A MicroRNA Catalog of Swine Umbilical Vein Endothelial Cells Identified by Deep Sequencing

MicroRNAs （miRNAs） are endogenous -22 nt RNAs that play important regulatory roles in targeting mRNAs for cleavage or translational repression. Despite the discovery of increasing numbers of human and mouse miRNAs, little is known about miRNAs from pig. In this study, we sought to extend the repertoire of porcine small regulatory RNAs using Solexa sequencing. We sequenced a library of small RNAs prepared from immortalized swine umbilical vein endothelial cells （SUVECs）. We produced over 13.6 million short sequence reads, of which 8 547 658 perfectly mapped to the pig genome. A bioinformatics pipeline was used to identify authentic mature miRNA sequences. We identified 154 porcine miRNA genes, among which 146 were conserved across species, and 8 were pig-specific miRNA genes. The 146 miRNA genes encoded 116 conserved mature miRNAs and 66 miRNA^＊. The 8 pig-specific miRNA genes encoded 4 mature miRNAs. Four potential novel miRNAs were identified. The secondary structures of the 154 miRNA genes were predicted; 13 miRNAs have 2 structures, and miR-9 and miR-199 have 4 and 3 structures, respectively. 36 miRNAs were organized into 19 compact clusters, miR-206, miR-21 and miR-378 were the relatively highly expressed miRNAs. In conclusion, Solexa sequencing allowed the successful discovery of known and novel porcine miRNAs with high accuracy and efficiency. Furthermore, our results supply new data to the somewhat insufficient pig miRBase, and are useful for investigating features of the blood-brain barrier, vascular diseases and inflammation.

Viscoelastic evaluation of fetal umbilical vein for reconstruction of middle cerebral artery

The transplantation of artificial blood vessels with 〈 6 mm inner diameter as substitutes for human arterioles or veins has not achieved satisfactory results. Umbilical vein has been substituted for ar- tery in vascular transplantation, but it remains unclear whether the stress relaxation and creep be- tween these vessels are consistent. In this study, we used the fetal umbilical vein and middle cere- bral artery from adult male cadavers to make specimens 15 mm in length, 0.196-0.268 mm in tu- nica media thickness, and 2.82-2.96 mm in outer diameter. The results demonstrated that the stress decrease at 7 200 seconds was similar between the middle cerebral artery and fetal umbilical vein specimens, regardless of initial stress of 18.7 kPa or 22.5 kPa. However, the strain increase at 7 200 seconds of fetal umbilical veins was larger than that of middle cerebral arteries. Moreover, the stress relaxation experiment showed that the stress decrease at 7 200 seconds of the fetal umbilical vein and middle cerebral artery specimens under 22.5 kPa initial stress was less than the decrease in these specimens under 18.7 kPa initial stress. These results indicate that the fetal umbilical vein has appropriate stress relaxation and creep properties for transplantation. These properties are advantageous for vascular reconstruction, indicating that the fetal umbilical vein can be transplanted to repair middle cerebral artery injury.

Martentoxin, a large-conductance Ca^(2+)-activated K^+ channel inhibitor, attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells

Martentoxin, a 4,046 Da polypeptide toxin purified from the venom of the scorpion Buthus martensii Karsch, has been demonstrated to block large-conductance Ca2＋-activated K＋ （BKca） channels; however, its biological roles are still largely unknown. In the present study, we investigated the pharmacological effects of martentoxin on regulating the production of nitric oxide induced by TNF-a in human umbilical vein endothelial cells （HU- VECs）. We found that, 1, 10 and 100 ~tmol/L martentoxin decreased nitric oxide production by HUVECs ex- posed to 10 ng/mL TNF for 6, 12 and 24 hours. We further demonstrated that martentoxin inhibited the activity of iNOS and retarded the down-regulation of eNOS mRNA induced by TNF-a. Therefore, martentoxin could be a potential therapeutic agent for vascular diseases.

