GRK2 inhibits Flt-1+ macrophage infiltration and its proangiogenic properties in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/- mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/- mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.

Estudo de associação entre polimorfismos genéticos e níveis plasmáticos do VEGF, FLT1, KDR e o risco para depressão / Study of association between genetic polymorphisms and plasma levels of VEGF, FLT1, KDR and the risk for depression

Introdução: a depressão é um transtorno mental comum, grave e incapacitante que afeta mais de 350 milhões de pessoas em todo o mundo. A depressão é caracterizada principalmente por sintomas como tristeza, perda de interesse, diminuição da energia, perda de confiança e autoestima, culpa inadequada, distúrbios do sono e do apetite, pensamentos de morte e suicídio. Além disso, essa patologia também tem um forte impacto na qualidade de vida dos indivíduos afetados e de suas famílias. Sabe-se que fatores genéticos interagem com as condições socioambientais de modo a influenciar a predisposição das pessoas ao adoecimento. Estudos identificaram polimorfismos de nucleotídeos simples (SNPs) que podem ser marcadores genéticos apropriados para prever inflamação sistêmica, por exemplo, e a atual tese teve como foco o efeito de SNPs na via do fator de crescimento endotelial vascular (VEGF). Esta proteína é uma potente molécula angiogênica e está envolvida na neurogênese do hipocampo, uma das principais estruturas límbicas afetadas em pessoas com depressão. O VEGF está implicado em uma das principais teorias que tentam explicar a fisiopatologia deste transtorno mental grave, a teoria neurotrófica, a qual diz que a diminuição ou desregulação da sinalização de neurotrofinas pode contribuir para a manifestação do transtorno depressivo (TD). Objetivo: avaliar se polimorfismos do VEGF e seus receptores, KDR e FLT1, estão associados à depressão e à gravidade dos sintomas, à ideação e tentativas de suicídio, independentemente tanto de um tratamento otimizado quanto da presença de estresse precoce (do inglês, early-life stress, ELS), também verificar se há efeito destes polimorfismos nas concentrações plasmáticas de proteínas expressas pelos seus respectivos genes e observar se existe correlação entre VEGF e seus inibidores, VEGF e s100ß. Metodologia: participaram do presente estudo 160 pacientes com depressão e 114 controles saudáveis. Foram aplicados durante entrevista questionários que avaliaram o perfil clínico dos pacientes como o MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI e foi registrado o número de tentativas de suicídio. Os controles passaram por uma entrevista para serem avaliados quanto aos critérios de inclusão e exclusão do grupo. A genotipagem dos participantes foi realizada através da técnica de Real Time PCR e as mensurações de proteínas por meio do ensaio ensaio imunoenzimático (ELISA). Resultados: indivíduos com depressão, homozigotos AA do polimorfismo rs699947, apresentaram maiores concentrações plasmáticas de VEGF (P-valor= 0.006) e se associaram a um maior número de tentativas de suicídio na análise direta (P-valor= 0.041) e na análise corrigida foi observada uma tendência para a confirmação deste resultado (P-valor= 0.076). O genótipo homozigoto GG do polimorfismo rs7993418 do FLT1 se associou à severidade de sintomas (P-valor= 0.040), bem como uma tendência de associação com um aumento nas tentativas de suicídio e uma maior pontuação na escala que avaliou ideação suicida. Entre os pacientes quanto maior foram as concentrações plasmáticas de VEGF, maior foram as de KDR, FLT1 e s100ß. Conclusão: os resultados sugerem que os polimorfismos da via VEGF estão associados ao número de tentativas de suicídio e severidade dos sintomas depressivos

