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Objective:To study the effects of Fangji Huangqitang（FJHQT） on migration， adhesion，invasion and tube formation of human umbilical vein endothelial cells （HUVECs） induced by vascular endothelial growth factor （VEGF）. Method:HUVECs were induced by VEGF （20 μg·L<sup>-1</sup>） <italic>in vitro</italic>. The effects of FJHQT （0.25，0.5，1 g·L<sup>-1</sup>） on HUVECs were detected by methyl thiazolyl tetrazolium（MTT）， scratch repair， transwell migration， adhesion， invasion and tube formation. Protein in HUVECs was extracted and protein expression levels of phosphorylated Janus kinase 1 （p-JAK1） were detected by Western blot. Result:Compared with control group， VEGF （20 μg·L<sup>-1</sup>） can increase the proliferation， scratch repair， transwell migration， adhesion， invasion and tube formation of HUVECs cells （<italic>P</italic><0.01）， compared with VEGF group， FJHQT （0.25，0.5，1 g·L<sup>-1</sup>） ，there is no significant effect on the proliferation of HUVECs induced by VEGF for 24 hours， but it can significantly reduce the scratch repair， migration， adhesion， invasion and tube formation of HUVECs induced by VEGF within 24 hours （<italic>P</italic><0.05）. Compared with blank group， VEGF could induce abnormal elevation of p-JAK1 in HUVECs （<italic>P</italic><0.01）， while FJHQT （0.25，0.5，1 g·L<sup>-1</sup>） could significantly reduce the expression levels of p-JAK1 （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:FJHQT can inhibit the migration， adhesion and invasion of HUVECs， the mechanism may be related to JAK1.
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The study investigates the mechanism by which Peganum harmala L. (Luotuopeng, LTP) inhibits tube formation in retinal vascular endothelial cells. Tube formation was induced by treatment of retinal vascular endothelial cells with glucose. The cells were divided into a normal group, model group, and an LTP group. The total length of tube formation was measured. The active components, targets, and pathway by which LTP acts in the treatment of diabetic retinopathy was explored by network pharmacology. The mRNA expression levels of targets [extracellular signal-regulated kinase 2 (ERK2), phosphoinositide 3 kinase catalytic alpha polypeptide (PIK3CA), serine/threonine-protein kinase 1 (AKT1)] related to the mitogen-activated protein kinase (MAPK) signaling pathway and vascular endothelial growth factor (VEGF) signaling pathway was measured by real-time PCR. The results of tube formation indicated that compared with the normal group, the total tube length increased in the model group (P < 0.01); after the treatment with LTP, the total tube length decreased compared with the model group (P < 0.01). Network pharmacology revealed that the targets of LTP included PIK3CA, AKT1, and ERK2, and the pathways involved the MAPK signaling pathway and the VEGF signaling pathway. Real-time PCR indicated that compared with the normal group, the mRNA expression levels of ERK2, PIK3CA and AKT1 were elevated in the model group (P < 0.05); after treatment with LTP, the mRNA expression levels of ERK2, PIK3CA and AKT1 decreased compared with the model group (P < 0.05). LTP may inhibit retinal vascular endothelial cell tube formation by regulating the MAPK signaling pathway and the VEGF signaling pathway. This study confirms the multi-targets and multi-pathways of LTP and provides a basis for its use in the treatment of diabetic retinopathy．
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BACKGROUND: The research on neovascularization in diabetic retinopathy is mostly limited to vascular endothelial growth factor, but 6-phosphofructoklnase-2/fructose-2,6-diphosphatase (PFKFB3) also plays a certain role. OBJECTIVE: To Investigate the effect of PFKFB3-SÍRNA on human umbilical vein endothelial cells (HUVECs) In high glucose environment. METHODS: HUVECs were divided Into four groups: Normal glucose control group (5.5 mmol/L glucose), normal glucose+PFKFB3-slRNA group (5.5 mmol/L glucose+PFKFB3-siRNA), high glucose group (30 mmol/L glucose), high glucose+PFKFB3-slRNA group (30mmol/L glucose+PFKFB3-slRNA). Western blot assay was used to detect the silencing effect of PFKFB3 expression. PFKFB3 with optimal silencing effect was selected for subsequent experiments. The tubule formation was detected by in vitro tubule formation assay. The expression of PFKFB3 mRNA was detected by real-time fluorescent quantitative PCR. The expression of PFKFB3 and AKT protein was detected by western blot. RESULTS AND CONCLUSION: PFKFB3-SÍRNA significantly inhibited the expression of PFKFB3 (P 0.05). Compared with the high glucose group, the tube formation ability of the high glucose+PFKFB3-siRNA group was significantly increased (P 0.05). Compared with the high glucose group, the ratio of p-AKT/AKT protein in the high glucose+PFKFB3-siRNA group was increased (P < 0.01). To conclude, siRNA silencing of PFKFB3 gene expression can inhibit the expression of PFKFB3 and improve tube formation in HUVECs. The mechanism may be related to the down-regulation of AKT expression.
