Effect and mechanism of Xihuang Pills on rats with precancerous lesions of breast / 中国中药杂志

The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P<0.05 or P<0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P<0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P<0.05 or P<0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P<0.05 or P<0.01), and the protein expression of Bcl-2 was lowered(P<0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P<0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P<0.01), while an increase in the mRNA expression of caspase-3(P<0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P<0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.

Research Progress of Targeted Ultrasound Contrast Agent BR55 / 中国医学科学院学报

BR55 is an ultrasound contrast agent targeting vascular endothelial growth factor receptor 2,which can be used to detect tumor neovascularization and improve the diagnostic accuracy.Overseas researchers have used BR55 for human ultrasound molecular imaging,which showed good safety and tolerance.We reviewed the research progress on BR55 applied in the evaluation of tumor neovascularization from the composition,characteristics,animal experiments,and clinical studies of BR55.

Immunohistochemical localization of vascular factors in tooth germ of amphibian (Cynops pyrrhogaster) / Localización inmunohistoquímica de factores vasculares en germen dentario de anfibios (Cynops pyrrhogaster)

SUMMARY: Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2, are known to regulate blood vessel endothelium growth. They play important role in human and rodents teeth development. In newt jaws, there are sequential developmental teeth germs following behind the mature teeth. We examined the immunohistochemical localization of VEGF and its receptor and showed the specific expression pattern of VEGF and VEGF receptor in Cynops pyrrhogaster sequential tooth development. The intensity of immunoreactivity for VEGF in the inner enamel epithelium was weaker than that in the outer enamel epithelium in the dentine matrix formation and mineralization stages. Finally, at the enameloid maturation and enamel-like matrix formation stage, immunoreactivity for VEGF in inner enamel epithelium was stronger than in the outer enamel epithelium. The intensity of immunoreactivity for VEGFR-2 was positive for the outer enamel epithelium throughout tooth development. The crown sides of the odontoblasts were stained especially strongly for VEGF and VEGFR-2 during the dentine matrix formation and mineralization stage of the enameloid maturation and enamel- like matrix formation stage. We postulate that the expression of VEGF in the inner enamel epithelium and odontoblast widely effects tooth development in newts, as well as in human and rodents.

RESUMEN: Se sabe que el factor de crecimiento endotelial vascular (VEGF) y su receptor, VEGFR-2, regulan el crecimiento del endotelio de los vasos sanguíneos. Desempeñan un papel importante en el desarrollo de los dientes humanos y de los roedores. En las mandíbulas de tritón, hay gérmenes dentales de desarrollo secuenciales que siguen a los dientes maduros. Examinamos la localización inmunohistoquímica de VEGF y su receptor y mostramos el patrón de expresión específico de VEGF y receptor de VEGF en el desarrollo secuencial de dientes de Cynops pyrrhogaster. La intensidad de la inmunorreactividad para VEGF en el epitelio interno del esmalte era más débil que en el epitelio externo del esmalte en las etapas de formación y mineralización de la matriz de dentina. Finalmente, en la etapa de maduración del esmalte y de formación de la matriz similar al esmalte, la inmunorreactividad para VEGF en el epitelio interno del esmalte fue más fuerte que en el epitelio externo del esmalte. La intensidad de la inmunorreactividad para VEGFR- 2 fue positiva para el epitelio externo del esmalte durante el desarrollo del diente. Los márgenes de la corona de los odontoblastos se tiñeron especialmente para VEGF y VEGFR-2 durante la etapa de formación de la matriz de dentina y mineralización de la etapa de maduración del esmalte y la etapa de formación de la matriz similar al esmalte. Postulamos que la expresión de VEGF en el epitelio interno del esmalte y odontoblastos afecta ampliamente el desarrollo de los dientes en tritones, así como en humanos y roedores.

