Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF α (1-250 U/ml) or IL 1...Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF α (1-250 U/ml) or IL 1β (0.1-50 U/ml) for 24 h. HUVEC were also cultured with cytokines, TNF α (100 U/ml) or IL 1β (10 U/ml), for 4-72 h, cell surface expression of adhesion molecules (ICAM 1 and VCAM 1) were detected and quantitated by immunocytochemical methods and computerized imaging analysis technique. Adhesion molecules expression were up regulated by TNF α, IL 1β in a concentration and time dependent manner. Some significant differences were observed between the effects of cytokines on the ICAM 1 and on VCAM 1 expression. Cytokines might directly induce the expression of ICAM 1 and VCAM 1 in vascular endothelial cells. Our observations indicate differential functions of the two adhesion molecules during the evolution of inflammatory responses in stroke.展开更多
INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7...INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7], in critically ill patients infections and sepsis are still associated with a high mortality[8,9].展开更多
Pingyangmycin (bleomycin A5 hydrochloride,PYM) is one of the anti-neoplastic agents which have been commonly used to treat venous malformations.However,the underlying mechanism by which PYM treats venous malformations...Pingyangmycin (bleomycin A5 hydrochloride,PYM) is one of the anti-neoplastic agents which have been commonly used to treat venous malformations.However,the underlying mechanism by which PYM treats venous malformations remains poorly understood.It was reported that venous endothelial cells could recruit neutrophils via adhesion molecules (E-selectin,ICAM-1,ICAM-3,VCAM-1) during the acute/chronic inflammation and subsequent histological fibrosis after sclerotherapy with PYM.This study explored if the expression of E-selectin,ICAM-1,ICAM-3 and VCAM-1 in human venous malformation endothelial cells could be affected by PYM.HVMECs were cultured from human venous malformation tissue.Expressions of E-selectin,ICAM-1,ICAM-3 and VCAM-1 on HVMECs in response to PYM were analyzed by cell ELISA.The relative levels of mRNA expression in the cells were semi-quantified.The results showed that PYM up-regulated the expressions of E-selectin,ICAM-3,VCAM-1 and ICAM-1 in both time-and concentration-dependent manner.Our findings suggested that PYM could induce the expression of adhesion molecules in HVMECs,which might be a possible mechanism by which sclerotherapy by intralesional injection of PYM treats venous malformations.展开更多
The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the po...The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the potential role of A.cinnamomea in cardiovascular diseases remains unexplored.Herein,using carotid arterial ligation models,we found that ethanol extract from A.cinnamomea(EEAC)signifi cantly inhibited neointimal hyperplasia in a dose-dependent manner,accompanied with the reduced expression of activated p65 and infl ammatory cytokines.We also show that EEAC ameliorated TNF-α-induced phosphorylation of p65 and pro-infl ammatory cytokine expression in both vascular smooth muscle cells(VSMCs)and macrophages in vitro.Mechanistically,EEAC suppressed expression levels of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule(VCAM-1)in VSMCs,which attenuates the ability of monocytes/macrophages adhesion to VSMCs.Furthermore,the expression level of these adhesion molecules and infi ltration of monocytes/macrophages were also decreased in neointimal VSMCs of arteries pretreated with EEAC.Altogether,our results reveal a novel function of A.cinnamomea in suppressing vascular infl ammation upon ligation injury during neointimal formation,likely through inhibition of infl ammatory cell infi ltration via downregulating the adhesion molecules in VSMCs.Thus,A.cinnamomea may offer a pharmacological therapy to slow down disease progression in patients with vascular injury.展开更多
Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating im...Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.展开更多
BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is associated with obesity,insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries.Current s...BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is associated with obesity,insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries.Current standard of care for patients varies according to disease stage,but includes lifestyle interventions common insulin sensitizers,antioxidants and lipid modifiers.However,to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials,and there is still no licensed therapy for NAFLD.Given the high prevalence,limited treatment options and significant screening costs for the general population,new treatments are urgently required.AIM To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1(VAP-1)to modify hepatic lipid accumulation in NAFLD.METHODS We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum.We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export.A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression.RESULTS We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression.Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake.This was recapitulated using hydrogen peroxide,and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3,FATP6,insulin receptor subunits and PPARα.Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis.This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1,and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet.Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4,FATP3-5,caveolin-1,VLDLR,PPARGC1 and genes associated with the inflammatory response.CONCLUSION Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis,metabolic disturbance and inflammation.This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.展开更多
BACKGROUND： Animal studies have confirmed that hyperbaric oxygen （HBO） therapy can reduce matrix metalloproteinase activity and blood brain barrier permeability, thereby exhibiting neuroprotective effects. However,...BACKGROUND： Animal studies have confirmed that hyperbaric oxygen （HBO） therapy can reduce matrix metalloproteinase activity and blood brain barrier permeability, thereby exhibiting neuroprotective effects. However, at present, consensus does not exist in terms of its clinical efficacy. OBJECTIVE： To validate the significance of changes in serum cellular adhesion molecule and MMP-9 levels in patients with cerebral infarction following HBO therapy. DESIGN, TIME AND SETTING： This randomized, controlled, neurobiochemical study was performed at the Department of Neurology, Affiliated Hospital of Qingdao University Medical College between December 2002 and March 2006. PARTICIPANTS： A total of 112 patients with acute cerebral infarction of internal carotid artery, comprising 64 males and 48 females, averaging （67 ±11） years, were recruited and randomized to a HBO group （n = 50） and a routine treatment group （n = 62）. An additional 30 gender- and age-matched normal subjects, consisting of 17 males and 13 females, averaging （63 ± 9） years, were enrolled as control subjects. METHODS： The routine treatment group received routine drug treatment and rehabilitation exercise. HBO treatment was additionally performed in the HBO group, once a day, for a total of 10 days. MAIN OUTCOME MEASURES： Serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9 were detected by enzyme linked immunosorbent assay. RESULTS： Upon admission, serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9 were significantly increased in patients with cerebral infarction, compared with control subjects （P 〈 0.01）. Following HBO and routine treatments, serum levels of the above-mentioned indices were significantly reduced in the HBO and routine treatment groups （P 〈 0.01）. Moreover, greater efficacy was observed in the HBO group, compared with the routine treatment group （P 〈 0.05 or P 〈 0.01）. CONCLUSION： Intergroup comparison and case-control results indicated that HBO noticeably reduced serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9.展开更多
Background Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important cytokines in inflammatory response, which may induce rolling and adhesion of both leukocytes and l...Background Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important cytokines in inflammatory response, which may induce rolling and adhesion of both leukocytes and lymphocytes, while modulating vascular permeability at the same time. These adhesion molecules usually serve as surrogate markers of activation and injury of vascular endothelial cells. Tumor necrosis factor-α (TNF-α) is a key factor to induce the expression and production of the above cell adhesion molecules. However, it remains to be elucidated whether exogenous ubiquitin exerts any effect on the cytokines in sepsis-induced ALI. Methods Sixty mice were devided randomly into five groups with twelve mice in each group, i.e. CLP group, SHAM group, UB1 group (10 mg/kg), UB2 group (5 mg/kg) and UB3 group(1 mg/kg). Mice of SHAM group underwent sham operation, and other four groups underwent CLP. Six hours after surgery, mice of three UB groups received ubiquitin by caudal vein injection while CLP and SHAM group received vehicle. Seven hours after surgery, blood and lungs of all mice were collected. ICAM-1, VCAM-1 and TNF-α level of 9% lung homogenate and serum TNF-α level were measured by ELISA. Results Pulmonary ICAM-1, VCAM-1 and TNF-α level of three UB groups were lower than CLP and SHAM group, and there were several comparisons with a statistically significant difference. Serum TNF-α level of three UB groups were slightly lower than CLP group, but far higher than SHAM group. Pulmonary ICAM-1 level, VCAM-1 level and serum TNF-α level of UB3 group were lower than UB1 and UB2 group, and there was a significant difference in VCAM-1 between UB3 and UB1 group. Pulmonary TNF-α level of UB3 group was slightly higher than UB1 and UB2 group.展开更多
