OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At...OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.展开更多
Corneal stem/progenitor cells are typical adult stem/progenitor cells.The human cornea covers the front of the eyeball,which protects the eye from the outside environment while allowing vision.The location and functio...Corneal stem/progenitor cells are typical adult stem/progenitor cells.The human cornea covers the front of the eyeball,which protects the eye from the outside environment while allowing vision.The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role.Corneal stem/progenitor cells include mainly corneal epithelial stem cells,corneal endothelial cell progenitors and corneal stromal stem cells.Since the discovery of corneal epithelial stem cells(also known as limbal stem cells)in 1971,an increasing number of markers for corneal stem/progenitor cells have been proposed,but there is no consensus regarding the definitive markers for them.Therefore,the identification,isolation and cultivation of these cells remain challenging without a unified approach.In this review,we systematically introduce the profile of biological characterizations,such as anatomy,characteristics,isolation,cultivation and molecular markers,and clinical applications of the three categories of corneal stem/progenitor cells.展开更多
AIM： To compare intraoperative phacoemulsification parameters and its effect on the corneal endothelium of eyes undergoing femtosecond laser-assisted cataract surgery（FLACS） versus conventional phacoemulsification...AIM： To compare intraoperative phacoemulsification parameters and its effect on the corneal endothelium of eyes undergoing femtosecond laser-assisted cataract surgery（FLACS） versus conventional phacoemulsification（CP） cataract surgery.METHODS： Two hundred eyes from one hundred patients were included in a prospective, non-blinded, randomized, controlled, intraindividual clinical study. One hundred eyes underwent FLACS while their one hundred fellow eyes underwent CP. All surgeries were performed using the Victus？ femtosecond laser platform and Infinity？ Vision System phacoemulsification machine. Primary outcome measure was endothelial cell density 6 mo after surgery. Secondary outcome measures included central corneal thickness（CCT）, average cell area, standard deviation, coefficient of variation and hexagonality before surgery and 6 mo after surgery and endothelial cell density loss during this period were also evaluated. Intraoperative efficiency parameters [cumulative dissipated energy（CDE）, total intraocular surgery time, total ultrasound time, total phacoemulsification time, total torsional energy time, total aspiration time, ultrasound energy, torsional amplitude and fluid required during surgery] were also collated. RESULTS： Data from these patients was not considered for analysis. Data from 92 patients were analysed. Postoperative endothelial cell density（cells/mm2） between groups（2211.88±392.49 CP; 2246.31±403.48 FLACS） was not statistically significant（P=0.869）. Total ultrasound time, torsional energy time, CDE and fluid requirements were significantly lower the FLACS group（P〈0.05）. Other parameters did not show statistically significant difference between FLACS and CP.CONCLUSION： FLACS displays significant improvements in phacoemulsification parameters in comparison to CP. There are no significant differences in corneal endothelium measures between FLACS and CP.展开更多
This study examined the association of expression of vascular endothelial growth factor(VEGF),a promoter of angiogenesis,with endothelium dysfunction in preeclampsia.The level of VEGF protein and mRNA in the placenta ...This study examined the association of expression of vascular endothelial growth factor(VEGF),a promoter of angiogenesis,with endothelium dysfunction in preeclampsia.The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry,real-time reverse transcriptase-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA),respectively.VEGF expression in the human umbilical vein endothelial cells(HUVECs) was blocked by small interfering RNAs(siRNAs).The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers.The cell proliferation and cell-secreted nitric oxide(NO) level were detected by MTT method and nitrate reductase assay,respectively.The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue.The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects,although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high.Moreover,VEGF deficit could lead to endothelium cell dysfunction,and the administration of VEGF could protect endothelium cells from injury.It was concluded that lack of VEGF contributes to endothelium dysfunction,which may lead to the occurrence and development of preeclampsia.展开更多