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ObjectiveThe relationship between glycosaminoglycans sulodexide (SDX) and HDP such as preeclampsia (PE) has not been reported. The purpose of this study is to observe the protective effect and molecular mechanism of SDX on the function damage of human umbilical vein endothelial cells induced by pregnancy serum of PE.Methodsthe indicated concentrations of SDX (0, 0.1, 0.3, 1, 3, 10, 30 LSU/mL) were used to interfere with HUVEC and Ea.hy926 cells. CCK8 and Matrigel methods were used to detect cell proliferation and tube formation. The normal pregnant women serum (NPS) or PE patients serum (PES) which collected at the 12 th week of pregnancy and the effective concentration of SDX were used to intervene the cells. Matrigel methods were used to observe the protective effect of SDX on endothelial function damage which induced by pathological serum. The secretion level of sFLT-1 and PlGF in supernatant were determined by ELISA.ResultsCompared with the control group, high concentration of SDX inhibited the proliferation of endothelial cells. SDX significantly promoted the tube formation activity wiht a peak at 0.3 LSU/mL (P<0.01). PES damaged the tube formation activity. 0.3 LSU/mL SDX protected cells from tube formation damage which induced by PES (P<0.01). PES promoted the secretion of sFLT-1 and inhibit the secretion of PlGF, while 0.3 LSU/mL SDX reversed the secretion of sFLT-1 and PlGF induced by PES (P<0.01).Conclusion0.3 LSU/mL SDX can protect endothelial cells from PES induced endothelial dysfunction, which is associated with the secretion balance regulation of sFLT-1 / PlGF.
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Objective: To investigate the effect and mechanism of cinnamaldehyde on the angiogenesis of diabetic retinopathy, and the effect of cinnamaldehyde on vascular endothelial growth factor (VEGF) induced proliferation, migration, tube formation and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway of EA.hy 926 cells were observed. Method:EA.hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (60, 90, 120, 150 μmol·L-1). The methyl thiazolyl tetrazolium (MTT) assay and scratch test were used to observe the effect of cinnamaldehyde on the proliferation and migration of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (90, 150 μmol·L-1). The tube formation experiment was used to observe the effect of cinnamaldehyde on the tube formation of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), VEGF+AG490 group (50 μmol·L-1), VEGF+cinnamaldehyde group (90 μmol·L-1), VEGF+cinnamaldehyde group (150 μmol·L-1), and VEGF+cinnamaldehyde group (150 μmol·L-1)+AG490 group (50 μmol·L-1). Western Blot method was used to explore the effect of cinnamaldehyde on the JAK2/STAT3 signaling pathway in EA.hy 926 cells induced by VEGF. Result:Compared with the control group, model group obviously promoted the proliferation and migration of EA.hy 926 cells(P-1) significantly suppressed VEGF-induced proliferation and migration of EA.hy 926 cells (P-1) showed an obvious inhibitory effect on the number of nodes, junctions and meshes of tubules (PPPP-1) significantly reduced the expressions of P-JAK2, P-STAT3, STAT3 proteins (P-1) obviously reduced the expressions of p-STAT3 and STAT3 proteins (PPConclusion:Cinnamaldehyde showed a significantly inhibitory effect on the proliferation, migration and tube formation of VEGF-induced EA.hy 926 cells, which was related to the inhibition of the activation of JAK2/STAT3 pathway.
