Analyzing the pharmacological substances and targets of Xuefu Zhuyu decoction in hypertensive vascular endothelial cells

Background:Xuefu Zhuyu decoction(XFZY)could significantly improve the function of hypertensive vascular endothelial cells,but the targets and mechanism are not clear.This study is to analyze the pharmacological substances and targets of Xuefu Zhuyu decoction in hypertensive vascular endothelial cells.Methods:This study used Xuefu Zhuyu decoction to intervene human umbilical vein endothelial cells incubated by hypertensive patients’serum,then detected the function of vascular endothelial cells.The aqueous extract of XFZY was analyzed and validated by liquid chromatography-mass spectrometry technology;Finally,macromolecular docking technology was used to analyze the potential active substances and targets of XFZY in the prevention and treatment of hypertension.Results:Compared with the model group,the XFZY group showed a significant increase in NO expression(P<0.01)and a significant decrease in ET-1 expression(P<0.001);and the expression of BIP,P-JNK,CHOP,and BAX in XFZY group cells was significantly decreased(P<0.001),while the expression of JNK and BCL2 was significantly increased(P<0.001).19 main compounds were identified in XFZY and there were 3 pairs of molecular complexes with high affinity for markers of the endoplasmic reticulum stress,including BIP-Hesperidin complex,BIP-HSYA complex and JNK-Naringin complex.Conclusion:This study analyzed the potential pharmacodynamic substance and targets of Xuefu Zhuyu decoction in improving the function of hypertensive vascular endothelial cells,which could provide a scientific basis for the future molecular mechanism of XFZY in treating hypertension.

Ferroptosis inhibition protects vascular endothelial cells and maintains integrity of the blood-spinal cord barrier after spinal cord injury

Maintaining the integrity of the blood-spinal cord barrier is critical for the recove ry of spinal cord injury.Ferro ptosis contributes to the pathogenesis of spinal cord injury.We hypothesized that ferroptosis is involved in disruption of the blood-s pinal cord barrier.In this study,we administe red the ferroptosis inhibitor liproxstatin-1 intraperitoneally after contusive spinal co rd injury in rats.Liproxstatin-1 improved locomotor recovery and somatosensory evoked potential electrophysiological performance after spinal cord inju ry.Liproxstatin-1 maintained blood-spinal cord barrier integrity by upregulation of the expression of tight junction protein.Liproxstatin-1 inhibited ferroptosis of endothelial cell after spinal cord injury,as shown by the immunofluorescence of an endothelial cell marker(rat endothelium cell antigen-1,RECA-1) and fe rroptosis markers Acyl-CoA synthetase long-chain family member 4 and 15-lipoxygenase.Liproxstatin-1reduced brain endothelial cell ferroptosis in vitro by upregulating glutathione peroxidase 4 and downregulating Acyl-CoA synthetase long-chain family member4 and 15-lipoxygenase.Furthermore,inflammatory cell recruitment and astrogliosis were mitigated after liproxstatin-1 treatment.In summary,liproxstatin-1im proved spinal cord injury recovery by inhibiting ferroptosis in endothelial cells and maintaining blood-s pinal co rd barrier integrity.

Inhibition of VEGF-A expression in hypoxia-exposed fetal retinal microvascular endothelial cells by exosomes derived from human umbilical cord mesenchymal stem cells