A comprehensive analysis of the angiogenesis-related genes in glioblastoma multiforme vs. brain lower grade glioma / Uma análise abrangente dos genes relacionados à angiogênese no glioblastoma multiforme vs. glioma cerebral de baixo grau

Abstract Brain tumors are one of the most common causes of cancer-related deaths around the world. Angiogenesis is critical in high-grade malignant gliomas, such as glioblastoma multiforme. Objective: The aim of this study is to comparatively analyze the angiogenesis-related genes, namely VEGFA, VEGFB, KDR, CXCL8, CXCR1 and CXCR2 in LGG vs. GBM to identify molecular distinctions using datasets available on The Cancer Genome Atlas (TCGA). Methods: DNA sequencing and mRNA expression data for 514 brain lower grade glioma (LGG) and 592 glioblastoma multiforme (GBM) patients were acquired from The Cancer Genome Atlas (TCGA), and the genetic alterations and expression levels of the selected genes were analyzed. Results: We identified six distinct KDR mutations in the LGG patients and 18 distinct KDR mutations in the GBM patients, including missense and nonsense mutations, frame shift deletion and altered splice region. Furthermore, VEGFA and CXCL8 were significantly overexpressed within GBM patients. Conclusions: VEGFA and CXCL8 are important factors for angiogenesis, which are suggested to have significant roles during tumorigenesis. Our results provide further evidence that VEGFA and CXCL8 could induce angiogenesis and promote LGG to progress into GBM. These findings could be useful in developing novel targeted therapeutics approaches in the future.

Resumo Os tumores cerebrais são uma das causas mais comuns de mortes relacionadas ao câncer em todo o mundo. A angiogênese tem caráter crítico em gliomas malignos de alto grau, como o glioblastoma multiforme. Objetivo: O objetivo deste estudo foi analisar comparativamente os genes relacionados à angiogênese, VEGFA, VEGFB, KDR, CXCL8, CXCR1 e CXCR2 em GBG vs. GBM para identificar distinções moleculares usando conjuntos de dados disponíveis no The Cancer Genome Atlas (TCGA). Métodos: Os dados de sequenciamento de DNA e expressão de mRNA para 514 pacientes com glioma cerebral de baixo grau (GBG) e 592 pacientes com glioblastoma multiforme (GBM) foram adquiridos do TCGA e as alterações genéticas e os níveis de expressão dos genes selecionados foram analisados. Resultados: Identificamos seis mutações KDR distintas nos pacientes GBG e 18 mutações KDR distintas nos pacientes GBM, incluindo mutações missense e nonsense, exclusão de mudança de quadro e região de emenda alterada. Além disso, VEGFA e CXCL8 foram significativamente super-expressos nos pacientes com GBM. Conclusões: VEGFA e CXCL8 são fatores importantes para a angiogênese, os quais parecem ter um papel significativo durante a tumorigênese. Nossos resultados fornecem evidências adicionais de que o VEGFA e o CXCL8 podem induzir a angiogênese e promover o GBG a progredir no GBM. Esses achados podem ser úteis no desenvolvimento de novas abordagens terapêuticas direcionadas no futuro.

Coexistence of proangiogenic potential and increased MMP-9, TIMP-1, and TIMP-2 levels in the plasma of patients with critical limb ischemia / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The objective of this study was to assess the angiogenic potential expressed as a quotient of vascular endothelial growth factor A (VEGF-A), as an indicator of proangiogenic activity, and the circulating receptors (soluble VEGF receptor protein R1 (sVEGFR-1) and sVEGFR-2), as indicators of the effect of angiogenic inhibition, depending on the concentrations of matrix metalloproteinase 2 (MMP-2) and MMP-9 and their tissue inhibitor 1 (TIMP-1) and TIMP-2 in the plasma of patients with lower extremity artery disease (LEAD). These blood parameters in patients with intermittent claudication (IC) and critical limb ischemia (CLI) were compared for select clinical and biochemical features. Stimulation of angiogenesis in the plasma of individuals with LEAD was evident as indicated by the significant increase in VEGF-A concentration along with reduced inhibition depending on circulating receptors sVEGFR-1 and sVEGFR-2. Critical ischemia was associated with higher VEGF-A, MMP-9, TIMP-1, and TIMP-2 concentrations than in the case of IC.