Introduction: Depression is a common, serious, and disabling mental disorder that affects more than 350 million people worldwide. Depression is mainly characterized by symptoms such as sadness, loss of interest, decreased energy, loss of confidence and self-esteem, inadequate guilt, sleep and appetite disturbances, thoughts of death and suicide. Furthermore, this pathology also has a strong impact on the quality of life of those affected and their families. It is known that genetic factors interact with social and environmental conditions to influence people's predisposition to illness. Studies have identified single nucleotide polymorphisms (SNPs) that may be appropriate genetic markers to predict systemic inflammation, for example, and the current thesis focused on the effect of SNPs on the vascular endothelial growth factor (VEGF) pathway. This protein is a potent angiogenic molecule and is involved in hippocampal neurogenesis, one of the main limbic structures affected in people with depression. VEGF is implicated in one of the main theories that try to explain the pathophysiology of this severe mental disorder, the neurotrophic theory, which says that the decrease or dysregulation of neurotrophin signaling can contribute to the manifestation of depressive disorder (DT). Objective: to assess whether polymorphisms of VEGF and its receptors, KDR and FLT1, are associated with depression and severity of symptoms, suicide ideation and attempts, regardless of both optimal treatment and the presence of early-life stress (ELS) in these associations. also check whether there is an effect of these polymorphisms on the plasma concentrations of proteins expressed by their respective genes and observe whether there is a correlation between VEGF and its inhibitors, VEGF and s100ß. Methodology: 160 patients with depression and 114 healthy controls participated in this study. Questionnaires that assessed the clinical profile of patients, such as the MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI, were applied during interviews, and the number of suicide attempts was recorded. The controls underwent an interview to be evaluated regarding the inclusion and exclusion criteria. The genotyping of the participants was performed using the Real Time PCR technique and protein measurements were performed using the enzyme-linked immunosorbent assay (ELISA). Results: individuals with depression, homozygous AA of the rs699947 polymorphism, had higher plasma concentrations of VEGF (P-value = 0.006) and a greater number of suicide attempts in the direct analysis (P-value = 0.041) and in the corrected analysis a trend towards confirmation of this result was observed (P-value = 0.076). The GG genotype of the FLT1 polymorphism rs7993418 was associated with symptom severity (P-value = 0.040), as well as with a trend for association with increase in suicide attempts and a higher score on the scale that evaluated suicidal ideation. The bigger the plasma concentrations of VEGF, the higher were those of KDR, FLT1 and s100ß. Conclusion: the results indicate that VEGF pathway polymorphisms are associated with the number of suicides and severity of depressive symptoms

Estudo de associação entre polimorfismos genéticos e níveis plasmáticos do VEGF, FLT1, KDR e o risco para depressão / Study of association between genetic polymorphisms and plasma levels of VEGF, FLT1, KDR and the risk for depression

Introdução: a depressão é um transtorno mental comum, grave e incapacitante que afeta mais de 350 milhões de pessoas em todo o mundo. A depressão é caracterizada principalmente por sintomas como tristeza, perda de interesse, diminuição da energia, perda de confiança e autoestima, culpa inadequada, distúrbios do sono e do apetite, pensamentos de morte e suicídio. Além disso, essa patologia também tem um forte impacto na qualidade de vida dos indivíduos afetados e de suas famílias. Sabe-se que fatores genéticos interagem com as condições socioambientais de modo a influenciar a predisposição das pessoas ao adoecimento. Estudos identificaram polimorfismos de nucleotídeos simples (SNPs) que podem ser marcadores genéticos apropriados para prever inflamação sistêmica, por exemplo, e a atual tese teve como foco o efeito de SNPs na via do fator de crescimento endotelial vascular (VEGF). Esta proteína é uma potente molécula angiogênica e está envolvida na neurogênese do hipocampo, uma das principais estruturas límbicas afetadas em pessoas com depressão. O VEGF está implicado em uma das principais teorias que tentam explicar a fisiopatologia deste transtorno mental grave, a teoria neurotrófica, a qual diz que a diminuição ou desregulação da sinalização de neurotrofinas pode contribuir para a manifestação do transtorno depressivo (TD). Objetivo: avaliar se polimorfismos do VEGF e seus receptores, KDR e FLT1, estão associados à depressão e à gravidade dos sintomas, à ideação e tentativas de suicídio, independentemente tanto de um tratamento otimizado quanto da presença de estresse precoce (do inglês, early-life stress, ELS), também verificar se há efeito destes polimorfismos nas concentrações plasmáticas de proteínas expressas pelos seus respectivos genes e observar se existe correlação entre VEGF e seus inibidores, VEGF e s100ß. Metodologia: participaram do presente estudo 160 pacientes com depressão e 114 controles saudáveis. Foram aplicados durante entrevista questionários que avaliaram o perfil clínico dos pacientes como o MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI e foi registrado o número de tentativas de suicídio. Os controles passaram por uma entrevista para serem avaliados quanto aos critérios de inclusão e exclusão do grupo. A genotipagem dos participantes foi realizada através da técnica de Real Time PCR e as mensurações de proteínas por meio do ensaio ensaio imunoenzimático (ELISA). Resultados: indivíduos com depressão, homozigotos AA do polimorfismo rs699947, apresentaram maiores concentrações plasmáticas de VEGF (P-valor= 0.006) e se associaram a um maior número de tentativas de suicídio na análise direta (P-valor= 0.041) e na análise corrigida foi observada uma tendência para a confirmação deste resultado (P-valor= 0.076). O genótipo homozigoto GG do polimorfismo rs7993418 do FLT1 se associou à severidade de sintomas (P-valor= 0.040), bem como uma tendência de associação com um aumento nas tentativas de suicídio e uma maior pontuação na escala que avaliou ideação suicida. Entre os pacientes quanto maior foram as concentrações plasmáticas de VEGF, maior foram as de KDR, FLT1 e s100ß. Conclusão: os resultados sugerem que os polimorfismos da via VEGF estão associados ao número de tentativas de suicídio e severidade dos sintomas depressivos.