Objective:This study aimed to investigate the potential of human umbilical cord mesenchymal stem cell(hucMSC)-derived exosomes(hucMSC-Exos)in inhibiting hypoxia-induced cell hyper proliferation and overexpression of vascular endothelial growth factor A(VEGF-A)in immature human fetal retinal microvascular endothelial cells(hfRMECs).Methods:Exosomes were isolated from hucMSCs using cryogenic ultracentrifugation and characterized through various techniques,including transmission electron microscopy,nanoparticle tracking analysis,bicinchoninic acid assays,and western blotting.The hfRMECs were identified using von Willebrand factor(vWF)co-staining and divided into four groups:a control group cultured under normoxic condition,a hypoxic model group,a hypoxic group treated with low-concentration hucMSC-Exos(75μg/mL)and a hypoxic group treated with high-concentration hucMSC-Exos(100μg/mL).Cell viability and proliferation were assessed using Cell Counting Kit-8(CCK-8)assay and EdU(5-ethynyl-2′-deoxyuridine)assay respectively.Expression levels of VEGF-A were evaluated using RT-PCR,western blotting and immunofluorescence.Results:Hypoxia significantly increased hfRMECs’viability and proliferation by upregulating VEGF-A levels.The administration of hucMSC-Exos effectively reversed this response,with the high-concentration group exhibiting greater efficacy compared to the lowconcentration group.Conclusion:In conclusion,hucMSC-Exos can dose-dependently inhibit hypoxia-induced hyperproliferation and VEGF-A overexpression in immature fetal retinal microvascular endothelial cells.

Small interfering RNA targeting PGC-1α inhibits VEGF expression and tube formation in human retinal vascular endothelial cells

AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O2) or hypoxic (1%, O2) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O2) or hypoxic (1%, O2) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.

A New Rat Model of Cerebral Infarction Based on the Injury of Vascular Endothelial Cell

Objective： A new rat model of cerebral infarction was developed to elucidate the contribution of vascular endothelial cell during focal cerebral infarction formation. Methods： Forty-eight Sprague-Dawley （SD） rats were randomly divided into the model group, sham operation group, and control group for indexes observation of triphenyltetrazolium chloride （TTC） dyeing, neurological deficit, plasma tissue-type plasminogen activator （tPA） activity, plasminogen activator inhibitor （PAl） activity, thromboxane B2（TXB2） content, and 6-keto-prostaglandin （6-keto-PGF1α） content. Results： （1） The highest neurological score appeared at 6 h after operation, descending significantly at sequential time. （2） Using TTC dyeing and optical microscope technique, pathological changes in brains were observed. （3） Compared with control group and sham operation groups, there was a decrease in tPA activity of model rats at the initial 12 h after injection of sodium laurate （P〈0.05）, PAl activity decreased markedly in the model group at 24 h after injection of sodium laurate. （4） In plasma TXB2 concentration reached the highest level compared at 6 h after injection of sodium laurate, but there were not obvious differences in plasma 6-keto-PGF1α concentration among all groups （P〉 0.05）. Conclusion. Focal cerebral infarction in rats could be induced by some sodium laurate, showing ischemic cerebrum necrosis, function disorder of vascular endothelium-platelet, fibrinolysis abnormality. This model could play an important role in researching the contribution of vascular endothelial cell during cerebral infarction development, preventing and curing by traditional Chinese medicine.

Size-dependent Biological Effects on Vascular Endothelial Cells Induced by Different Particulate Matters

Summary： The contribution of particles to cardiovascular mortality and morbidity has been enlightened by epidemiologic and experimental studies. However, adverse biological effects of the particles with different sizes on cardiovascular cells have not been well recognized. In this study, sub-cultured human umbilical vein endothelial cells （HUVECs） were exposed to increasing concentrations of pure quartz particles （DQ） of three sizes （DQPM1, 〈1 μm; DQPM3-5, 3-5 μm; DQPM5, 5 μm） and carbon black particles of two sizes （CB0.1, 〈0.1 μm; CB 1, 〈 1 μm） for 24 h. Cytotoxicity was estimated by measuring the activity of lactate dehydrogenase （LDH） and cell viability. Nitric oxide （NO） generation and cyto- kines （TNF-α and IL-1β） releases were analyzed by using NO assay and enzyme-linked immunoabsorbent assay （ELISA）, respectively. It was found that both particles induced adverse biological effects on HUVECs in a dose-dependent manner. The size of particle directly influenced the biological activity. For quartz, the smaller particles induced stronger cytotoxicity and higher levels of cytokine responses than those particles of big size. For carbon black particles, CB0.1 was more capable of inducing adverse responses on HUVECs than CB 1 only at lower particle concentrations, in contrast to those at higher concentrations. Meanwhile, our data also revealed that quartz particles performed stronger cell damage and produced higher levels of TNF-α than carbon black particles, even if particles size was similar. In conclusion, particle size as well as particle composition should be both considered in assessing vascular endothelial cells injury and inflammation responses induced by particles.