This study determined the levels of serum soluble intercellular adhesion molecule-1 （sI-CAM-l） and soluble vascular cell adhesion molecular-1 （sVCAM-1） in patients with different types of Keshan disease （KD）, ex...This study determined the levels of serum soluble intercellular adhesion molecule-1 （sI-CAM-l） and soluble vascular cell adhesion molecular-1 （sVCAM-1） in patients with different types of Keshan disease （KD）, examined the relationship between Coxsackie B virus-specific IgM antibody （CBV-IgM） and slCAM-1 or sVCAM-1 in KD patients, and investigated the role of these adhesion molecules in the pathogenesis of KD and their clinical implications. The levels of serum slCAM-1, sVCAM-1 and CBV-IgM were measured by using enzyme-linked immunosorbent assay in 22 patients with chronic Keshan disease （CKD）, 27 with latent Keshan disease （LKD） and 28 healthy controis. The subjects in different groups were adjusted for sex and age. Echocardiography was adopted to determine left ventricular ejection fraction （LVEF） in 22 patients with CKD. The results showed that CKD patients had significantly higher levels of slCAM-1 and sVCAM-1 than LKD patients and healthy controls （P〈0.01 for all）. And there was significant difference in the levels of the 2 adhesion molecules between LKD patients and healthy controls （P〈0.05）. A negative correlation was found between LVEF and slCAM-1 or sVCAM-1 in CKD patients. The percentage of CBV-specific IgM positive individuals in KD patients was significantly higher than that of healthy controls. In CVB-specific IgM positive patients, the levels of serum slCAM-1 and sVCAM-1 were significantly greater than those in CBV-specific IgM negative counterpart. It was concluded that the increase in the levels of slCAM-1 and sVCAM-1 suggests the progression of inflammation in KD. slCAM-1 and sVCAM-1 can promote the development of myocardial pathology and lead to poor myocardial function. The increased serum slCAM-1 and sVCAM-1 in KD patients may be related to CBV infection.展开更多
Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is prop...Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.展开更多
The effects of hydrogen peroxide（H<sub>2</sub>O<sub>2</sub>）on endothelial-polymorphonuclear leuko-cyte（EC-PMN）adhesion and their mechanisms were studied in cultured bovine pulmonaryartery ...The effects of hydrogen peroxide（H<sub>2</sub>O<sub>2</sub>）on endothelial-polymorphonuclear leuko-cyte（EC-PMN）adhesion and their mechanisms were studied in cultured bovine pulmonaryartery endothelial monolayers in vitro.H<sub>2</sub>O<sub>2</sub> at various concentrations(10<sup>-3</sup>,10<sup>-2</sup>,10<sup>-1</sup>mol/Lrespectively)stimulated EC-dependent PMN adhesion,of which 10<sup>-2</sup>mol/L H<sub>2</sub>O<sub>2</sub> was the mostpotent one,increasing adhesion to 2.3 times that of the control.Pretreatment of PMNs with SRI63-441,a platelet-activating factor（PAF）receptor antagonist,had no inhibition effect on H<sub>2</sub>O<sub>2</sub>induced EC-PMN adhesion.Pretreatment of ECs with SRI 63-441 before H<sub>2</sub>O<sub>2</sub> exposure signifi-cantly decreased PMN adherence to ECs.Pretreatment of ECs with phospholipase A<sub>2</sub> inhibitorp-bromophenacyl-bromide or calmodulin antagonist chlorpromazine and calcium ion chelate EG-TA obviously decreased H<sub>2</sub>O<sub>2</sub> induced increment of EC-PMN adhesion.These results suggestthat H<sub>2</sub>O<sub>2</sub> may activate ECs,causing the inflow of extracellular calcium or the release of calciumfrom intracellular deposits.Increased intracellar Ca<sup>2+</sup>may bind with calmodulin to activate phos-pholipase A<sub>2</sub>,thus initiating PAF synthesis and promoting EC-PMN adhesion.展开更多
文摘Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF α (1-250 U/ml) or IL 1β (0.1-50 U/ml) for 24 h. HUVEC were also cultured with cytokines, TNF α (100 U/ml) or IL 1β (10 U/ml), for 4-72 h, cell surface expression of adhesion molecules (ICAM 1 and VCAM 1) were detected and quantitated by immunocytochemical methods and computerized imaging analysis technique. Adhesion molecules expression were up regulated by TNF α, IL 1β in a concentration and time dependent manner. Some significant differences were observed between the effects of cytokines on the ICAM 1 and on VCAM 1 expression. Cytokines might directly induce the expression of ICAM 1 and VCAM 1 in vascular endothelial cells. Our observations indicate differential functions of the two adhesion molecules during the evolution of inflammatory responses in stroke.