Study on Flk1 Cells during Mouse Early Embryogenesis by Lineage Tracing / 中国实验血液学杂志

OBJECTIVE@#To understand the differentiation of mesoderm-derived Flk1 cells on different locations of the early mouse embryonic development and to explore the potential of Flk1 cells to differentiate into mesenchymal lineage, like pericytes during vascular development.@*METHODS@#Based on the Cre-LoxP system conditional knockout study strategy, the Flk1-Cre mice and ROSA26 reporter mice were used for lineage-tracing studies. The fate of the Flk1 progenitor cells was traced with the GFP population. The detection of mesoderm marker Flk1, hematopoietic cell-specific marker CD45, endothelial cell-specific markers CD31, CD144, and Emcn (endomucin), pericyte specific markers PDGFRβ and NG2, using the methods of immunohistochemistry, immunofluorescence, and flow cytometry should be combined to solve the concerned problems.@*RESULTS@#Immunohistochemical staining of different fractions of E8.5-10.5 in the early embryogenesis of Flk1-Cre; ROSA26-EYFP mouse lineage showed that there were multiple populations in the Flk1 cell-derived GFP population surrounding hematopoietic sites, such as dorsal aortas, limb buds and yolk sac. In addition to hematopoietic cells, the CD31/Emcn typical endothelial cells distributed specifically along the blood vessel wall, there were many types of cell populations, such as mesenchymal-like cells. The immunofluorescence demonstrated that the cells of this group are neither hematopoietic, non-endothelial cells around the blood vessels, which are NG2+ pericytes. FACS analysis also confirmed that Flk1 cells contributed to pericytes. In addition, in different hematopoietic sites of the embryo, a small population of CD31+CD140B+ cell populations with a mesenchymal-like morphology was observed in the GFP population.@*CONCLUSION@#In the early stages of embryogenesis, mesoderm-derived Flk1 populations not only contribute to hematopoietic, endothelial, and muscle lineages, but also have a differentiation potential for mesenchymal lineage, like pericytes. The presumably observed CD31CD140B cell population may be a group of endothelial cells differentiated from Flk1 progenitor cells and undergoing an endothelium-to-mesenchymal transition, EndMT, gradually losing the endothelial surface-specific marker and also starting to express a pericyte surface-specific marker.

Expression and significance of hypoxia-inducible factor-1α and vascular endothelial growth factor and receptor 2 in stage 3 pressure injury of rats / 中国应用生理学杂志

OBJECTIVE@#To analyze the expression and relationship of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and kinase insert domain receptor (KDR) in local skin tissues of pressure injury and investigate the possible mechanism of stage 3 pressure injury refractory wound.@*METHODS@#Forty male SD rats were randomly divided into normal control group, compressed 3 d, 5 d, 7 d, and 9 d groups. Stage 3 pressure injury animal model were established by magnet compression. The morphology of skin was observed by HE staining. The expression of VEGF was detected by immunohistochemistry. The expression levels of HIF-1α, VEGF and KDR protein in skin tissue were detected by Western blot. One-way analysis of variance and LSD test were performed on the data.@*RESULTS@#①The HE results showed that compared with the normal control group, the epidermis of the compressed group was gradually thickened, the number of blood vessels was decreased, the collagen arrangement disordered and inflammatory cells infiltration were increased. ②Immunohistochemical results showed that the expression of VEGF protein in the 3 d group was significantly higher than that in the normal control group (P＜0.01). The expression of VEGF protein in the skin tissue of 5 d, 7 d and 9 d groups was lower than that in normal control group (P＜0.05). WB results were consistent with immunohistochemistry results. ③WB results showed that the expression of HIF-1α in the skin tissues of the rats in 3 d, 5 d and 7 d groups was higher than that in the normal control group (P＜0.01 or P＜0.05). The expression of KDR protein was lower than that of the normal control group (P＜0.05 or P＜0.01).@*CONCLUSION@#HIF-1α mediated reduction of VEGF and KDR protein expression and decreased tissue angiogenesis may be one of the important causes of chronic dysfunction of stage 3 pressure injury.