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Objective To compare the protective effects of pharmacological batch RC28-E1 and pilot batch RC28-E2 on retinal vascular endothelial cells (RF/6A) under the stimulation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).Methods RF/6A cells were divided into normal control group,VEGF + FGF group and RC28-E1 groups with different concentrations.The optimal concentration of RC28-E1 was determined by cell counting kit-8 (CCK-8) method.Cells were divided into normal control group,VEGF+FGF group,RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group,and cultured with serum-free culture medium,serum-free culture medium containing VEGF+FGF,serum-free culture medium containing VEGF+FGF+RC28-E1,serum-free culture medium containing VEGF+FGF+RC28-E2,and serum-free culture medium containing VEGF+FGF+ conbercept,serum-free medium containing VEGF+FGF+FGF trap,respectively.Cell proliferation rate was measured by CCK-8 method,cell migration ability was detected by Transwell test,and tube formation ability was detected by Matrigel assay.Results The cell proliferation rate of 0.080 mg/ml RC28-E1 group was significantly lower than that of VEGF+FGF group (P＜0.05).The cell proliferation rate of RC28-E1 group,RC28-E2 group and FGF trap group were significantly lower than that of VEGF+FGF group (P＜0.05).The number of migrated cells in RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P=0.000).The numbers of meshes formed by retinal vascular endothelial cells in RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P =0.003,0.001,0.009,0.018).The number of tube formation in FGF trap group was significantly higher than those in RC28-E1 group,RC28-E2 group,conbercept group and normal control group (P =0.014,0.000,0.008,0.014).Conclusions Under the stimulation of VEGF + FGF,the inhibitory effect of RC28-E on the proliferation of retinal vascular endothelial cells is greater than that of conbercept,and its inhibitory effect on the tube formation is superior to that of FGF trap.There is no significant difference in the effects of different batches of recombinant decoy receptor innovative drugs on retinal vascular endothelial cells.
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Natural products are rapidly becoming the primary sources of novel antimicrobial agents, as resistance to existing antimicrobial agents is increasing. Apart from determining the antimicrobial activity of natural products, it is also important to understand their effects on the virulence factors of microorganisms. This study aimed to determine the antimicrobial activity of Sternbergia species prevalent in Turkey and investigate their role in the inhibition of germination tube and biofilm formation, both of which are known to be important virulence factors of Candida albicans. The antimicrobial activities of the plant extracts were evaluated using bore-plate and broth microdilution method. The extracts' capacity to inhibit the formation of the germ-tube was also evaluated. The findings of our study revealed that Sternbergia lutea, Sternbergia vernalis possessed antimicrobial activities, with MIC values ranging between 0.048 mg/mL and 0.39 mg/mL. The highest antimicrobial activity was observed against Candida dubliniensis (0.048 mg/mL). While evaluating the inhibition of fungal germination activities, S. vernalis extract (at a concentration of 0.09 mg/mL) was found to be the most effective against C. albicans ATCC 90028 strain. The results also indicated that S. vernalis extracts at sub-MIC levels inhibited germ tube formation and modulated the tail-length of germinated cells, both of which are important virulence factors of C. albicans. Furthermore, the inhibition of biofilm-formation was also investigated, and it was found that two Sternbergia spp. extracts at or below MIC levels inhibited biofilm formation.
Subject(s)
Biofilms/drug effects , Amaryllidaceae/classification , Anti-Infective Agents/analysis , Candida albicans , Plant Extracts/adverse effects , Virulence FactorsABSTRACT
OBJECTIVE:To investigate the effect of KCa3.1 channel on the function of EPCs. METHODS: The gene expression of KCa3.1, vWF and CD31 on EPCs were detected by qRT-PCR. CCK-8 kit, cell adherent method and Matrigel were used to detect the changes of cell proliferation, adhesion and in vitro angiogenesis; cell immunofluorescence or fluorescence activated cell sorter(FACS) was used to detect the protein expression of KCa3.1, vWF, CD31, integrin β1, integrin β3 separately. RESULTS: Blocking the function of KCa3.1 or interfering with the expression of KCa3.1 can attenuated EPC function of proliferation, adhesion and angiogenesis, but it can promote the differentiation of EPCs. Overexpression or activation of KCa3.1 channel can enhance EPCs proliferation, adhesion and angiogenesis but decrease the level of differentiation. The expression of integrin β1 on EPCs was attenuated with blocking KCa3.1 channel, but the expression effect was reversible by the activator. CONCLUSION: The alteration of KCa3.1 channel function or expression affect the biological characteristics and differentiation of EPCs.
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Notopterygium incisum (QH) has been used for the treatment of rheumatoid arthritis (RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis (AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner (2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils (5, 15, and 45 μg·mL) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.