Co-culture of Mesenchymal Stem Cells with Umbilical Vein Endothelial Cells under Hypoxic Condition

By co-culturing humm mesenchymal stem cells (hMSCs) and human umbilical rein endothelial cells (HUVECs) under hypoxia and creating a microenvironment similar to that of transplanted hMSCs for the treatment of avascular ni ANFH, the effect of hMSCs on survival, apoptosis, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the hypoxic condition were investigated in vitro. hMSCs and HUVECs were cultured and identified in vitro. Three kinds of conditioned media, CdM-CdMNOR, CdM-CdMHYP and HUVEC-CdMHYP were prepared. HUVECs were cultured with these conditioned media under hypoxia. The survival rate, apoptosis rate, migration and angiogenesis of HUVECs were respectively detected by CCK-8, flow cytometry, Transwell and tube formation assay. The content of SDF-1α, VEGF and IL-6 in CdM was determined by ELISA. Our results showed that hMSCs and HUVECs were cultured and identified successfully. Compared with MSC-CdMNOR and HUVEC-CdMHYP groups, the survival rate, migra-tion and angiogenesis of HUVECs in MSC-CdMHYP group were significantly increased while the apoptosis rate was declined (P<0.05). Moreover, the expression of SDF-1α, VEGF and IL-6 in MSC-CdMHYP group was up-regulated. Under hypoxia, the apoptosis of HUVECs was inhibited while survival, migration and angiogenesis were improved by co-culture of hMSCs and HUVECs. The underlying mechanism may be that hMSCs could secrete a number of cytokines and improve niche, which might be helpful in the treatment of femoral head necrosis.

Knockdown of Ezrin Suppresses the Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells In Vitro

Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells（ECs） are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin（ERM） family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells（HUVECs） in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.

Sonographic features of umbilical vein recanalization for a Rex shunt on cavernous transformation of portal vein in children

BACKGROUND The Rex shunt was widely used as the preferred surgical approach for cavernous transformation of the portal vein(CTPV)in children that creates a bypass between the superior mesenteric vein and the intrahepatic left portal vein(LPV).This procedure can relieve portal hypertension and restore physiological hepatopetal flow.However,the modified procedure is technically demanding because it is difficult to make an end-to-end anastomosis of a bypass to a hypoplastic LPV.Many studies reported using a recanalized umbilical vein as a conduit to resolve this problem.However,the feasibility of umbilical vein recanalization for a Rex shunt has not been fully investigated.AIM To investigate the efficacy of a recanalized umbilical vein as a conduit for a Rex shunt on CTPV in children by ultrasonography.METHODS A total of 47 children who were diagnosed with CTPV with prehepatic portal hypertension in the Second Hospital,Cheeloo College of Medicine,Shandong University,were enrolled in this study.Fifteen children received a recanalized umbilical vein as a conduit for a Rex shunt surgery and were enrolled in group I.Thirty-two children received the classic Rex shunt surgery and were enrolled in group II.The sonographic features of the two groups related to intraoperative and postoperative variation in terms of bypass vessel and the LPV were compared.RESULTS The patency rate of group I(60.0%,9/15)was significantly lower than that of group II(87.5%,28/32)7 d after(on the 8th d)operation(P<0.05).After clinical anticoagulation treatment for 3 mo,there was no significant difference in the patency rate between group I(86.7%,13/15)and group II(90.6%,29/32)(P>0.05).Moreover,3 mo after(at the beginning of the 4th mo)surgery,the inner diameter significantly widened and flow velocity notably increased for the bypass vessels and the sagittal part of the LPV compared to intraoperative values in both shunt groups(P<0.05).However,there was no significant difference between the two surgical groups 3 mo after surgery(P>0.05).CONCLUSION For children with hypoplastic LPV in the Rex recessus,using a recanalized umbilical vein as a conduit for a Rex shunt may be an effective procedure for CTPV treatment.