Effects of Vascular Endothelial Cell Growth Factor on Fibrovascular Ingrowth into Rabbit's Hydroxyapatite Orbital Implant

Summary: The effects of different concentrations of vascular endothelial cell growth factor (VEGF) on the fibrovascular ingrowth into rabbits hydroxyapatite orbital implant were investigated. Twelve New Zealand white rabbits were divided into 3 groups and received hydroxyapatite orbital implant surgery in their right eyes. Before and after the operation, the implants were treated with 10 ng/ml VEGF, 100 ng/ml VEGF, or normal saline as control group. The animals received technetium bones scan at 2, 4, and 6 weeks postoperatively. The mean radioactivity counts within region of interest (ROI) of the surgery eye (R) and the non-surgery eye (L) in the same animal were tested, and the R/L ratios were calculated. The implants were harvested at 6th weeks and examined histopathologically. The results showed that at second week, there was no significant difference in mean R/L ratios between VEGF group and control group (F=2.83, P=0.111); At 4th week (F=7.728, P=0.011) and 6th week (F=7.831, P=0.011) postoperatively, the mean ratios in VEGF groups were significantly higher than that in control group. At 6th week postoperatively, the fibrovascularization rates in VEGF groups were higher than in control group significantly (F=8.711, P=0.008). It was suggested that VEGF could promote the fibrovascular ingrowth into hydroxyapatite orbital implant, thus might shorten the time required for complete vascularization of the HA orbital implant.

Different Responses of Cell Cycle between Rat Vascular Smooth Muscle Cells and Vascular Endothelial Cells to Paclitaxel

Summary： Although previous reports showed dmg-eluting stent （DES） could effectively inhibit neointima formation, in-stent restenosis （ISR） remains an important obstacle. The purpose of this study was to investigate different effects of paclitaxel on proliferation and cell cycle regulators between vascular smooth muscle cells （VSMCs） and vascular endothelial cells （VECs） of rats in vitro. The cultured VSMCs and VECs of rats from the same tissues were examined by using immunohistochemistry, flow cytometry and Western blotting in control and paclitaxel-treated groups. The results showed paclitaxel could effectively inhibit proliferation of VSMCs and VECs. However, as compared with VECs, prolif- eration of VSMCs in paclitaxel-treated group decreased less rapidly. The percentage of cells in G0-G1 and G2-M phases was reduced, and that in S phase increased after treatment for 72 h. The expression of cyclin D1 and B1, p27 and PCNA in VSMCs of paclitaxel-treated group was up-regulated, but that of p21 down-regulated as compared with VECs. It is concluded that there are significant differences in the expression of cell cycle regulators and proliferation rate between paclitaxel-treated VSMCs and paclitaxel-treated VECs, suggesting that the G1 S checkpoint regulated by paclitaxel may play a critical role in the development of complications of DES, which provides new strategies for treatments of ISR.

TIR/BB-loop mimetic AS-1 protects vascular endothelial cells from injury induced by hypoxia/reoxygenation

Morphological and functional abnormalities of vascular endothelial cells(VECs) are risk factors of ischemiareperfusion in skin flaps.Signaling pathway mediated by interleukin-1 receptor(IL-1 R) is essential to hypoxia/reoxygenation(H/R) injury of VECs.While the TIR/BB-loop mimetic(AS-1) disrupts the interaction between IL-1 R and myeloid differentiation primary-response protein 88(MyD88),its role in the VECs dysfunction under H/R is unclear.In this study,we first showed that there was an infiltration of inflammatory cells and the apoptosis of VECs by using a skin flap section from patients who received flap transplantation.We then showed that the H/R treatment induced apoptosis and loss of cell migration of endothelial cell line H926 were attenuated by AS-1.Furthermore,our data suggested that AS-1 inhibits the interaction between IL-1 R and MyD88,and subsequent phosphorylation of IκB and p38 pathway,as well as the nuclear localization of NF-κB subunit p65/p50.Thus,this study indicated that the protective role of AS-1 in H/R induced cellular injury may be due to the AS-1 mediated down-regulation of IL-1 R signaling pathway.