Subject(s)
Animals , Male , Mice , Rats , Angiogenesis Inhibitors , Chemistry , Anti-Inflammatory Agents , Chemistry , Apiaceae , Chemistry , Arthritis, Experimental , Drug Therapy , Allergy and Immunology , Cell Proliferation , Drugs, Chinese Herbal , Chemistry , Gas Chromatography-Mass Spectrometry , Nitric Oxide , Allergy and Immunology , Oils, Volatile , Chemistry , Rats, Sprague-DawleyABSTRACT
Notopterygium incisum (QH) has been used for the treatment of rheumatoid arthritis (RA), and volatile oils may be its mainly bioactive constituents. The present study was designed to analyze the volatile compounds in QH and to determine the anti-arthritic capacity of Notopterygium volatile oils and the potential mechanism of action. The volatile compounds analysis was conducted by GC-MS. The anti-arthritic capacity test of the volatile oils was conducted on adjuvant-induced arthritis (AIA) rats. The anti-inflammatory property was tested in NO release model in RAW 264.7 cells. Endothelial cells were used to evaluate the anti-proliferative and anti-tube formative effects. 70 compounds were analyzed by GC-MS in the volatile oils. Notopterygium volatile oils weakened the rat AIA in a dose-dependent manner (2, 4, and 8 g crude drug/kg). The NO production by RAW 264.7 was decreased by more than 50% in Notopterygium volatile oils (5, 15, and 45 μg·mL) pretreated groups. Notopterygium volatile oils also inhibited EAhy926 cell proliferation and further delayed EAhy926 cell capillary tube formation in a concentration-dependent manner. The anti-NO productive, anti-proliferative, and anti-tube formative effects of Notopterygium volatile oils strongly suggested that the therapeutic effect of QH in AIA might be related to the potent anti-inflammatory and anti-angiogenic capacities of the volatile oils.
Subject(s)
Animals , Male , Mice , Rats , Angiogenesis Inhibitors , Chemistry , Anti-Inflammatory Agents , Chemistry , Apiaceae , Chemistry , Arthritis, Experimental , Drug Therapy , Allergy and Immunology , Cell Proliferation , Drugs, Chinese Herbal , Chemistry , Gas Chromatography-Mass Spectrometry , Nitric Oxide , Allergy and Immunology , Oils, Volatile , Chemistry , Rats, Sprague-DawleyABSTRACT
Objective To compare the protective effects of pharmacological batch RC28.E1 and pilot batch RC28.E2 on retinal vascular endothelial cells ( RF/6A) under the stimulation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Methods RF/6A cells were divided into normal control group, VEGF + FGF group and RC28.E1 groups with different concentrations. The optimal concentration of RC28.E1 was determined by cell counting kit.8 (CCK.8) method. Cells were divided into normal control group,VEGF+FGF group, RC28.E1 group,RC28.E2 group,conbercept group and FGF trap group,and cultured with serum.free culture medium, serum.free culture medium containing VEGF+FGF,serum.free culture medium containing VEGF+FGF+RC28.E1, serum.free culture medium containing VEGF+FGF+RC28.E2,and serum.free culture medium containing VEGF+FGF+conbercept,serum.free medium containing VEGF+FGF+FGF trap,respectively. Cell proliferation rate was measured by CCK.8 method, cell migration ability was detected by Transwell test, and tube formation ability was detected by Matrigel assay. Results The cell proliferation rate of 0. 080 mg/ml RC28.E1 group was significantly lower than that of VEGF+FGF group (P<0. 05). The cell proliferation rate of RC28.E1 group,RC28.E2 group and FGF trap group were significantly lower than that of VEGF+FGF group (P<0. 05). The number of migrated cells in RC28.E1 group,RC28.E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P=0. 000). The numbers of meshes formed by retinal vascular endothelial cells in RC28.E1 group,RC28.E2 group, conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group ( P=0. 003,0. 001, 0. 009,0. 018). The number of tube formation in FGF trap group was significantly higher than those in RC28.E1 group,RC28.E2 group, conbercept group and normal control group ( P = 0. 014, 0. 000, 0. 008, 0. 014 ). Conclusions Under the stimulation of VEGF+FGF,the inhibitory effect of RC28.E on the proliferation of retinal vascular endothelial cells is greater than that of conbercept,and its inhibitory effect on the tube formation is superior to that of FGF trap. There is no significant difference in the effects of different batches of recombinant decoy receptor innovative drugs on retinal vascular endothelial cells.