IOP-lowering effects for the application of human umbilical vein in non-penetrating deep sclerostomy in rabbits

AIM: To estimate the effects of human umbilical vein (HUV) implanted under the sclera of glaucoma model on intraocular pressure (IOP) lowering and to investigate its related mechanisms METHODS: A total of 20 human umbilical veins (HUV) were collected from healthy fetus umbilical core. After the establishment of glaucoma model in rabbits, human freeze-dried umbilical vein was implanted under the sclera during NPDS, while for control group, sclerostomy was performed without implant. The formation of the filtration bleb and TOP were detected every 24 hours before surgery and on day 3, 7, 10 and 14 after surgery. Handheld pen-type Tono-pen II tonometer was used to measure TOP after topical anesthesia treatment. Each measurement has three duplicates. The incision recovery, filtration, conjunctiva congestion and anterior chamber inflammation were observed everyday after surgery. RESULTS: IOP was decreased dramatically with less inflammation than traditional sclerostomies with the application of HUV. The significant differences of IOP between the NPDS with and without HUV implant groups were shown up from 10 days after surgery. The average TOP in NPDS without HUV implant was 14.25mmHg, while for NPDS with HUV implant group, it was 12.30mmHg. This structure of filtration bleb, which allowed the aqueous humor to leave the eye, was formed for any type of surgery. However, 1-2 weeks later, filtration bleb was still Existed in the group of sclerostomy with HUV implant and more stable than that of the surgery without HUV implant. Histological observations were performed on day 3, 7 and 14 after surgery. For the eyes under sclerostomy with HUV implant, HUV lumina was shown up on 3 days after surgery with few fibroblast cells near the sclera. On 7 days after surgery, HUV lumina was stably maintained but with obvious fibroblast cells and inflammatory cell. On 14 days after surgery, HUV lumina was still clearly observed but with scarring formation, which suggests that the IOP lowering effects might result from an effective drainage structure formation. CONCLUSION: HUV might be an alternative material to make the drainage pathway for non-penetrating deep sclerostomy.

Vein transplantation using human umbilical cord blood stem cells in the treatment of stroke sequela

BACKGROUND: Transplanted mononuclear cell (MNC) of umbilical blood can survive in central nervous system (CNS) of host through blood brain barrier, differentiate into nerve cells, migrate to damaged site and integrate morphological structure and function with nerve cells of host so as to improve deficiencies of sensatory function, motor function and cognitive function and influence on stroke sequela. OBJECTIVE: To observe the vein transplantation of human umbilical cord blood stem cells (HUCBSC) for improving neurological function, limb function and activity of daily living of patients with stroke and evaluate the reliability. DESIGN: Self-controlled study. SETTING: Department of Neurosurgery, the Second People's Hospital of Zhengzhou City; Red-crossed Blood Center of Henan Province; Department of Neurosurgery, the Fist Affiliated Hospital of Zhengzhou University. PARTICIPANTS: A total of 10 patients with stroke sequela were selected from Department of Cerebral Surgery, the Second People's Hospital of Zhengzhou City from April to December 2005. There were 9 males and 1 female aged from 35 to 75 years with the mean age of 56 years. All of them were diagnosed with CT and MRI examination and coincidence with diagnostic criteria of stroke established by the Fourth National Academic Meeting for Cerebrovascular Disease. All patients provided informed consent. METHODS: 80-140 mL umbilical blood of term birth of newborn was selected hermetically and maintained in sterile plastic bag. And then, the blood was centrifugated at the speed of 1 500 r/min for 30 minutes at 22 ℃ in order to separate MNC, i.e., HUCBSC. In addition, after final diagnosis during hospitalization, stroke patients were perfused with HUCBSC through superficial vein of back of the hand. Each patient was averagely perfused with 6 portions of HUCBSC (cellular numbers ≥ 1×108/portion) and the interval between each portion was 1-7 days with the mean interval of 4 days. MAIN OUTCOME MEASURES: ① Neurological function of stroke patients was evaluated with neurological function deficiency (NFD) before treatment and at 3 months after treatment. The scale includes consciousness, level fix function, facial paralysis, language, muscle force of upper limbs, muscle force of lower limb and step function. The total scores ranged from 0 to 45; meanwhile, the lower the scores were, the better the neurological function was. ② Motor function of injured limbs was evaluated with Fugl-Meyer Assessment (FMA), including motor function of upper limbs, motor function of lower limbs, balance ability, sensory function and motion of joint. The total scores ranged from 0 to 226; meanwhile, the higher the scores were, the better the motor function of limbs was. ③ Activities of daily living (ADL) was evaluated with Barthel Index (BI), including having meals, taking a bath, dressing oneself, putting on clothes, walking in balance and stair activity. The total scores ranged from 0 to 100; meanwhile, the higher the scores were, the stronger the ADL was. RESULTS: A total of 10 patients were involved in the final analysis. After treatment, NFD of stroke patients was (10.9±5.09) points, which was lower than that before treatment [(25.4±6.09) points, t =8.213, P < 0.01]. In addition, after treatment, FMA and BI of stroke patients were (80.9±25.00) points and (81.1±15.93) points, respectively, which were higher than those before treatment [(31.9±21.85) points, (36.2±19.41) points, t =13.024, 13.670, P < 0.01]. Immuno-suppressive drugs were not used during the whole therapeutic procedure; moreover, immunological rejection and allergic reaction were not observed during the same period. CONCLUSION: Transplanting HUCBSC through superficial vein of back of the hand is regarded as a simple and safe method for the treatment of stroke sequela.