The role of KDR in intrauterine adhesions may involve the TGF-ß1/Smads signaling pathway

The aim of this study was to investigate the role of kinase-insert domain-containing receptor (KDR) in intrauterine adhesions (IUA) and its mechanism. The Case group consisted of 92 patients diagnosed with IUA, and the Control group included 86 patients with uterine septum who had normal endometrium verified with an uteroscope. In addition, 50 rats were randomly assigned into Control, Sham, Model, NC-siRNA, and KDR-siRNA groups. Rats in the Model, NC-siRNA, and KDR-siRNA groups were induced by uterine curettage and lipopolysaccharide (LPS) treatment to establish the IUA model. Then, immunohistochemistry was applied for detection of VEGF and KDR expression, HE staining was used for observation of the endometrial morphology and gland counting, Masson staining for measurement of the degree of endometrial fibrosis, and qRT-PCR and western blot for the expression of KDR, VEGF, MMP-9, as well as TGF-β1/Smads pathway-related proteins. Compared with the Control group, the mRNA and protein expressions of KDR were significantly higher in IUA endometrial tissues, and the expression of KDR was positively correlated to the severity of IUA. In addition, the injection of si-KDR increased the number of endometrial glands, reduced the area of fibrosis, inhibited mRNA and protein expression of KDR and VEGF, up-regulated the expression of MMP-9 and Smad7, and decreased the expression level of TGF-β1, p-Smad2, p-Smad3, and Smad4 in rats with IUA. Highly-expressed KDR was related to patients' severity of IUA, and silencing KDR may prevent the occurrence and development of IUA via TGF-β1/Smads signaling pathway and up-regulating the expression of MMP-9.

Is the effect of melatonin on vascular endothelial growth factor receptor-2 associated with angiogenesis in the rat ovary?

OBJECTIVES Vascular endothelial growth factor (VEGF) and its receptors play important roles in angiogenesis. Melatonin plays an important role in gonadal development; thus, its effect on the reproductive system is evident. We investigated the influence of melatonin on the expression of VEGF, vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2), as well as on changes in oxidative stress markers and follicle numbers in rat ovaries. METHODS For this purpose, 45 Wistar rats were separated into the following groups: Group 1, control; Group 2, vehicle; and Group 3, melatonin. Rats in Group 3 were treated with melatonin at 50 mg/kg/day for 30 days. The effects of melatonin on the expression of VEGF, VEGFR1 and VEGFR2 were established by immunohistochemistry analysis. The effects of melatonin on antioxidant enzyme activities were demonstrated by spectrophotometric analysis. RESULTS Based on immunohistochemistry analysis, VEGFR2 was predominantly localized to theca cells in the ovary. Our data indicate that melatonin treatment can significantly increase VEGF and VEGFR1 expression in the ovary ( p <0.05). Additionally, the number of degenerated follicles significantly decreased with melatonin treatment ( p <0.05). Melatonin administration also led to significant increases in antioxidant enzyme levels in the ovary. CONCLUSION Melatonin treatment exerts protective effects on follicles against increased lipid peroxidation through modulating tissue antioxidant enzyme levels. These effects may be related to angiogenesis and antioxidant activities.