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The tube formation assay is a widely used in vitro experiment model to evaluate angiogenic properties by measuring the formation of tubular structures from vascular endothelial cells (ECs). in vitro experimental results are crucial when considered the advisability of moving forward to in vivo studies. Thus, the additional attentions to the in vitro assay is necessary to improve the quality of the pre-clinical data, leading to better decision-making for successful drug discovery. In this study, we improved the tube formation assay system in three aspects. First, we used human endothelial colony forming cells (ECFCs), which are endothelial precursors that have a robust proliferative capacity and more defined angiogenic characteristics compared to mature ECs. Second, we utilized a real-time cell recorder to track the progression of tube formation for 48 hours. Third, to minimize analysis error due to the limited observation area, we used image-stitching software to increase the microscope field of view to a 2×2 stitched area from the 4× object lens. Our advanced tube formation assay system successfully demonstrated the time-dependent dynamic progression of tube formation in the presence and absence of VEGF and FGF-2. Vatalanib, VEGF inhibitor, was tested by our assay system. Of note, IC₅₀ values of vatalanib was different at each observation time point. Collectively, these results indicate that our advanced tube formation assay system replicates the dynamic progression of tube formation in response to angiogenic modulators. Therefore, this new system provides a sensitive and versatile assay model for evaluating pro- or anti-angiogenic drugs.
Subject(s)
Humans , Angiogenesis Inhibitors , Attention , Drug Discovery , Endothelial Cells , Fibroblast Growth Factor 2 , In Vitro Techniques , Vascular Endothelial Growth Factor AABSTRACT
Background Tumstatin is the most active endogenous angiogenesis inhibitor,which has a marked inhibitory effect on pathological neovascularization,and Tum5 is an angiogenesis inhibitors fragment of fulllength tumstatin.Objective This study was to investigate the effects of adenovirus-mediated overexpression of recombinant Tum5 gene on the proliferation,migration and tube formation of human umbilical vein endothelial cells (HUVECs) in physiological status.Methods The empty adenoviral vector expressing green fluorescent protein (rAd-GFP) and the viral vector expressing recombinant Tum5 gene were constructed.The HUVECs cultured in RPMI1640 medium were divided into normal control group,empty vector group (rAd-GFP group) and Tum5 gene infection group (rAd-GFP-Tum5 group).The rAd-GFP and rAd-GFP-Tum5 adenoviral particles at the density of 1 × 1010/ml were added into the medium to infect the cells for 48 hours.The proliferation of the cells was assayed at 24,48 and 72 hours by cell counting kit-8 (CCK-8) to evaluate the proliferative rate;the migration number of the cells was detected at 48 hours after infection by Transwell chamber;the tube formation number of the cells were detected by Matrigel method.The concentration of vascular endothelial growth factor (VEGF) in cell supernatants was assayed by ELISA at 24,48,and 72 hours following adenoviral infection.Results The cultured cells showed green fluorescence in the rAd-GFP group and rAd-GFP-Tum5 group under the inverted fluorescence microscope,and the infection efficiency of rAd-GFP and rAd-GFP-Tum5 was 55.13％ and 50.31％,respectively.No significant difference was found in cell proliferative rate among normal control group,rAd-GFP group and rAd-GFP-Tum5 group both at 24 and 48 hours after infection (both at P＞0.05),and the cell proliferative rate was significantly lower in the rAd-GFP-Tum5 group than that in the normal control group and rAd-GFP group at 72 hours after infection (both at P＜0.01).The migration number of the cells at 48 hours after infection was 2 260.25-±930.44,2 370.00±441.06 and 723.75± 363.80 in the normal control group,rAd-GFP group and rAd-GFP-Tum5 group,showing a significant difference among the groups (F =8.524,P =0.008),and the migrated cells were evidently decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group and the normal control group (both at P＜ 0.01).The tube number at 48 hours after infection was 95.67±5.86,88.00±4.58 and 20.67±3.51 in the normal control group,rAd-GFP group and rAdGFP-Tum5 group,showing a significant difference among the groups (F=226.498,P＜0.01),and the tube number in the rAd-GFP-Tum5 group was significantly reduced in comparison with the normal control group and rAd-GFP group (both at P＜ 0.01).The considerably differences in VEGF concentration in the cell supernatants were found in different groups and various time points (Fgroup =73.260,P＜0.01;Ftime =73.477,P＜0.01),and VEGF concentration in the cell supernatants was significantly decreased in the rAd-GFP-Tum5 group compared with the rAd-GFP group at both 48 hours and 72 hours (both at P＜0.01).Conclusions The overexpression of the recombinant Tum5 can inhibit the proliferation,migration and tube formation of the HUVECs in physiological status,which may be associated with Tum-5-mediated down-regulation of VEGF protein in the cell supernatant.