Breakdown of blood-brain barrier,formed mainly by brain microvascular endothelial cells(BMECs),represents the major cause of mortality during early phases of ischemic strokes.Hence,discovery of novel agents that can e...Breakdown of blood-brain barrier,formed mainly by brain microvascular endothelial cells(BMECs),represents the major cause of mortality during early phases of ischemic strokes.Hence,discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine.Although endothelial progenitor cells(EPCs)represent one such agents,their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells(OECs)as a result.Functional analyses of these cells,in the present study,demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel,prominent endothelial cell and angiogenesis markers,respectively.Further analyses by flow cytometry demonstrated that OECs expressed specific markers for sternness(CD34),immaturity(CD133)and endothelial cells(CD31)but not for hematopoietic cells(CD45).Like BMECs,OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes,suggesting their capacity to form tight junctions.Ischemic injury mimicked by concurrent deprivation of oxygen and glucose(4 hours)or deprivation of oxygen and glucose followed by reperfusion(20 hours)affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux,respectively.Wound scratch assays comparing the vasculoreparative capacity of cells revealed that,compared to BMECs,OECs possessed a greater proliferative and directional migratory capacity.In a triple culture model of BBB established with astrocytes,pericytes and BMEC,exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions.Taken together,these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain(neuro)vascular homeostasis during or after a cerebral ischemic injury.展开更多
Objective To clarify the mechanism and provide the basis for prevention and treatme nt of the early injuries of kidney after severe burn in rats.We observed the expression of intercellular adhesion molecule 1and inter...Objective To clarify the mechanism and provide the basis for prevention and treatme nt of the early injuries of kidney after severe burn in rats.We observed the expression of intercellular adhesion molecule 1and interleukin 6and the early p athological changes in different time.Method Early pathological changes in the kidney were observed by LM and EM.The ex pression of ICAM-1were observed by i mmunohis-tochemistry,in situ hybridization.The expression of IL-6was also obse rved.Result From 5min to 72h after burn,the early changes in the kidney included edema,hemorrhage,and congestion,injury of capillary epithelium cells.2ICAM-1and IL-6w ere higher in the kidney 30min after burn,and from 2h to 24h,they we re strongest positive,but on 72h,th ey were negative.Conclusion ICAM-1and IL-6may play important roles in mechanisms of kid ney injury,and the major target cell s may be the endothelium cells.展开更多
The experimental models of cultured porcine aortic endothelial cells(EC) in vitro were established. 6-keto-prostaglandin F1a(6-keto-PGF1a), thromboxane B2(TXB2) content, plasminogen activator(P A), plasminogen activat...The experimental models of cultured porcine aortic endothelial cells(EC) in vitro were established. 6-keto-prostaglandin F1a(6-keto-PGF1a), thromboxane B2(TXB2) content, plasminogen activator(P A), plasminogen activator inhibitor(PAI) activity in cultured medium and cyclic adenosine monophosphate(cAMP) level in EC were measured with radioimmunoassay (RIA), chromogenic substrates methods, in order to assess the effect and mechanism of captopril(CP) on antithrombus function of EC. The results showed that after administration of CP, the contents of 6-keto-PGF1a and cAMP and PA activity were significantly higher, PAI activity were remarkably lower than those of control group. These effects were dose-dependent. Our finding indicated that CP might act as a prospective drug for antithrombosis through promoting anticoagulation and fibrinolysis function and increasing antithrombus action of EC.展开更多
Objective To study the effects of quercetin (Que) on the release of endothelin-1 (ET-1) and prostacylin(PGI2) by normal human vascuiar endothelial cell (VEC). Methods Radioimmunoassay(RIA) was used to assess the amoun...Objective To study the effects of quercetin (Que) on the release of endothelin-1 (ET-1) and prostacylin(PGI2) by normal human vascuiar endothelial cell (VEC). Methods Radioimmunoassay(RIA) was used to assess the amount of ET-1 and PGI2 produced by VEC. VEC prollferation was assessed by tetrazolium(MTT) assay. Results Que increased the normal VEC prollferation at the concentration or 5, 2o, 4o, 8o, 1oompol/L and increased the production of PG12 and inhibits the release of ET by the normal VEC at the concentratiou or 5, 2o and 8ompol/L. Que at the concentration of 5, 2o and 8omol/L had no direct effect on morphology of the normal VEC. ConcIusion Que can stimulate the proliferation of VEC and inhibit tbe reIease of ET-1 and increase the formation of PGI2. The data suggest that Que might be beneficial for the prevention and treatment of vascular endothelial injury-related cardiovascular diseases, such as atherosclerosls and thromboembolism diseases.展开更多