Fetal umbilical vein transplantation for the repair of middle cerebral artery injury

It is necessary to investigate the longitudinal tensile mechanical characteristics of the middle cere- bral artery and the fetal umbilical vein prior to applying fetal umbilical vein transplantation for repair of injured middle cerebral artery. Fifteen fresh fetal umbilical vein specimens and 15 normal human fresh cadaver middle cerebral artery specimens were collected for longitudinal tensile testing at the speed of 0.5 mm/min and at normal human temperature. The results showed that under 16.0 kPa physiological stress, the strain value of fetal umbilical vein specimens was larger, while the maximal stress and elastic modulus values were less than those of middle cerebral artery specimens. Our findings indicate that fetal umbilical vein has good elastic properties and the stress-strain curve of the fetal umbilical vein is similar to that of the middle cerebral artery. Fetal umbilical vein transplan- tation can, therefore, potentially repair the injured middle cerebral artery.

Comparison of conventional and directional freezing for the cryopreservation of human umbilical vein endothelial cells

AIM:To compare conventional slow equilibrium cooling and directional freezing(DF) by gauze package for cryopreservation of human umbilical vein endothelial cells(HUVECs).METHODS:HUVECs were randomly assigned to conventional freezing(CF) and DF by gauze package group. The two groups of HUVECs were incubated with a freezing liquid consisting of 10% dimethylsulfoxide(DMSO), 60% fetal bovine serum(FBS) and 30%Dulbecco’s modified Eagle’s medium(DMEM) and then put into cryopreserved tubes. CF group, slow equilibrium cooling was performed with the following program:precool in 4℃ for 30 min,-20℃ for 1h, and then immersion in-80℃ refrigerator. DF group, the tubes were packaged with gauze and then directional freezing in-80℃ refrigerator straightly. One month later, the vitality of HUVECs were calculated between two groups.RESULTS:There was no significant difference in the survival rate and growth curve between CF and DF groups. The DF group was significantly better than CFgroup in adherent rates, morphological changes and proliferative ability.CONCLUSION:In the conventional cryopreserved method, cells are slow equilibrium cooling by steps(4℃,-20℃ and finally-80℃), which is a complicated and time-consuming process. But the improved DF by gauze package method is better than conventional method, for which is convenient and easy to operate.