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To investigate the effects of cryptotanshinone (an active ingredient of Salvia Miltiorrhiza) inhibition of angiogenesis, the toxicity of cryptotanshinone was assayed in human hepatic sinusoidal endothelial cells (HHSEC) by CCK8 method. Max dose without toxicity is 10 μmol·L-1. The proliferation of HHSEC were induced by the endothelial cell growth supplement (ECGS), with 2.5 μmol·L-1 sorafenib as the positive control. Cell proliferation was analyzed by EdU assay. Cell viability was analyzed by CCK8 method. The expression of vWF was analyzed by immunofluorescence method. Fluorescence probe method was used to detect the intracellular nitric oxide (NO) levels. Tube formation of HHSEC and transgenic zebrafish were also observed to evaluate the effects of cryptotanshinone against angiogenesis. Compared with normal control, there is a proliferation of HHSEC induced by ECGS. The expression of vWF and the NO levels increased significantly. Cryptotanshinone inhibited the proliferation, down regulated the expression of vWF and the NO levels. Further, cryptotanshinone inhibited the tube formation of HHSEC and reduced the number of fu nctional vessels in transgenic zebrafish. The results suggest that cryptotanshinone could inhibit angiogenesis by regulating the HHSEC cell function.
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Objective: To investigate the effects of miR-24 on endothelial nitric oxide synthase (eNOS) gene expression with regulation and endothelial cell proliferation, migration, tube formation in human umbilical vein endothelial cells (HUVECs). Methods: Constructed high expression plasmid of miR-24 and miR-24 antisense sequence were introduced into HUVECs and the cells included in 3 groups: Control group, miR-24 group and miR-24 inhibitor group. HUVEC proliferation was detected by MTT test, migration was measured by Scratching and Transwell methods, tube formation was examined by Matrigel assay; mRNA and protein expressions of eNOS and Sp1were determined by RT-PCR and Western blot analysis respectively. Results:①Compared with Control group, miR-24 group had decreased cell proliferation by 45.45% as (0.36 ± 0.04) vs (0.66 ± 0.08),P<0.05; miR-24 group had lower speed of cell migration, decreased number of cell migration by 74.75% as (30.25±3.78) vs (119.80±10.94),P<0.01 and there was no obvious tube formation.②Compared with Control group, miR-24 group showed reduced eNOS mRNA expression by 46.2% as (0.49±0.02) vs (0.91±0.01),P<0.05, reduced protein expression by 49.07% as (0.55±0.05) vs (1.08±0.05),P<0.05; meanwhile, decreased Sp1 mRNA expression by 44.9% as (0.49±0. 01) vs (0. 89±0.02)P<0.05, decreased protein expression by 54.90% as (0.46±0.02) vs (1.02±0.04),P<0.05. In miR-24 inhibitor group, the above indexes were lower than Control group but higher than miR-24 group, the amount of tube formation and the length of tubes were similar between Control group and miR-24 inhibitor group. Conclusion: MiR-24 may inhibit HUVECs proliferation, migration, tube formation and suppress eNOS expression; Sp1 might be one of the important regulators.
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Aim To study the effect of activating Sonic hedgehog( Shh) pathway on the function of endothelial progenitor cells ( EPCs ) in type 1 diabetic mice. Methods EPCs were isolated and cultured by density gradient method from diabetic mice. The effects of Shh N-terminal peptide and agonist SAG on EPCs prolifera-tion were evaluated by using the MTT colorimetric as-say. EPCs migration was measured by Transwell meth-od. EPCs tube formation ability was estimated by Matrigel . EPCs senescence activity was determined by β-galactosidase staining. Results Compared with control mice, the function of EPCs in type 1 diabetic mice was impaired. The proliferation, migration and tube formation of diabetic EPCs could be promoted by Shh peptide and agonist SAG. The senescence of dia-betic EPCs could be decreased by Shh peptide and ag-onist SAG. Conclusion Activating Shh signaling pathway can improve the impared function of diabetic EPCs in type 1 diabetic mice.