Construction of human VEGF165 gene eukaryotic expression plasmid and its effect on proliferation of vascular endothelial cells

After organ transplantation, rapid repair of injured vascular endothelial cell (VEC) is a key to prevent graft chronic dysfunction besides control of immunological rejection. Many studies have confirmed that vascular endothelial growth factor 165 (VEGF165) could accelerate the repair of VEC injury, decrease thrombosis and thrombotic occlusion, and inhibit hyperplasia of the intima. This study was designed to construct eukaryotic expression plasmid pBudCE4.1/VEGF165, and observe its effect on the prolife ration of VEC. METHODS:The VEGF165 gene cloned from human heart tissue by RT-PCR was cloned into eukaryotic expression plasmid pBudCE4.1. The recombinant expression plasmid pBudCE4.1/VEGF165 was identified by restriction enzyme (Hind III and BamH I) digestion analysis, and was sequenced. The pBudCE4.1/VEGF165 was introduced into VEC through lipofection transfection. The VEGF165 mRNA expression by Northern blot and VEGF165 protein expression was detected by immunocytochemical staining. The effect of expression protein on VEC proliferation was detected by flow cytometry. RESULTS:The RT-PCR product of the VEGF165 gene was about 576bp. Sequencing analysis revealed that the sequence of the amplified VEGF165 gene was identical with that in GenBank. Restrictive enzyme digestion analysis showed that recombinant expression plasmid pBudCE4.1/ tVEGF165 had been constructed successfully. The expression of VEGF165 at mRNA and protein levels in the transformed VSMCs had been demonstrated by Northern blot and immunocytochemical staining respectively. The expressed product of VEGF165 could notably accelerate the proliferation of VECs. CONCLUSIONS:pBudCE4.1/VEGF165 is successfully cons- tructed and is expressed in VECs. Expressed VEGF165 can accelerate the VEC proliferation. The present study has laid a foundation for potential use of VEGF165 gene transfection to prevent and treat vascular stenosis in the transplanted organ.

Inhibition of proliferation of retinal vascular endothelial cells more effectively than choroidal vascular endothelial cell proliferation by bevacizumab

AIM： To evaluate the differential inhibitory effects of bevacizumab on cell proliferation of vascular endothelial growth factor （VEGF）-stimulated choroidal vascular endothelial cells （CVECs） and retinal vascular endothelial cells （RVECs） in vitro.METHODS： VEGF （400 ng/mL） enriched CVECs and RVECs were treated with escalating doses of bevacizumab （0.1, 0.5, 1, 1.5 and 2 mg/mL）. Cell proliferation changes were analyzed with WST-1 assay and trypan blue exclusion assay at 48, 72h and 1wk. Morphological changes were recorded with bright field microscopy.RESULTS： VEGF enriched RVECs showed significantly more decline of cell viability than CVECs after bevacizumab treatment. One week after treatment, RVEC cell proliferation decreased by 29.7%, 37.5%, 52.8%, 35.9% and 45.6% at 0.1, 0.5, 1.0, 1.5 and 2 mg/mL bevacizumab respectively compared to CVEC proliferation decrease of 4.1%, 7.7%, 2.4%, 4.1% and 17.7% （P〈0.05） by WST-1 assay. Trypan blue exclusion assay also revealed similar decrease in RVEC proliferation of 20%, 60%, 73.3%, 80% and 93.3% compared to CVEC proliferation decrease of 4%, 12%, 22.9%, 16.7% and 22.2% respectively （P〈0.05）. The maximum differential effect between the two cell types was observed at bevacizumab doses of 1.0 and 1.5 mg/mL at all time points. RVECs were 22 fold more sensitive （P〈0.01） compared to CVECs （52.8% vs 2.4%） at concentration of 1.0 mg/mL, and 8.7 fold more at 1.5 mg/mL （35.9% vs 4.1%） 1wk after treatment （P〈0.05 respectively）.CONCLUSION： VEGF-enriched RVECs are more susceptible to bevacizumab inhibition than CVECs at clinically used dosage of 1.25 mg and this differential sensitivity between two cell types should be taken into consideration in dosage selection.