Introduction: Depression is a common, serious, and disabling mental disorder that affects more than 350 million people worldwide. Depression is mainly characterized by symptoms such as sadness, loss of interest, decreased energy, loss of confidence and self-esteem, inadequate guilt, sleep and appetite disturbances, thoughts of death and suicide. Furthermore, this pathology also has a strong impact on the quality of life of those affected and their families. It is known that genetic factors interact with social and environmental conditions to influence people's predisposition to illness. Studies have identified single nucleotide polymorphisms (SNPs) that may be appropriate genetic markers to predict systemic inflammation, for example, and the current thesis focused on the effect of SNPs on the vascular endothelial growth factor (VEGF) pathway. This protein is a potent angiogenic molecule and is involved in hippocampal neurogenesis, one of the main limbic structures affected in people with depression. VEGF is implicated in one of the main theories that try to explain the pathophysiology of this severe mental disorder, the neurotrophic theory, which says that the decrease or dysregulation of neurotrophin signaling can contribute to the manifestation of depressive disorder (DT). Objective: to assess whether polymorphisms of VEGF and its receptors, KDR and FLT1, are associated with depression and severity of symptoms, suicide ideation and attempts, regardless of both optimal treatment and the presence of early-life stress (ELS) in these associations. also check whether there is an effect of these polymorphisms on the plasma concentrations of proteins expressed by their respective genes and observe whether there is a correlation between VEGF and its inhibitors, VEGF and s100ß. Methodology: 160 patients with depression and 114 healthy controls participated in this study. Questionnaires that assessed the clinical profile of patients, such as the MINI-International Neuropsychiatric Interview, GRID-HAMD21, CTQ, BSI, were applied during interviews, and the number of suicide attempts was recorded. The controls underwent an interview to be evaluated regarding the inclusion and exclusion criteria. The genotyping of the participants was performed using the Real Time PCR technique and protein measurements were performed using the enzyme-linked immunosorbent assay (ELISA). Results: individuals with depression, homozygous AA of the rs699947 polymorphism, had higher plasma concentrations of VEGF (P-value = 0.006) and a greater number of suicide attempts in the direct analysis (P-value = 0.041) and in the corrected analysis a trend towards confirmation of this result was observed (P-value = 0.076). The GG genotype of the FLT1 polymorphism rs7993418 was associated with symptom severity (P-value = 0.040), as well as with a trend for association with increase in suicide attempts and a higher score on the scale that evaluated suicidal ideation. The bigger the plasma concentrations of VEGF, the higher were those of KDR, FLT1 and s100ß. Conclusion: the results indicate that VEGF pathway polymorphisms are associated with the number of suicides and severity of depressive symptoms.

Tc-99m and Fluorescence-Labeled Anti-Flt1 Peptide as a Multimodal Tumor Imaging Agent Targeting Vascular Endothelial Growth Factor-Receptor 1 / 대한핵의학회잡지

PURPOSE: We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-GNQWFI, to target tumor cells, and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model.METHODS: TAMRA-GHEG-ECG-GNQWFI was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-GNQWFI with Tc-99m was done using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with U87MG tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy.RESULTS: After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-GNQWFI complexes were prepared in high yield (> 95%). The K(d) of Tc-99m TAMRA-GHEG-ECG-GNQWFI determined by saturation binding was 29.5 ± 4.5 nM. Confocal microscopy images of U87MG cells incubated with TAMRA-GHEG-ECG-GNQWFI showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI in tumors. Tumor uptake was effectively blocked by the co-injection of an excess concentration of GNQWFI. Specific uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI was assessed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies.CONCLUSION: In vivo and in vitro studies revealed substantial and specific uptake of Tc-99m TAMRA-GHEG-ECG-GNQWFI in tumor cells. Tc-99m TAMRA-GHEG-ECG-GNQWFI could be a good candidate dual-modality imaging agent for tumors.