基金Supported by the National Natural Science Foundation of China, No. 39870796
文摘INTRODUCTIONMultiple organ dysfunction syndrome (MODS) isthought to be a frequent consequence of sepsis[1-3].Despite substantial advances in our knowledge and understanding of the basic pathophysiologic mechanisms[4-7], in critically ill patients infections and sepsis are still associated with a high mortality[8,9].
基金supported by the Natural Science Foundation of Hubei province,China (No.2010CDB07907)
文摘Pingyangmycin (bleomycin A5 hydrochloride,PYM) is one of the anti-neoplastic agents which have been commonly used to treat venous malformations.However,the underlying mechanism by which PYM treats venous malformations remains poorly understood.It was reported that venous endothelial cells could recruit neutrophils via adhesion molecules (E-selectin,ICAM-1,ICAM-3,VCAM-1) during the acute/chronic inflammation and subsequent histological fibrosis after sclerotherapy with PYM.This study explored if the expression of E-selectin,ICAM-1,ICAM-3 and VCAM-1 in human venous malformation endothelial cells could be affected by PYM.HVMECs were cultured from human venous malformation tissue.Expressions of E-selectin,ICAM-1,ICAM-3 and VCAM-1 on HVMECs in response to PYM were analyzed by cell ELISA.The relative levels of mRNA expression in the cells were semi-quantified.The results showed that PYM up-regulated the expressions of E-selectin,ICAM-3,VCAM-1 and ICAM-1 in both time-and concentration-dependent manner.Our findings suggested that PYM could induce the expression of adhesion molecules in HVMECs,which might be a possible mechanism by which sclerotherapy by intralesional injection of PYM treats venous malformations.
基金This work was supported by the National Key Research Project of China(2019YFC1606400)Major Public Welfare Projects in Henan Province(201300110200)+4 种基金National Key Research Project of Hebei Province(20375502D)Natural Science Foundation of Hebei Province(H2019206212)High-level Talent Funding Project of Hebei Province(A201905006)Fund of National R&D Center for Edible Fungus Processing Technology,Henan University(20200109)the Open Fund from Beijing Advanced Innovation Center for Food Nutrition and Human Health(20182025).
文摘The increased vascular infl ammation is a key event in the development of atherosclerotic lesions.Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing infl ammation.However,the potential role of A.cinnamomea in cardiovascular diseases remains unexplored.Herein,using carotid arterial ligation models,we found that ethanol extract from A.cinnamomea(EEAC)signifi cantly inhibited neointimal hyperplasia in a dose-dependent manner,accompanied with the reduced expression of activated p65 and infl ammatory cytokines.We also show that EEAC ameliorated TNF-α-induced phosphorylation of p65 and pro-infl ammatory cytokine expression in both vascular smooth muscle cells(VSMCs)and macrophages in vitro.Mechanistically,EEAC suppressed expression levels of intercellular adhesion molecule-1(ICAM-1)and vascular cell adhesion molecule(VCAM-1)in VSMCs,which attenuates the ability of monocytes/macrophages adhesion to VSMCs.Furthermore,the expression level of these adhesion molecules and infi ltration of monocytes/macrophages were also decreased in neointimal VSMCs of arteries pretreated with EEAC.Altogether,our results reveal a novel function of A.cinnamomea in suppressing vascular infl ammation upon ligation injury during neointimal formation,likely through inhibition of infl ammatory cell infi ltration via downregulating the adhesion molecules in VSMCs.Thus,A.cinnamomea may offer a pharmacological therapy to slow down disease progression in patients with vascular injury.