Pretreatment with Dingnaofang reduces vascular endothelial cell apoptosis induced by cerebral ischemia/perfusion injury

BACKGROUND： Vascular endothelial cell apoptosis participates in cerebral ischemia/reperfusion injury, and the method of Qi-supplementation and blood-activation has remarkable neuroprotective effects against cerebral ischemia/reperfusion injury. OBJECTIVE： To explore the molecular mechanism that Dingnaofang （Chinese herbs for supplementing Qi and activating blood circulation） inhibits vascular endothelial cell apoptosis induced by cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETFING： A randomized controlled animal experiment was performed at the Department of Central Laboratory, Third Xiangya Hospital, Central South University, China, between October 2007 and December 2008. MATERIALS： Dingnaofang consisted of Huangqi （Milkvetch Root; Radix Astragah）, Chuanxiong （Szechwan Lovage Rhizome; Rhizoma Chuanxiong）, Yinxingye （ginkgo leaf; Fofium Ginkgo）, Dilong （earthworm; Pheretima）, Danggui （Chinese Angelica; Radix Angelicae Sinensis）, Tianqi （Radix Notoginseng）, and Gancao （Radix Glycyrrhizae; Radix Glycytthizae）, with a proportion of 5：2：2： 1： 1： 1： 1. METHODS： A total of 130 Sprague Dawley rats were randomly divided into sham-surgery （n = 10）, cerebral ischemia/reperfusion （n = 40）, cerebral ischemia pretreatment （n = 40） and Dingnaofang pretreatment groups （n = 40）. Middle cerebral artery occlusion was used to induce cerebral ischemic injury. The bilateral common carotid artery in the cerebral ischemia pretreatment group was blocked for 10 minutes on days 7, 3 and 1 prior to ischemia/reperfusion injury, while rats in Dingnaofang pretreatment group were intragastrically administrated with 4 g Dingnaofang 1 week prior to cerebral ischemia once per day, for 7 successive days. MAIN OUTCOME MEASURES： Apoptosis ratios in vascular endothelial cells were measured using Hoechst 33258 staining and flow cytometry; apoptosis was detected by monitoring DNA gradient bands and the activation of caspase-3, 8, 9 and Bid using Western blot. RESULTS： Following cerebral ischemiaJreperfusion injury, the number of apoptotic vascular endothelial cells in the middle cerebral artery significantly increased （P 〈 0.01）; however, cerebral ischemia pretreatment and Dingnaofang pretreatment groups significantly reduced apoptosis induced by cerebral ischemia/reperfusion injury （P 〈 0.01）. In particular, DNA gradient bands were not observed following Dingnaofang pretreatment. At 24 hours after cerebral ischemia/reperfusion injury, cleaved fragments of caspase-3, 8 and 9 were detected at 11 kD （P 11）, 20 kD （P 20） and 10 kD （P 10）, respectively, following Western blot. Bid was also cleaved into its truncated form （tBid; 15 kD）. Gray scale analysis indicated that P 11, P 20, P 10 and tBid band values in the Dingnaofang pretreatment group were significantly less than in the cerebral ischemia/reperfusion and cerebral ischemia pretreatment groups （P 〈 0.01 or P 〈 0.05）. CONCLUSION： Dingnaofang inhibits vascular endothelial cell apoptosis induced by cerebral ischemia/reperfusion injury via inhibition of apoptotic signal transduction pathways.