AIM:To clarify whether the vasoconstrictory response is impaired and to study vascular function in patients with migraine during the headache attack.METHODS:We studied vascular reactivity in the resistance arteries by...AIM:To clarify whether the vasoconstrictory response is impaired and to study vascular function in patients with migraine during the headache attack.METHODS:We studied vascular reactivity in the resistance arteries by using the forearm perfusion technique associated with plethysmography.We measuredforearm blood flow by strain-gauge plethysmography during intra-brachial infusion of acetylcholine,sodium nitroprusside or norepinephrine in 11 controls and 13patients with migraine,11 of them(M) in the interval between the migraine attacks and 4 during a headache attack(MH).Written informed consent was obtained from patients and healthy controls,and the study was approved by the Ethics Committee of the University Federico Ⅱ.RESULTS:Compared to healthy control subjects,in patients with migraine studied during the interictal period,the vasodilating effect of acetylcholine,that acts through the stimulation of endothelial cells and the release of nitric oxide,was markedly reduced,but became normal during the headache attack(P<0.05by analysis of variance).The response to nitroprusside,which directly relaxes vascular smooth muscle cells(VSMCs),was depressed in patients with migraine studied during the interictal period,but normal during the headache attack(P<0.005).During norepinephrine infusion,forearm blood flow decreased in control subjects(-40% ± 5%,P<0.001).In contrast,in patients with migraine,either when studied during or free of the headache attack forearm blood flow did not change compared to the baseline value(-3%±13% and-10.4%±15%,P>0.05).CONCLUSION:In migrainers,the impaired relaxation of VSMCs is restored during the headache attack.The vasoconstrictory response is impaired and remains unchanged during the migraine attack.展开更多
Sickle cell anemia (SCA) is an autosomal-recessive hemoglobinopathy with a highly variable phenotype. Multiple clinical complications are characteristic of SCA including inflammatory and oxidant damage to both small a...Sickle cell anemia (SCA) is an autosomal-recessive hemoglobinopathy with a highly variable phenotype. Multiple clinical complications are characteristic of SCA including inflammatory and oxidant damage to both small and large blood vessels, hemolysis, vasoocclusion, and premature mortality. The overall severity of SCA is affected by multiple genetic modifier loci, including ARFGEF2, a gene known to modify TNF-α receptor release from human endothelial cells. In this report, we examine the effect of siRNA mediated knockdown of ARFGEF2 inhuman pulmonary artery endothelial cells and report that TNF-α induced expression of ICAM1 and VCAM1, both important mediators of endo-thelial-leukocyte adhesion, is significantly enhanced after ARFGEF2 knockdown. Levels of ICAM-1 protein are also increased in TNF-α treated endothelial cells after ARFGEF2 knockdown;the increased ICAM-1 appears to be localized in the cytoplasm. IL-1β stimulation of endothelial cells without ARFGEF2 produced enhanced ICAM1 expression only. Additionally, ARFGEF2 knockdown distinctly altered endothelial cell morphology. Large-vessel pathology in SCA is believed to begin with endothelial activation by inflammatory cytokines and adhesion of sickle erythrocytes and leukocytes, leading to a progressive vasculopathy characterized by smooth muscle cell migration and proliferation. Understanding how variability in the function of ARFGEF2 alters the response of pulmonary vasculature to TNF-α might suggest new targets for SCA treatment.展开更多
Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there aremany challeng...Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there aremany challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.展开更多
AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cho...AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.展开更多
基金The project supported by Central Public Scientific Research Institution Fundamental Project(2016CX09)by National Natural Science Foundation of China(81573645)
文摘OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.