Changes in Human Umbilical Vein Endothelial Cells Induced by Endothelial Nitric Oxide Synthase Traffic Inducer

This study investigated the changes in human umbilical vein endothelial cells （HUVECs） induced by overexpression of endothelial nitric oxide synthase traffic inducer （NOSTRIN） and its role in cellular injury. Recombinant NOSTRIN-expressing and empty vectors were transfected into cultured HUVECs, and factor Ⅷ-related antigen was examined by using immunohistochemical analysis. Growth curves were generated for both transfected and untransfected cells and these indicated that the prolifera- tive ability of cells overexpressing NOSTRIN was significantly decreased. The expression of NOSTRIN and eNOS proteins was detected by using Western blot analysis, endothelial NOS （eNOS） activity was assayed by using spectrophotometry, and NO2-/NO3- levels were measured usin~ nitrate reductase. Immunohistochemical analysis demonstrated that all groups expressed NOSTRIN in the plasma mem- brane and cytoplasm, and Western blot analysis confirmed that NOSTR1N levels were significantly higher in cells transfected with the NOSTR1N plasmid （P〈0.01）. The activity of eNOS and the levels of NO2-/NO3 were significantly decreased in NOSTRIN overexpressing cells as compared with empty vector and untransfected cells （P〈0.01 and P〈0.01, respectively）. Morphological and ultrastructural changes were observed under light and electron microscopy, and it was found that NOS- TRIN-overexpressing cells were elongated with deformities of the karyotheca, injury to the plasma membrane, increased lipids in the cytoplasm, and shortened microvilli. This study showed that overex- pression of NOSTRIN had a significant effect on eNOS activity in HUVECs and resulted in significant cellular damage.

Effects of miR-21 antisense oligonucleotides on proliferation,migration and autophagy of human umbilical vein endothelial cells

Objective:To investigate the effects of microRNA-21 antisense nucleotide(AS-miR-21)on the proliferation,migration and autophagy of human umbilical vein endothelial cells(HUVECs).Methods:HUVECs were treated with1,000 nmol/L rapamycin for 6 h(rapamycin group)or ASmiR-21 transfection followed by 1,000 nmol/L rapamycin for6 h(AS-miR-21+rapamycin group).HUVECs without any treatment were defined as control group.The proliferation and migration abilities of HUVECs were detected by methyl thiazolyl tetrazolium(MTT)assay,scratch wound healing assay and transwell test,respectively.The expressions of microtubule-associated protein light chain 3 Ⅱ/Ⅰ(LC3 Ⅱ/Ⅰ)and Becline-1 were determined by western blotting.Results:The rapamycin group showed decreased OD value and migration rate,an increased ratio of LC3 Ⅱ/Ⅰ and up-regulated expression of Beclin-1 compared with the control group(P<0.05).The AS-miR-21+rapamycin group demonstrated lower OD value,migration rate,the number of migrated cells,and significantly higher ratio of LC3 Ⅱ/Ⅰ and Beclin-1 protein expression level than the control group and the rapamycin group(P<0.05).Conclusion:AS-miR-21 suppressed the autophagy,proliferation and migration in the HUVECs model of autophagy induced by rapamycin.

The Effects and Mechanism of GSA on Expression of MCP-1 in Cultured Human Umbilical Vein Endothelial Cells

