACE2 protein increased.These plantarum results suggested that NC8-pSIP409-alr-ACEIP protects against H_(2)O_(
文摘Objective To investigate the cellular effects of cigarette smoke extract (CSE) on primarily cultured human umbilical vein endothelial cells (HUVEC). Methods The effects of CSE (5%-20%) and nicotine (10-4 mol/L) on HUVEC viability, proliferation, angiogenesis and apoptosis were observed. Results CSE decreased HUVEC survival rate and angiogenesis after 24 h as well as its proliferation after 48 h in a dose-dependent manner. Moreover, CSE induced apoptosis of HUVEC as indicated in condensation of nuclear chromatin and the presence of hypodiploid DNA. HUVEC incubated with CSE for 24 h gave a significant decrease in the expression of Bcl-2 as well as the decline in the Bcl-2/Bax ratio accompanied with the loss of mitochondrial membrane potential and excess cytosolic calcium. Our study also observed that p53 protein level decreased, rather than increased in cells treated with CSE. Nicotine had no discernible inhibitory effects on the above indices of HUVEC. Conclusion Exposure to CSE other than nicotine causes inhibition of viability, proliferation and differentiation of HUVEC. CSE-induced HUVEC injury is mediated in part through accelerated apoptosis but independent of p53 pathway. It appears that mitochondria have played a key role in the apoptosis of HUVEC induced by CSE.