文摘Corneal stem/progenitor cells are typical adult stem/progenitor cells.The human cornea covers the front of the eyeball,which protects the eye from the outside environment while allowing vision.The location and function demand the cornea to maintain its transparency and to continuously renew its epithelial surface by replacing injured or aged cells through a rapid turnover process in which corneal stem/progenitor cells play an important role.Corneal stem/progenitor cells include mainly corneal epithelial stem cells,corneal endothelial cell progenitors and corneal stromal stem cells.Since the discovery of corneal epithelial stem cells(also known as limbal stem cells)in 1971,an increasing number of markers for corneal stem/progenitor cells have been proposed,but there is no consensus regarding the definitive markers for them.Therefore,the identification,isolation and cultivation of these cells remain challenging without a unified approach.In this review,we systematically introduce the profile of biological characterizations,such as anatomy,characteristics,isolation,cultivation and molecular markers,and clinical applications of the three categories of corneal stem/progenitor cells.
文摘AIM： To compare intraoperative phacoemulsification parameters and its effect on the corneal endothelium of eyes undergoing femtosecond laser-assisted cataract surgery（FLACS） versus conventional phacoemulsification（CP） cataract surgery.METHODS： Two hundred eyes from one hundred patients were included in a prospective, non-blinded, randomized, controlled, intraindividual clinical study. One hundred eyes underwent FLACS while their one hundred fellow eyes underwent CP. All surgeries were performed using the Victus？ femtosecond laser platform and Infinity？ Vision System phacoemulsification machine. Primary outcome measure was endothelial cell density 6 mo after surgery. Secondary outcome measures included central corneal thickness（CCT）, average cell area, standard deviation, coefficient of variation and hexagonality before surgery and 6 mo after surgery and endothelial cell density loss during this period were also evaluated. Intraoperative efficiency parameters [cumulative dissipated energy（CDE）, total intraocular surgery time, total ultrasound time, total phacoemulsification time, total torsional energy time, total aspiration time, ultrasound energy, torsional amplitude and fluid required during surgery] were also collated. RESULTS： Data from these patients was not considered for analysis. Data from 92 patients were analysed. Postoperative endothelial cell density（cells/mm2） between groups（2211.88±392.49 CP; 2246.31±403.48 FLACS） was not statistically significant（P=0.869）. Total ultrasound time, torsional energy time, CDE and fluid requirements were significantly lower the FLACS group（P〈0.05）. Other parameters did not show statistically significant difference between FLACS and CP.CONCLUSION： FLACS displays significant improvements in phacoemulsification parameters in comparison to CP. There are no significant differences in corneal endothelium measures between FLACS and CP.
基金The researches described in this article were partially supported by grants from the National Natural Science Foundation of China （No. 81570271 and 81400357） and NIH （UL1 RR024996）. We are very grateful to John R Lee （Assistant Professor of Medicine, Weill Comell Medical College, New York）, and Jeff J Zhu （Research Manager, Weill Comell Medical College, New York） for critical review of the article. The authors have nothing to disclosure.