Dynamic modeling and control of extracellular ATP concentration on vascular endothelial cells via shear stress modulation

A new dynamic model for cell-deformation-induced adenosine triphosphate （ATP） release from vascular endothelial cells （VECs） is proposed in this paper to quantify the relationship between the ATP concentration at the surface of VECs and blood flow-induced shear stress. The simulation results demonstrate that ATP concentration at the surface of VECs predicted by the proposed new dynamic model is more consistent with the experimental observations than those by the existing static and dynamic models. Furthermore, it is the first time that a proportional-integral-derivative （PID） feedback controller is applied to modulate extracellular ATP concentration. Three types of desired ATP concentration profiles including constant, square wave and sinusoid are obtained by regulating the wall shear stress under this PID control. The systematic methodology utilized in this paper to model and control ATP release from VECs via adjusting external stimulus opens up a new scenario where quantitative investigations into the underlying mechanisms for many biochemical phenomena can be carded out for the sake of controlling specific cellular events.

Mesenchymal stem cell-derived exosomes inhibit the VEGF-A expression in human retinal vascular endothelial cells induced by high glucose

AIM:To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(hUCMSCs)on the expression of vascular endothelial growth factor A(VEGF-A)in human retinal vascular endothelial cells(HRECs).METHODS:Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy,Western blotting and nanoparticle tracking analysis.HRECs were randomly divided into a normal control group(group A),a high glucose model group(group B),a high glucose group with 25μg/mL(group C),50μg/mL(group D),and 100μg/mL exosomes(group E).Twenty-four hours after coculture,the cell proliferation rate was detected using flow cytometry,and the VEGF-A level was detected using immunofluorescence.After coculture 8,16,and 24h,the expression levels of VEGF-A in each group were detected using PCR and Western blots.RESULTS:The characteristic morphology(membrane structured vesicles)and size(diameter between 50 and 200 nm)were observed under transmission electron microscopy.The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis(NTA).The exosomal markers CD9,CD63,and HSP70 were strongly detected.The proliferation rate of the cells in group B increased after 24h of coculture.Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes(F=39.03,P<0.01).The upregulation of VEGF-A protein(group C:F=7.96;group D:F=17.29;group E:F=11.89;8h:F=9.45;16h:F=12.86;24h:F=42.28,P<0.05)and mRNA(group C:F=4.137;group D:F=13.64;group E:F=22.19;8h:F=7.253;16h:F=16.98;24h:F=22.62,P<0.05)in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes(P<0.05).CONCLUSION:hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.

A mathematical model for ATP-mediated calcium dynamics in vascular endothelial cells induced by fluid shear stress

In consideration of the mechanism for shear-stress-induced Ca^2＋ influx via ATP（adenosine triphosphate）-gated ion channel P2X4 in vascular endothelial cells, a modified model is proposed to describe the shear-stress-induced Ca^2＋ influx. It is affected both by the Ca^2＋ gradient across the cell membrane and extracellular ATP concentration on the cell surface. Meanwhile, a new static ATP release model is constructed by using published experimental data. Combining the modified intracellular calcium dynamics model with the new ATP release model, we establish a nonlinear Ca^2＋ dynamic system in vascular endothelial cells. The ATP-mediated calcium response in vascular endothelial cells subjected to shear stresses is analyzed by solving the governing equations of the integrated dynamic system. Numerical results show that the shear-stress-induced calcium response predicted by the proposed model is more consistent with the experimental observations than that predicted by existing models.