基金supported by NIH grant RO1 NS093985 (to DS, NZ, XW) and RO1 NS101955 (to DS)the VCU Microscopy Facility,supported,in part,by funding from NIH-NCI Cancer Center Support Grant P30 CA016059。
文摘Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.
文摘BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is associated with obesity,insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries.Current standard of care for patients varies according to disease stage,but includes lifestyle interventions common insulin sensitizers,antioxidants and lipid modifiers.However,to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials,and there is still no licensed therapy for NAFLD.Given the high prevalence,limited treatment options and significant screening costs for the general population,new treatments are urgently required.AIM To assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1(VAP-1)to modify hepatic lipid accumulation in NAFLD.METHODS We have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum.We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export.A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression.RESULTS We confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression.Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake.This was recapitulated using hydrogen peroxide,and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3,FATP6,insulin receptor subunits and PPARα.Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis.This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1,and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet.Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4,FATP3-5,caveolin-1,VLDLR,PPARGC1 and genes associated with the inflammatory response.CONCLUSION Our data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis,metabolic disturbance and inflammation.This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.
文摘BACKGROUND： Animal studies have confirmed that hyperbaric oxygen （HBO） therapy can reduce matrix metalloproteinase activity and blood brain barrier permeability, thereby exhibiting neuroprotective effects. However, at present, consensus does not exist in terms of its clinical efficacy. OBJECTIVE： To validate the significance of changes in serum cellular adhesion molecule and MMP-9 levels in patients with cerebral infarction following HBO therapy. DESIGN, TIME AND SETTING： This randomized, controlled, neurobiochemical study was performed at the Department of Neurology, Affiliated Hospital of Qingdao University Medical College between December 2002 and March 2006. PARTICIPANTS： A total of 112 patients with acute cerebral infarction of internal carotid artery, comprising 64 males and 48 females, averaging （67 ±11） years, were recruited and randomized to a HBO group （n = 50） and a routine treatment group （n = 62）. An additional 30 gender- and age-matched normal subjects, consisting of 17 males and 13 females, averaging （63 ± 9） years, were enrolled as control subjects. METHODS： The routine treatment group received routine drug treatment and rehabilitation exercise. HBO treatment was additionally performed in the HBO group, once a day, for a total of 10 days. MAIN OUTCOME MEASURES： Serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9 were detected by enzyme linked immunosorbent assay. RESULTS： Upon admission, serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9 were significantly increased in patients with cerebral infarction, compared with control subjects （P 〈 0.01）. Following HBO and routine treatments, serum levels of the above-mentioned indices were significantly reduced in the HBO and routine treatment groups （P 〈 0.01）. Moreover, greater efficacy was observed in the HBO group, compared with the routine treatment group （P 〈 0.05 or P 〈 0.01）. CONCLUSION： Intergroup comparison and case-control results indicated that HBO noticeably reduced serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9.
文摘Background Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are two important cytokines in inflammatory response, which may induce rolling and adhesion of both leukocytes and lymphocytes, while modulating vascular permeability at the same time. These adhesion molecules usually serve as surrogate markers of activation and injury of vascular endothelial cells. Tumor necrosis factor-α (TNF-α) is a key factor to induce the expression and production of the above cell adhesion molecules. However, it remains to be elucidated whether exogenous ubiquitin exerts any effect on the cytokines in sepsis-induced ALI. Methods Sixty mice were devided randomly into five groups with twelve mice in each group, i.e. CLP group, SHAM group, UB1 group (10 mg/kg), UB2 group (5 mg/kg) and UB3 group(1 mg/kg). Mice of SHAM group underwent sham operation, and other four groups underwent CLP. Six hours after surgery, mice of three UB groups received ubiquitin by caudal vein injection while CLP and SHAM group received vehicle. Seven hours after surgery, blood and lungs of all mice were collected. ICAM-1, VCAM-1 and TNF-α level of 9% lung homogenate and serum TNF-α level were measured by ELISA. Results Pulmonary ICAM-1, VCAM-1 and TNF-α level of three UB groups were lower than CLP and SHAM group, and there were several comparisons with a statistically significant difference. Serum TNF-α level of three UB groups were slightly lower than CLP group, but far higher than SHAM group. Pulmonary ICAM-1 level, VCAM-1 level and serum TNF-α level of UB3 group were lower than UB1 and UB2 group, and there was a significant difference in VCAM-1 between UB3 and UB1 group. Pulmonary TNF-α level of UB3 group was slightly higher than UB1 and UB2 group.