Efecto del praziquantel en los receptores angiogénicos, citocinas inflamatorias y anti-inflamatorias en la esquistosomiasis intestinal murina / The effect of praziquantel in the angiogenic receptors, inflammation and inflammatory cytokines on the murine intestinal schistosomiasis

Los estudios en torno al papel patogénico del Schistosoma mansoni se enfocan en el daño hepático y la respuesta inmune en el hospedador definitivo, en contraste a la escasa información en relación a la patología intestinal. A tal fin, se evaluó el efecto del praziquantel en los receptores angiogenicos, citocinas inflamatorias y anti-inflamatorias en la esquitosomiasis intestinal murina. La respuesta inflamatoria granulomatosa intestinal se midió en secciones histológicas teñidas con H&E; la detección de TNF-α, IL-10, TGF-ß, el VEGF y sus receptores (FLK1 y FLT1) mediante inmunohistoquímica y ELISA, en suero e intestino de ratones BALB/c infectados con S. mansoni a las 8 (RI8S) y 20 semanas (RI20S), y ratones con 8 semanas de infección a los quince días de post-tratamiento, con praziquantel 40 µgr/gr (RPT). En RI8S se observaron granulomas pequeños (11304 µm2 en promedio), sin bordes definidos ni zonas marcadas dentro del granuloma, y granulomas de gran tamaño con bordes definidos (70650 µm2) sin zonas definidas dentro del granuloma, ambos con predominio de macrófagos y presencia de plasmocitos. En RI20S, los granulomas presentan una zona interna constituida por abundantes macrófagos y escasos plasmocitos, y una externa constituida solo por macrófagos (11985 µm2). En RPT no se apreciaron granulomas, solo algunos focos inflamatorios cercanos a la muscularis mucosae y las glándulas de Lieberkuhn. La Inmunolocalización en el intestino solo fue positiva en RPT para VEGF y sus receptores, TGP-ß e IL-10; Los resultados muestran una discreta respuesta en cuanto a tamaño del granuloma, celularidad y la expresión de citocinas a nivel intestinal.

The studies on the pathogenic role of the Schistosoma mansoni focus on the hepatic damage and the immune response of the final host, in constrast to the limited information regarding intestinal lesion. This work evaluates. Intestinal granulomatous inflammation was measured in histological sections the effect of praziquantel in the angiogenic receptors (FLK1 and FLT1), inflammation and inflammatory cytokines on the murine intestinal schistosomiasis stained with H&E; the detection of TNF-α, IL-10, TGF-ß, VEGF and its receptors was carried out by immunohistochemistry and ELISA, in serum and intestine sections of BALB/c mice infected with S. mansoni at 8 (RI8S) and 20 (RI20S) weeks post-infection, and mice infected for 8 weeks and evaluated 15 days post-treatment with praziquantel 40 µg/(RPT). In RI8S small granulomas (11.304 µm2 average), without sharp or marked edges within the granuloma; and large granulomas (70.650 µm2) with defined borders without defined zones within the granuloma, In both groups of mice macrophages predominated and plasma cells were present. In RI20S, granulomas have an inner zone composed by abundant macrophages and few plasma cells, and external zone constituted only by macrophages (11.985 µm2). In RPT, granulomas were not observed, only a few inflammatory foci nearby the muscularis mucosae and the Lieberkuhn glands. Localization of immune molecules in the intestine was only positive in RPT for VEGF, its receptors, TGF-ß and IL-10; the results show a discrete response over the granuloma size, cellularity and cytokine expression at intestinal level.