文摘This study examined the association of expression of vascular endothelial growth factor(VEGF),a promoter of angiogenesis,with endothelium dysfunction in preeclampsia.The level of VEGF protein and mRNA in the placenta and peripheral blood samples of 30 preeclampsia patients and 30 normotensive pregnant women was measured by immunohistochemistry,real-time reverse transcriptase-polymerase chain reaction(RT-PCR) and enzyme-linked immunosorbent assay(ELISA),respectively.VEGF expression in the human umbilical vein endothelial cells(HUVECs) was blocked by small interfering RNAs(siRNAs).The monolayer barrier function of HUVECs was determined by measuring the fluorescence intensity of BSA that crossed the HUVEC monolayers.The cell proliferation and cell-secreted nitric oxide(NO) level were detected by MTT method and nitrate reductase assay,respectively.The results showed that VEGF was expressed in the syncytiotrophoblasts and endothelial cells of vessels and capillaries in the placenta tissue.The serum level of VEGF in the preeclampsia patients was significantly decreased as compared with that in normal pregnant subjects,although VEGF mRNA expression in the placenta tissue of preeclampsia patients remained still high.Moreover,VEGF deficit could lead to endothelium cell dysfunction,and the administration of VEGF could protect endothelium cells from injury.It was concluded that lack of VEGF contributes to endothelium dysfunction,which may lead to the occurrence and development of preeclampsia.
基金Part of this study has been supported by a grant to Dr Bayraktutan from The Dunhill Medical Trust(R459/0216)
文摘Breakdown of blood-brain barrier,formed mainly by brain microvascular endothelial cells(BMECs),represents the major cause of mortality during early phases of ischemic strokes.Hence,discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine.Although endothelial progenitor cells(EPCs)represent one such agents,their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells(OECs)as a result.Functional analyses of these cells,in the present study,demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel,prominent endothelial cell and angiogenesis markers,respectively.Further analyses by flow cytometry demonstrated that OECs expressed specific markers for sternness(CD34),immaturity(CD133)and endothelial cells(CD31)but not for hematopoietic cells(CD45).Like BMECs,OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes,suggesting their capacity to form tight junctions.Ischemic injury mimicked by concurrent deprivation of oxygen and glucose(4 hours)or deprivation of oxygen and glucose followed by reperfusion(20 hours)affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux,respectively.Wound scratch assays comparing the vasculoreparative capacity of cells revealed that,compared to BMECs,OECs possessed a greater proliferative and directional migratory capacity.In a triple culture model of BBB established with astrocytes,pericytes and BMEC,exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions.Taken together,these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain(neuro)vascular homeostasis during or after a cerebral ischemic injury.
文摘Objective To clarify the mechanism and provide the basis for prevention and treatme nt of the early injuries of kidney after severe burn in rats.We observed the expression of intercellular adhesion molecule 1and interleukin 6and the early p athological changes in different time.Method Early pathological changes in the kidney were observed by LM and EM.The ex pression of ICAM-1were observed by i mmunohis-tochemistry,in situ hybridization.The expression of IL-6was also obse rved.Result From 5min to 72h after burn,the early changes in the kidney included edema,hemorrhage,and congestion,injury of capillary epithelium cells.2ICAM-1and IL-6w ere higher in the kidney 30min after burn,and from 2h to 24h,they we re strongest positive,but on 72h,th ey were negative.Conclusion ICAM-1and IL-6may play important roles in mechanisms of kid ney injury,and the major target cell s may be the endothelium cells.
文摘The experimental models of cultured porcine aortic endothelial cells(EC) in vitro were established. 6-keto-prostaglandin F1a(6-keto-PGF1a), thromboxane B2(TXB2) content, plasminogen activator(P A), plasminogen activator inhibitor(PAI) activity in cultured medium and cyclic adenosine monophosphate(cAMP) level in EC were measured with radioimmunoassay (RIA), chromogenic substrates methods, in order to assess the effect and mechanism of captopril(CP) on antithrombus function of EC. The results showed that after administration of CP, the contents of 6-keto-PGF1a and cAMP and PA activity were significantly higher, PAI activity were remarkably lower than those of control group. These effects were dose-dependent. Our finding indicated that CP might act as a prospective drug for antithrombosis through promoting anticoagulation and fibrinolysis function and increasing antithrombus action of EC.