文摘This study determined the levels of serum soluble intercellular adhesion molecule-1 （sI-CAM-l） and soluble vascular cell adhesion molecular-1 （sVCAM-1） in patients with different types of Keshan disease （KD）, examined the relationship between Coxsackie B virus-specific IgM antibody （CBV-IgM） and slCAM-1 or sVCAM-1 in KD patients, and investigated the role of these adhesion molecules in the pathogenesis of KD and their clinical implications. The levels of serum slCAM-1, sVCAM-1 and CBV-IgM were measured by using enzyme-linked immunosorbent assay in 22 patients with chronic Keshan disease （CKD）, 27 with latent Keshan disease （LKD） and 28 healthy controis. The subjects in different groups were adjusted for sex and age. Echocardiography was adopted to determine left ventricular ejection fraction （LVEF） in 22 patients with CKD. The results showed that CKD patients had significantly higher levels of slCAM-1 and sVCAM-1 than LKD patients and healthy controls （P〈0.01 for all）. And there was significant difference in the levels of the 2 adhesion molecules between LKD patients and healthy controls （P〈0.05）. A negative correlation was found between LVEF and slCAM-1 or sVCAM-1 in CKD patients. The percentage of CBV-specific IgM positive individuals in KD patients was significantly higher than that of healthy controls. In CVB-specific IgM positive patients, the levels of serum slCAM-1 and sVCAM-1 were significantly greater than those in CBV-specific IgM negative counterpart. It was concluded that the increase in the levels of slCAM-1 and sVCAM-1 suggests the progression of inflammation in KD. slCAM-1 and sVCAM-1 can promote the development of myocardial pathology and lead to poor myocardial function. The increased serum slCAM-1 and sVCAM-1 in KD patients may be related to CBV infection.
基金supported by a grant from the Russian Science Foundation,No. 16-15-00010 (to RRI)funded by government assignment for FRC Kazan Scientific Center of RAS
文摘Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.
文摘The effects of hydrogen peroxide（H<sub>2</sub>O<sub>2</sub>）on endothelial-polymorphonuclear leuko-cyte（EC-PMN）adhesion and their mechanisms were studied in cultured bovine pulmonaryartery endothelial monolayers in vitro.H<sub>2</sub>O<sub>2</sub> at various concentrations(10<sup>-3</sup>,10<sup>-2</sup>,10<sup>-1</sup>mol/Lrespectively)stimulated EC-dependent PMN adhesion,of which 10<sup>-2</sup>mol/L H<sub>2</sub>O<sub>2</sub> was the mostpotent one,increasing adhesion to 2.3 times that of the control.Pretreatment of PMNs with SRI63-441,a platelet-activating factor（PAF）receptor antagonist,had no inhibition effect on H<sub>2</sub>O<sub>2</sub>induced EC-PMN adhesion.Pretreatment of ECs with SRI 63-441 before H<sub>2</sub>O<sub>2</sub> exposure signifi-cantly decreased PMN adherence to ECs.Pretreatment of ECs with phospholipase A<sub>2</sub> inhibitorp-bromophenacyl-bromide or calmodulin antagonist chlorpromazine and calcium ion chelate EG-TA obviously decreased H<sub>2</sub>O<sub>2</sub> induced increment of EC-PMN adhesion.These results suggestthat H<sub>2</sub>O<sub>2</sub> may activate ECs,causing the inflow of extracellular calcium or the release of calciumfrom intracellular deposits.Increased intracellar Ca<sup>2+</sup>may bind with calmodulin to activate phos-pholipase A<sub>2</sub>,thus initiating PAF synthesis and promoting EC-PMN adhesion.