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OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent ...
Subject(s)
Animals , Humans , Male , Rats , Angiogenesis Inhibitors/pharmacology , Anti-Inflammatory Agents/pharmacology , Cinnamates/pharmacology , Plant Extracts/pharmacology , Vascular Endothelial Growth Factor A/drug effects , Zingiberaceae/chemistry , Analysis of Variance , Angiogenesis Inhibitors/isolation & purification , Anti-Inflammatory Agents/isolation & purification , Cell Proliferation/drug effects , Enzyme-Linked Immunosorbent Assay , Human Umbilical Vein Endothelial Cells/drug effects , Interleukin-1/analysis , Rats, Sprague-Dawley , Reproducibility of Results , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/drug effects , /drug effects , Vascular Endothelial Growth Factor A/analysisABSTRACT
Objective To explore the effect of epidermal growth factor (EGF) on tube formation of HUVEC induced by the secretion of angiogenesis factors of adipose-derived stem cells (ADSCs).Methods ADSCs were primarily cultured by enzyme digestion method.The flow cytomertry was performed to detect the expression of cell surface marker.ELISA was used to detect the expression of VEGF,HGF,and SDF-1 after given different doses of EGF.Tube formation assay was used to examine the effect of EGF on the tube formation induced by ADSCs.Results ADSCs were successfully isolated and cultured from human liposuction tissue and specific markers were expressed on ADSCs.EGF promoted the secretion of angiogenesis factors VEGF,HGF,and SDF-1,which were secreted by ADSCs.EGF pretreatment increased the ability of tube formation of HUVECs induced by ADSCs.Conclusions ADSCs induce the secretion of angiogenesis factors in vitro,and thus increase the ability of tube formation of HUVECs.EGF promotes the secretion ability of ADSCs,and the best concentration is 15 mg/L.
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In this study, we investigated the effect of methanolic extract isolated from the root of Lycoris aurea (LA) on the growth of cancer cells and the tube formation activity of endothelial cells. Various cancer cells were treated with LA at doses of 0.3, 1, 3, 10 or 30 microg/ml and LA significantly suppressed the growth of several cancer cell lines, including ACHN, HCT-15, K-562, MCF-7, PC-3 and SK-OV-3, in a dose-dependent manner. We also found that LA induced cell cycle arrest at G2/M phase in ACHN renal cell adenocarcinoma cells. Further study demonstrated that LA concentration-dependently inhibited the tube formation, which is a widely used in vitro model of reorganization stage of angiogenesis, in human umbilical vein endothelial cells. Collectively, these results show that LA inhibits the growth of cancer cells and tube formation of endothelial cells and the growth-inhibitory effect of LA might be mediated, at least in part, by blocking cell cycle progression.
Subject(s)
Carcinoma, Renal Cell , Cell Cycle , Cell Cycle Checkpoints , Cell Line , Endothelial Cells , Human Umbilical Vein Endothelial Cells , Lycoris , MethanolABSTRACT
BACKGROUND: Angiogenesis mediated by VEGF constitutes a new target for anti-cancer therapy which has explored through different ways of intervention aiming at the blocking of the tumoral angiogenesis. In the present study, we developed the assays by which efficacies of anti-VEGF inhibitor candidates are evaluated at the various levels. METHODS & RESULTS: First, we developed two sandwich ELISAs using coated anti-VEGF Ab and soluble Flt-1 receptor fusion protein (sFlt-1/Fc). As low as 200 pg/ml of hVEGF diluted in human sera was detectable by these assays. In addition, we found that VEGF inhibitors (2 microngram/ml of either anti-VEGF Ab or sFlt-1/Fc) completely block 5 ng/ml VEGF in these ELISAs. Subsequently, two bioassays, wound healing and HUVEC tube formation assays, revealed that anti-VEGF Ab (1 microngram/ml) & sFlt-1/Fc Ab (1 microngram/ml), or SU5416 (VEGFR tyrosine kinase inhibitor, 1 micronM) prevents the activity of VEGF (1~10 ng/ml). Finally, secretion of MMP-9 by VEGF-stimulated macrophages was abolished by treatment of anti-VEGF Ab (1 microngram/ml) in gelatin zymography. CONCLUSION: ELISAs together with bioassays developed in this study are appropriate for evaluation of the efficacy of inhibitors of VEGF.