Regulation of opticin on bioactivity of retinal vascular endothelial cells cultured in collagen

AIM:To investigate the effects of collagen and opticin on the bioactivity of human retinal vascular endothelial cells(hR VECs),and explore its regulations by integrins and RhoA/ROCK1 signal pathway.METHODS:h RVECs were cultured in collagen and treated by opticin,and cell-based bioactivity assays of cell proliferation,migration,and adhesion were performed.The expression of integrinα2,integrinβ1,Rho A and ROCK1 were examined with real-time PCR and Western blotting.RESULTS:Collagen could promote cell viability of proliferation and migration(all P<0.05),and enhance the m RNA expression of integrinα2,integrinβ1,Rho A and ROCK1(all P<0.05).Opticin could inhibit proliferation and migration ability of hR VECs cultured in collagen,and reduce the mR NA expression of integrinα2,integrinβ1,RhoA and ROCK1(all P<0.05).CONCLUSION:Collagen and opticin can affect bioactivity of hR VECs,which may be regulated byα2-,β1-integrins and RhoA/ROCK1 signal pathway.

Application of vascular endothelial cells in stem cell medicine

Stem cell medicine is gaining momentum in the development of therapy for various end-stage diseases.The search for new seed cells and exploration of their application prospects are topics of interest in stem cell medicine.In recent years,vascular endothelial cells(VECs)have attracted wide attention from scholars.VECs,which form the inner lining of blood vessels,are critically involved in many physiological functions,including permeability,angiogenesis,blood pressure regulation,immunity,and pathological development,such as atherosclerosis and malignant tumors.VECs have significant therapeutic effects and broad application prospects in stem cell medicine for the treatment of various refractory diseases,including atherosclerosis,myocardial infarction,diabetic complications,hypertension,coronavirus disease 2019,and malignant tumors.On the one hand,VECs and their extracellular vesicles can be directly used for the treatment of these diseases.On the other hand,VECs can be used as therapeutic targets for some diseases.However,there are still some obstacles to the use of VECs in stem cell medicine.In this review,advances in the applications and challenges that come with the use of these cells are discussed.

INTERACTIONS BETWEEN THE HUMAN GASTRIC CARCINOMA CELL AND THE HUMAN VASCULAR ENDOTHELIAL CELL

Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co culture or direct contact co culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.

CREG regulates vascular endothelial cell migration mediated by ILK-β-parvin signal pathway

Background To investigate the effects and mechanisms of cellular repressor of ElA stimulated genes (CREG) on endothelial cell(EC) migration.Methods vascular endothelial cells(VE),CREG overexpression VEs, CREG suppression VEs and VEs transfected with CREG gene modified adenovirus(Ad-CREG) were cultured with dulbecco’s modified eagle’s medium contained 10%fetal calf serum. Western blot was used to detect the protein level of CREG and integrin-linked kinase(ILK) in the four kind ECs.Tran-swell migration model was applied to compare the migration cell number of the four kind ECs.Two kinds of ILK mutant plasmids;PCXN2-flag-ILK wt-IRES-GFP(wild-type ILK)and PCXN2-flag-ILK p-parvin-IRES-GFP(P-parvin-binding mutant) were used to transfect VS and VE respectively,then the two kind transfection ECs were named as VS-wtILK and VE-P -parvin which were selected by G418(600ng/ml)for 2 weeks;Transwell migration model was applied to compare migration capability before and after ILK plasmids transfecting VE and VS.Results Western blot analysis showed that CREG overexpression promoted ILK expression in ECs,on the contrary,ILK expression was down-regulated in CREG silent ECs(P【0.05).Further more,ILK expression was up-regulated obviously in VE transfected with Ad-CREG(P【 0.05);Transwell migration model showed that EC’s migration capability was positively correlated with the expression level of CREG in EC,that is,CREG overexpression induced VE migration and CREG silent suppressed VE migration, moreover,Ad-CREG transfecting VE showed better migration capability accompanied with CREG expression increase by transwell migration model(P【0.05).In order to know the relationship between ILK expression and cell migration,we obtained stable transfection cell strains of VS-wtILK and VE-Pparvin, transwell migration model demonstrated that VS-wtILK remarkably corrected the poor migration capability of VS(P【 0.01),butβ-parvin combining site mutation in ILK genes inhibited VE migration markedly(P【0.01).Conclusions ILKp -parvin signal pathway mediated vascular endothelial cell migration induced by CREG.