Objectives To investigate the effects and mechanism of glycated serum albumin(GSA) on expression of Monocyte chemoattratant protein-1(MCP-1) in Endothelial Cells. Methods Human Umbilical Vein Endothelial Cells (HUVEC)are cultured with GSA of different concentrations and interfered by glycosylation products inhibitor Aminoguanidine (AG) and anti-oxidant N-acetylcy-steine (NAC), The expression of MCP-1 are evaluated by Immunocytochemistry and Sandwich ELISA. MDA content and SOD activity are determined by the technique of TBA and XOD respectively. Results GSA can stimulate MCP-1 production and secretion. Immunocytochemistry showed that after HUVECs were cultured with 50 mg/L GSA, expression of MCP-1 in group 4hrs, 8hrs and 12hrs was 1.3, 1.9 and 2.8 fold as much as that in control group (P < 0.01), and there was significant difference among the experiment groups(P < 0.01). Sandwich ELISA showed that expression of MCP-1 in three different groups was 1.6, 2.4 and 3.0 fold as much as that in control group(P < 0.01), and there was significant difference among the experiment groups(P < 0.01); GSA can cause the decrease of SOD activity(P < 0.05) and increase of MDA content(P < 0.01); AG and NAC can restrain obviously the expression of MCP-1 of HUVECs stimulated by GSA(P < 0.01); NAC can restrain the effect of GSA on SOD activity and MDA content in HUVECs (P < 0.05). Conclusions GSA can stimulate the expression of MCP-1 of endothelial cells by inducing endothelial cells oxidative stress.

Clinical Application of Fetal Umbilical Vein Doppler Parameters in Maternal Preeclampsia

Objective: To observe the changes of fetal umbilical vein (UV) Doppler parameters in pregnant women with preeclampsia (PE) and analyze their predictive values for maternal PE. Methods: Forty-six patients with PE who underwent systematic ultrasound examination in our hospital from December 2017 to May 2021 were included as the subjects, which were divided into two groups according to the severity of the disease (23 cases in each group). And 120 normal pregnant women who underwent health examination in our hospital during the same period were enrolled as the control group. Color Doppler ultrasonography was used to monitor the umbilical vein flow (QUV), left portal vein flow (QLPV), venous catheter flow (QDV), left portal vein (LPV) shunt rate and venous catheter (DV) shunt rate. And the sensitivity and specificity of the related indexes were calculated and analyzed according to the gold standard for clinical diagnosis of PE. Results: As the severity of PE increased, the fetal QUV, QLPV and LPV shunt rates decreased, and the QDV and DV shunt rates increased, with statistically significant differences compared with the control group (P < 0.05). The specificity and sensitivity of the combination of fetal QUV, QLPV, QDV, LPV shunt rate and DV shunt rate in predicting PE were higher than those of the indexes used alone (P < 0.05). Conclusion: The fetal umbilical vein Doppler parameters QUV, QLPV, QDV, LPV shunt rate, and DV shunt rate have some value in predicting PE, but their combination showed greater value, as well as higher diagnostic and clinical significance.

LncRNA HCG27 Promotes Glucose Uptake Ability of HUVECs by MiR-378a-3p/MAPK1 Pathway