Effects of Acupuncture-rehabilitation Therapy on Neurological Function and Expression of Vascular Endothelial Growth Factor Receptors, Flt-1 and Flk-1, after Focal Cerebral Ischemia in Rats / 中国康复理论与实践

Objective To explore the effect of acupuncture-rehabilitation therapy on neurological function and expression of Flt-1 and Flk-1, members of vascular endothelial growth factor receptors, after permanent focal cerebral ischemia in rats. Methods Ninety male Sprague-Dawley rats were divided into five groups, namely sham group, model group, acupuncture group, rehabilitation group and acupunc-ture-rehabilitation group, and each group was further divided into 3-day, 7-day and 14-day subgroups, equally. Their middle cerebral arteries were occluded except those of sham group. The sham and model groups accepted no treatment, while the acupuncture group accepted clus-ter needling of scalp acupuncture, the rehabilitation group accepted treadmill training, and the acupuncture-rehabilitation group accepted both acupuncture and treadmill training. They were assessed with modified Neurologic Severity Score (mNSS) 3, 7 and 14 days after model-ing, while the expression of Flt-1 and Flk-1 were determined with Western blotting. Results The mNSS score reduced in all the treatment groups (P<0.05) compared with that of the model group at every time point, and was the least in the acupuncture-rehabilitation group (P<0.05) 7 and 14 days after modeling among the treatment groups. Meanwhile, the expression of Flt-1 and Flk-1 protein increased in all the treatment groups (P<0.05), and was the most in the acupuncture-rehabilitation group (P<0.05). Conclusion Acupuncture-rehabilitation thera-py can promote the neurological function recovery in rat with permanent focal cerebral ischemia, which may be associated with the continu-ous inducement of Flt-1, Flk-1 protein expression in ischemic penumbra cortex.

Expression of vascular endothelial cell growth factor and its receptors in vitreous of patients with diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

To investigate the expression of vascular endothelial cell growth factor (VEGF) and its receptors in vitreous of patients with diabetic retinopathy ( DR), and to discuss its role in the development of DR. ●METHODS: Selected 13 patients (16 eyes) with DR and 15 healthy people (15 eyes), the expression of VEGF and its receptors (fms-like tyrosine kinase-1, Flt-1 and kinase insert domain containing receptor, KDR) were evaluated by immunohistochemistry in vitreous. The levels of VEGF, the Flt-1 and KDR in vitreous of patients with DR were examined with enzyme linked immunosorbent assay (ELlSA). ● RESULTS: lmmunohistochemical staining showed that the expression of VEGF, Flt-1 and KDR in vitreous vessel membrane of patients with DR was increased significantly. And the levels of VEGF, Flt - 1 and KDR in vitreous of patients with DR were obviously higher than that in the control group (P ● CONCLUSlON: VEGF, Flt - 1 and KDR were widely expressed in vitreous of patients with DR, and were positively related to micro-angiogenesis of DR patients. lt proved that VEGF and its receptors played important roles in the occurrence and development of DR.

Expression of VEGF and its Receptors, Flk-1 and Flt-1 in Postnatal Murine Cerebellum / 체질인류학회지

VEGF and its receptors, flk-1 and flt-1 have been characterized as critical factors in angiogenesis and neurogenesis during development. Here we investigated the expression of VEGF and its receptors in postnatal murine cerebellum in terms of time-dependency and regional distribution. Immunofluorescence staining showed that the expression of VEGF was restricted only to Purkinje cells and was increased in their processes on the postnatal development. Flk-1 was expressed in Purkinje cellular bodies on postnatal day (P) 8, 11, 18. Flt-1 was expressed in Purkinje cells on P8 but gradually disappeared in all of cerebellar layers on the postnatal development. These results suggest that VEGF may contribute to postnatal development of cerebellum via its receptors. And they suggest that changes in the expression of VEGF and its receptors related to the difference in maturation and proliferation of Purkinje cells in the cerebellum.

Correlation between the levels of vascular endothelial growth factor and its receptor Flt-1 and KDR in human brain glioma / 肿瘤研究与临床

Objective To evaluate the serum changes in glioma patients, and in relations with clinicopathalogy parameters response to treatment and survival in patients with glioma via the examination of the significance of serum VEGF, Flt-1 and KDR levels. Methods Serum VEGF,Flt-1 and KDR levels were analyzed in patients of glioma before and after treatment, and patients with brain metastasis, as well as in healthy controls. Serum VEGF, Flt-1 and KDR levels were assessed by Enzyme-Linked Immunosorbent Assay(ELISA) and the data were processed by SPSS 11.5 for t-test and relevant analyses. Results Serum VEGF, Flt-1 and KDR levels were higher in patients with glioma than in healthy control (P =0.001, P =0.043 and P =0.045 respectively). Significant difference on levels of VEGF was found (P =0.032) comparing glioma group with brain metastasis group. The pretherapeutic serum levels of VEGF and Flt-1 were significantly different with disease recurrence or persistence after treatment (P =0.026 and P =0.038). There was significant correlation between the serum levels of VEGF correlated and that of Flt-1 and KDR (r =0.456 P