文摘Objective To study the effects of quercetin (Que) on the release of endothelin-1 (ET-1) and prostacylin(PGI2) by normal human vascuiar endothelial cell (VEC). Methods Radioimmunoassay(RIA) was used to assess the amount of ET-1 and PGI2 produced by VEC. VEC prollferation was assessed by tetrazolium(MTT) assay. Results Que increased the normal VEC prollferation at the concentration or 5, 2o, 4o, 8o, 1oompol/L and increased the production of PG12 and inhibits the release of ET by the normal VEC at the concentratiou or 5, 2o and 8ompol/L. Que at the concentration of 5, 2o and 8omol/L had no direct effect on morphology of the normal VEC. ConcIusion Que can stimulate the proliferation of VEC and inhibit tbe reIease of ET-1 and increase the formation of PGI2. The data suggest that Que might be beneficial for the prevention and treatment of vascular endothelial injury-related cardiovascular diseases, such as atherosclerosls and thromboembolism diseases.
文摘AIM:To clarify whether the vasoconstrictory response is impaired and to study vascular function in patients with migraine during the headache attack.METHODS:We studied vascular reactivity in the resistance arteries by using the forearm perfusion technique associated with plethysmography.We measuredforearm blood flow by strain-gauge plethysmography during intra-brachial infusion of acetylcholine,sodium nitroprusside or norepinephrine in 11 controls and 13patients with migraine,11 of them(M) in the interval between the migraine attacks and 4 during a headache attack(MH).Written informed consent was obtained from patients and healthy controls,and the study was approved by the Ethics Committee of the University Federico Ⅱ.RESULTS:Compared to healthy control subjects,in patients with migraine studied during the interictal period,the vasodilating effect of acetylcholine,that acts through the stimulation of endothelial cells and the release of nitric oxide,was markedly reduced,but became normal during the headache attack(P<0.05by analysis of variance).The response to nitroprusside,which directly relaxes vascular smooth muscle cells(VSMCs),was depressed in patients with migraine studied during the interictal period,but normal during the headache attack(P<0.005).During norepinephrine infusion,forearm blood flow decreased in control subjects(-40% ± 5%,P<0.001).In contrast,in patients with migraine,either when studied during or free of the headache attack forearm blood flow did not change compared to the baseline value(-3%±13% and-10.4%±15%,P>0.05).CONCLUSION:In migrainers,the impaired relaxation of VSMCs is restored during the headache attack.The vasoconstrictory response is impaired and remains unchanged during the migraine attack.
文摘Sickle cell anemia (SCA) is an autosomal-recessive hemoglobinopathy with a highly variable phenotype. Multiple clinical complications are characteristic of SCA including inflammatory and oxidant damage to both small and large blood vessels, hemolysis, vasoocclusion, and premature mortality. The overall severity of SCA is affected by multiple genetic modifier loci, including ARFGEF2, a gene known to modify TNF-α receptor release from human endothelial cells. In this report, we examine the effect of siRNA mediated knockdown of ARFGEF2 inhuman pulmonary artery endothelial cells and report that TNF-α induced expression of ICAM1 and VCAM1, both important mediators of endo-thelial-leukocyte adhesion, is significantly enhanced after ARFGEF2 knockdown. Levels of ICAM-1 protein are also increased in TNF-α treated endothelial cells after ARFGEF2 knockdown;the increased ICAM-1 appears to be localized in the cytoplasm. IL-1β stimulation of endothelial cells without ARFGEF2 produced enhanced ICAM1 expression only. Additionally, ARFGEF2 knockdown distinctly altered endothelial cell morphology. Large-vessel pathology in SCA is believed to begin with endothelial activation by inflammatory cytokines and adhesion of sickle erythrocytes and leukocytes, leading to a progressive vasculopathy characterized by smooth muscle cell migration and proliferation. Understanding how variability in the function of ARFGEF2 alters the response of pulmonary vasculature to TNF-α might suggest new targets for SCA treatment.
文摘Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there aremany challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.
基金Supported by the Austrian Science Fund,No.P20116-B13 and No.P22838-B13
文摘AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.