Objective:Gestational diabetes mellitus(GDM)is the most common metabolic disorder during pregnancy.LncRNA HLA complex group 27(HCG27)plays a crucial role in various metabolic diseases.However,the relationship between lncRNA HCG27 and GDM is not clear.This study aimed to verify a competing endogenous RNA(ceRNA)interaction regulation axis of miR-378a-3p/mitogen-activated protein kinase 1(MAPK1)regulated by HCG27 in GDM.Methods:LncRNA HCG27 and miR-378a-3p were detected by RT-qPCR.The expression of MAPK1 in umbilical vein endothelial cells(HUVECs)was detected by RT-qPCR and that in the placenta by Western blotting.To explore the relationship among lncRNA HCG27,miR-378a-3p,MAPK1 and the glucose uptake ability of HUVECs,vector HCG27,si-HCG27,miR-378a-3p mimic and inhibitor were transfected to achieve overexpression and inhibition of HCG27 or miR-378a-3p.The interaction between miR-378a-3p and lncRNA HCG27 or MAPK1 was confirmed by the dual-luciferase reporter assay.Besides,glucose consumption by HUVECs was detected by the glucose assay kit.Results:HCG27 expression was significantly decreased in both the placenta and primary umbilical vein endothelial cells,while the expression of miR-378a-3p was significantly increased in GDM tissues,and the expression of MAPK1 was decreased in GDM tissues.This ceRNA interaction regulation axiswas proved to affect the glucose uptake function of HUVECs.The transfection of si-HCG27 could significantly reduce the expression of the MAPK1 protein.If the MAPK1 overexpression plasmid was transfected simultaneously with si-HCG27 transfection,the reduced glucose uptake in HUVECs resulting from the decrease in lncRNA HCG27 was reversed.MiR-378a-3p mimic can significantly reduce the mRNA expression of MAPK1 in HUVECs,whereas miR-378a-3p inhibitor can significantly increase the mRNA expression of MAPK1.The inhibition of miR-378a-3p could restore the decreased glucose uptake of HUVECs treated with si-HCG27.Besides,overexpression of lncRNA HCG27 could restore the glucose uptake ability of the palmitic acid-induced insulin resistance model of HUVECs to normal.Conclusion:LncRNA HCG27 promotes glucose uptake of HUVECs by miR-378a-3p/MAPK1 pathway,which may provide potential therapeutic targets for GDM.Besides,the fetal umbilical cord blood and umbilical vein endothelial cells collected from pregnant women with GDM after delivery could be used to detect the presence of adverse molecular markers of metabolic memory,so as to provide guidance for predicting the risk of cardiovascular diseases and health screening of offspring.

Expression of Soluble Vascular Endothelial Growth Factor Receptor-2 and Its Effect on Proliferation of Vascular Endothelial Cells

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.

RO-heparin Inhibits L-Selectin-mediated Neutrophils Adhesion to Vascular Endothelium Under Flow Conditions

Selectins are carbohydrate-binding cell adhesion molecules that play a major role in the initiation of inflammatory responses. Accumulaed evidence has suggested that heparin＇s anti-inflammatory effects are mainly mediated by blocking L-or P-selectin-initiated cell adhesion. Recently, we have reported that periodate-oxidized, borohydridereduced heparin （RO-heparln） can inhibit P-selectin-mediated acute inflammation. Here we further examined the effect of RO-heparin on the adhesion of L-selectin-mediated leukocytes to vascular endothelium under flow conditions in vivo and in vitro. The results show that RO-heparin with a low anticoagulant activity can effectively reduce leucocyte roiling on thioglycoUate-induced rat mesenterlc venules and L-selectin-metadiated neutrophil roiling on TNF-α-induced human umbilical vein endothelial cells（HUVECs） under flow conditions. Our findings suggest that the effect of RO-heparin on inflammatory responses is mainly a result of its inhibiting the interaction between P- or L-selectin and its ligands. The findings also suggest that RO-heparin may be useful in preventing inflammation diseases.

Effect of cytokeratin 17 on retinal pigment epithelium degeneration and choroidal neovascularization