Effects of hVEGF genetically engineering membrane on the expression of flt-1 and TSP-1 during the healing process of full-thickness cutaneous defect / 上海第二医科大学学报

Objective To study the effects of hVEGF on the expression of fms-like tyrosine kinase-1(flt-1)and thrombospondin 1(TSP-1)during the healing process of rats' full-thickness cutaneous defect which covered by genetically membrane planted by fibroblast integrated with hVEGF recombinant.Methods Wounds with full-thickness cutaneous defect were produced on the dorsum of the SD rats.Eighty rats were divided into four groups at random,20 rats each.For the rats in the experimented group A,the wounds were covered with hVEGF genetically engineering membrane and Tegaderm membrane.In the control group B,the wounds were covered with membrane planted by the fibroblasts integrated with blank plasmid and Tegaderm membrane.In the control group C,the wounds were covered with blank membrane and Tegaderm membrane.In control group D,the wounds were only covered with Tegadern membrane.Specimens were obtained at the 3,7,14 and 29 days after injury,immunohistochemistry and image analysis were used to observe the expression of flt-1 and TSP-1.Results The expression of flt-1 in vascular endothelial cells was increased obviously in the experimental group.There was significant difference between the experimental group and other groups at 3,7 and 14d after injury(P

Suppression of retinal blood vessel leakage by inhibition of vascular endothlial growth factor using soluble-Flt in experimental diabetic rats / 眼科新进展

Objective　Vascular endothelial growth factor (VEGF) can cause potent vascular permeation. More and more findings have demonstrated that VEGF is increased in the diabetic retinopathy. The objective of this study is to investigate whether blocking the VEGF using soluble-Flt can suppress the retinal blood vessel leakage.Methods　Sprague-Dawley rats weighing approximately 200 grams were made diabetic by injection of streptozotocin (STZ). After the animals were diabetic for 1 week, they were intravenously injected with either PBS+50g*L-1Glycerol, interleukin-6 receptor monoclonal antibody (IL-6R), or soluble-Flt. On the eighth day, Evans blue method was used to check the leakage. Animals treated with citrate sodium buffer and STZ treated non-diabetic animals served as the control group. Results　There was no significant difference of the retinal blood vessel leakage between the two control groups (P>0.05). The retinal blood vessel leakage of 8 day diabetic animals was significantly higher than that of total control groups (P<0.05). The leakage of diabetic animals treated with PBS+Glycerol or IL-6R remains the same (P>0.05). But, the leakage of 25 mg*kg-1 soluble-Flt treated diabetic animals was remarkably lower than that before treatment, PBS+Glycerol treated or IL-6R treated diabetic animals (P<0.000 1), and there was no difference compared with total non-diabetic control groups (P>0.05). Conclusion　This study shows that the VEGF plays a very important role in the onset of the diabetic retinopathy and suggests by suppressing the action of VEGF, the retinal blood vessel leakage at the early stage of diabetic retinopathy could be reduced.

Production of Soluble VEGF Receptor Mutants for Inhibition of Angiogenesis / 대한암학회지

PURPOSE: Vascular endothelial growth factor (VEGF) is a potent angiogenic factor of many solid tumors, promoting vascularization and formation of metastases. In an attempt to generate effective VEGF inhibitors, the authors constructed the VEGF receptor mutants, expressed in E. coli and Sf9 insect cells, and examined their binding to VEGF. MATERIALS AND METHODS: The cDNA fragment encoding FLT-1 extracellular domain was cloned from human umbilical vein endothelial (HUVE) cell total RNA using RT-PCR. PCR- subcloning was performed using this template, in order to generate the deletion mutants by introducing FLT-1 partial sequences into E.coli expression vector pET-21d and baculovirus transfer vactors, pBAC-1 and pBAC-3. Two mutant proteins from baculovirus-infected insect cells were purified by heparin sepharose chromatography and immobilized into nitrdegrees Cellulose membrane followed by 125I-VEGF binding assay. RESULTS: Two mutant receptors, sFLT (1~7) and sFLT (2~4) expressed in E.coli appeared in inclusion body as insoluble proteins. The soluble mutant receptors were produced in low yield by baculovirus/insect cell expression system. Both immobilized mutant receptors, sFLT (1~7) and sFLT (2~4) were able to bind VEGF. CONCLUSION: These results suggest that a small soluble mutant receptor, sFLT (2~4), as well as sFLT (1~7) may be used effectively for bldegrees Cking angiogenic function of VEGF.

Effect of insulin on the secretion of VEGF and its receptor in serum-free cultured bovine thoracic aortic endothelial cells / 中国病理生理杂志

AIM: This study was designed to investigate the secretion of VEGF and its receptor (flt-1 or flk-1/KDR) protein by cultured bovine thoracic aortic endothelial cells treated with various insulin concentrations. METHODS: Endothelial cells was isolated from bovine thoracic aorta, and cultured in serum-free medium, then incubated with different insulin concentrations (30 mU/L, 300 mU/L, 3 000 mU/L). The level of VEGF and its receptor (flt-1 or flk-1/KDR) protein were detected by immunohistochemical staining. RESULTS: As compared with no insulin group, the expression of VEGF protein in low insulin concentration (30 mU/L and 300 mU/L) groups were significantly increased (P0.05) was observed. CONCLUSION: Low concentration insulin up-regulates the VEGF protein expression, while high concentration insulin down-regulates the VEGF protein expression in bovine thoracic aortic endothelial cells, but insulin had no directly effect on the VEGF receptor (flt-1 or flk-1/KDR) protein expression in bovine thoracic aortic endothelial cells. [

Gene Cloning of Soluble VEGF Receptor FLT-1 and the Effect of Its Recombinant Protein on Angiogenesis / 中国肿瘤生物治疗杂志

Objective: To clone and express cDNA fragment of domains 1 and 3 of soluble VEGF receptor FLT-1 in E. coli and investigate effect of its recombinant protein on endothelial proliferattion and angiogenesis. Methods: Total RNA from the umbelical venous endothelial cells was obtained. From it, soluble FLT-1(sFLT-1) cDNA fragment of domians 1 and 3 was cloned and expressed in QIA expressionist. The recombinant protein was purified by chromatography and refolded. The methods of MTT and angiogenesis test of chick chorioallantoic membrance were used to determine bioactivities of sFLT-1 recombinant protein. Results: The expression system could express the sFLT-1 cDNA fragment with a low expression level. After purification and renaturation, the recombinant protein could specifically bind to 125 I-VEGF. 1 ?g recombinant protein sFLT-1 could suppress endothelial cell proliferation induced by 10 ng VEGF and angiogenesis of chick chorioallantoic membrance induced by 20 ng VEGF.Conclusions: sFLT-1 cDNA fragment can be expressed in QIAexpressionist. After being refolded, it can bind to VEGF and serve as an antagonist of VEGF.

Effect of insulin on expression of VEGF receptor (flt-1、flk-1/KDR) protein in serum-free cultured bovine thoracic aortic endothelial cells and its relation to the NOS_3 activation of endothelium / 中国介入心脏病学杂志

0 05) between the insulin incubation groups both with and without L NAME incubations Conclusion Insulin has no direct effect on the expression of VEGF receptor (flt 1? flk 1/KDR) protein in bovine thoracic aortic endothelial cells The NOS 3 activation of endothelium is not the main cause that could affect the expression of VEGF receptor (flt 1?flk 1/KDR) protein of endothelium

Changes of expression levels of vascular endothelial growth factor and its receptors in diabetes rats with cerebral infarction / 临床神经病学杂志

Objective To study the changes of expression levels of vascular endothelial growth factor(VEGF) and its receptors(FLT-1,FLK-1) mRNA in equal area of penumbra in diabetes rats with cerebral infarction(DMCI).Methods Middle cerebral artery occlusion(MACO) model was made in diabetes rats.The expression levels of VEGF and FLT-1,FLK-1 mRNA in equal area of penumbra were measured with situ hybridization in different times after MACO(1 h,3 h,6 h,12 h,24 h).The difference was compared between DMCI group and no diabetes cerebral infarction(NDMCI) group.Results The expression level of VEGF mRNA was lower in DMCI group than that of NDMCI group at every time point(P