AIM： To study the effects of cytokeratin 17 （CK17） on sodium iodate （NalOs） induced rat retinal pigment epithelium （RPE） degeneration, laser induced rat choroidal neovascularization （CNV）, and oxidative stress of human retinal pigment epithelium cells （ARPE-19） and human umbilical vein endothelial cell （HUVEC）. METHODS： Thirty 8-week-old male Brown Norway rats were randomly divided into 3 groups, 10 rats in control group treated with solvent alone; 10 rats in NalOs group treated with solvent and 35 mg/kg NalO3 injection through hypoglossal vein and 10 rats in CK17 ＋NaIOs group treated with 1% CK17 eye drop 3 times a day for lwk before and 4wk after NalOs injection. RPE function was measured with c-wave of electroretinogram （ERG）. Another 20 rats were randomly divided into 2 groups. Of them 10 rats in CK17 group were anesthetized to receive Nd：YAG laser and given 1% CK17 eye drop before same as above; 10 rats in control were received Nd：YAG and treated with solvent. The development of choroidal neovascularization （CNV） was determined by fundus fluorescein angiography （FFA） performed on 4wk after laser. Methylthiazoly tetrazolium （MTT） assay was used to study effect of CK17 on various oxidants induced injury in ARPE-19 and HUVEC /n vitro RESULTS： Four weeks after NalOs injection, the c- wave amplitude of ERG was 0.393±0.02 V in the control group, 0.184±0.018 V in NalOs group and 0.3±0.01 V in CK17＋NalOs group. There was a significant reversal of the c-wave by CK17 as compared to NalOs group （P〈0.01）. Four weeks after laser, the size of the CNV lesion was 2.57±0.27 mm2 in control group and 1.64 ±0.08 mm2 in CK17 group. The lesion size significantly diminished in CK17 group （P〈0.01）. The inn vitro results showed CK17 also reversed the various oxidants induced injuries in ARPE-19 at the dose of 100 μg/mL and enhanced the injury in HUVECs at different concentrations. CONCLUSION： CK17 can significantly protect RPE from NalOs induced degeneration in vivo and /n vito and also could reverse the various oxidants induced injuries in vitro. It inhibits the development of CNV in rat model, interfered with vascular endothelial cell proliferation in ivtro.

The Active Metabolite of Leflunomide A771726 Inhibits Corneal Neovascularization

The effects of A771726, the active metabolite of leflunomide, on experimental rat corneal neovascularization （NV） in vivo and on cultured human umbilical vein endothelial cells in vitro were studied. The corneal NV was induced by alkali burn in 40 SD rats. The rats were randomly divided into 4 groups with 10 rats in each group. Group A was treated with 0.9% sodium chloride （control group）, and group B, group C and group D were given different concentrations of A771726 eye drops （0.5%, 1.0%, 2.0% respectively） 4 times daily during days 0--28. The occurrence and development of corneal NV were observed at 4, 7, 14, 21 and 28 day after alkali burn by a slit lamp microscope. The cultured human umbilical vein endothelial cells （ECV-304） were incubated with A771726 solution at different concentrations （20, 40, 80, 160, 320 μmol/L） for 36 h. The proliferation of cells was assessed by methyl thiazolyl tetrazolium （MTT）, and the expression of proliferating cell nuclear antigen （PCNA） in cells was detected by using immunofluorescence under the laser confocal microscope. The rat model showed that the onset of corneal NV was delayed and progression of corneal NV was inhibited in the groups C and D. The corneal NV areas in groups C and D were significantly smaller than in groups A and B （P〈0.01）. No significant difference was found in corneal NV areas between groups C and group D （P〉0.05）. A771726 solution （940 μmol/L） could inhibit proliferation of human umbilical vein endothelial cells and decrease the expression of PCNA in cells significantly, A771726, as the active metabolite of leflunomide, strongly prevented corneal NV induced by alkali burn in the in vivo model, and inhibited proliferation of human umbilical vein endothelial cells in the in vitro model. Therefore, A771726 may serve as an angiogenic inhibitor in the treatment of corneal NV.

The Effect of Brain-derived Neurotrophic Factor on Angiogenesis

To investigate the in vitro and in vivo proangiogenic effects of brain-derived neurotrophic factor （BDNF）, human umbilical vein endothelial cells （HUVECs） were isolated and cultured in primary culture. The effect of BDNF on the proliferation of HUVECs was examined by MTT assay. The effects of BDNF on HUVEC migration and tube formation were studied by modified Boyden cham- ber assay and tube formation assay, respectively. Matrigel plug assay and chorioallantoic membrane assay were used to evaluate the effects of BDNF on angiogenesis in vivo. Our results showed that BDNF substantially stimulated the migration and tube formation of HUVECs in vitro, although it did not induce HUVEC proliferation. BDNF also induced angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. In conclusion, BDNF can promote angiogenesis both in vitro and in vivo, and may be a proangiogenic factor.