Expresión de VEGF-A y VEGFR2 en Miocardio de Pollos Tratados con Ácido Acetilsalicílico (AAS) / VEGF-A and VEGFR2 Expression in Chicken Myocardium Treated with Acetylsalicylic Acid (AAS)

RESUMEN: Los niveles de VEGF y su unión a sus receptores son etapas claves en la regulación de la angiogénesis. El ácido acetilsalicílico (AAS), ampliamente utilizado en tratamiento post infarto al miocardio ha mostrado poseer un efecto antiangiogénico en modelos tumorales. Este efecto potencialmente contraproducente requiere ser estudiado en miocardio. El objetivo del presente trabajo es cuantificar el efecto de AAS y de ácido salicílico (AS) sobre la vascularización en membrana alantocoriónica (MAC) y sobre los niveles de VEGF-A y VEGFR2 en miocardio de embriones de pollo. Para ello, treinta fetos de pollo White Leghorn fueron instilados a los 10 días de gestación con 60 µL de DMSO 0,1 % (control) o conteniendo además 0,3 µmol de AAS o AS. A las 48 horas se realizó procesamiento histológico de MAC para recuento de vasos sanguíneos y de tejido cardíaco para cuantificar VEGF-A y VEGFR2 por inmunohistoquímica. La inmunorreactividad fue cuantificada mediante Image J. Tanto AAS como AS disminuyeron la densidad microvascular de MAC. En miocardio, AAS aunque no AS, disminuyó la concentración de VEGFR2. No hubo efecto sobre VEGF-A. En nuestro modelo experimental, fetos de pollo a los 10 días de gestación también se observó el efecto inhibidor de AAS sobre la angiogénesis en MAC. La disminución de VEGFR2 en cardiomiocitos sugiere que AAS también afecta la angiogénesis en miocardio sano, modificando la disponibilidad del receptor a VEGF. Estos hallazgos nos permiten postular que AAS podría interferir con la regeneración de tejido, en situaciones como post infarto al miocardio.

SUMMARY: The VEGF levels and its binding to its receptors are key stages in the regulation of angiogenesis. Acetylsalicylic acid (ASA), widely used in post-myocardial infarction treatment, has been shown to have an anti-angiogenic effect in tumor models. This potentially counterproductive effect requires to be studied in myocardium. The aim of this study is to quantify the effect of ASA and salicylic acid (SA) on the vascularization in chick allantochorionic membrane (CAM) and on the levels of VEGF-A and VEGFR2 in myocardium of chicken embryos. Thirty White Leghorn chicken fetuses were instilled at 10 days of gestation with 60 mL of 0.1 % DMSO (control) or also containing 0.3 mmol of ASA or SA. After 48 hours, CAM histological processing was performed to count blood vessels and heart tissue to quantify VEGFA and VEGFR2 by immunohistochemistry. Immunoreactivity was quantified by Image J. Both ASA and SA decreased CAM microvascular density. In myocardium, AAS, although not SA, decreased the concentration of VEGFR2. There was no effect on VEGF-A. In our experimental model, chicken fetuses at 10 days of gestation, the inhibitory effect of ASA on angiogenesis in CAM were also observed. The decrease in VEGFR2 in cardiomyocytes suggests that ASA also affects angiogenesis in healthy myocardium, modifying the availability of the receptor to VEGF. These findings allow us to postulate that ASA could interfere with tissue regeneration, when it is required, as post myocardial infarction.

Fatores prognósticos de resposta à quimioterapia em tumores avançados do colo uterino: o papel da neoangiogênese / Prognostic factors for response to chemotherapy in advanced tumors of the uterine cervix: the role of neoangiogenesis

RESUMO Objetivo: analisar a expressão do Fator de Crescimento do Endotélio Vascular (VEGF), seu receptor (VEGFR-2), idade e tipo histológico de carcinomas avançados de colo uterino com relação à resposta clínica à quimioterapia neoadjuvante. Métodos: foram incluídas 40 pacientes com diagnóstico de carcinoma de colo uterino (IB2 e IVA), com biópsias prévias ao tratamento. Todas as pacientes foram submetidas à quimioterapia neoadjuvante e avaliadas quanto à resposta clínica e à expressão do VEGF. Considerou-se boa resposta clínica uma regressão tumoral total ou maior do que 50%. Resultados: em relação à resposta à quimioterapia, 18 pacientes (45%) apresentaram boa resposta e 22 (55%), má resposta. Quanto à expressão do VEGF, em 16 pacientes foi considerada positiva e em 24, negativa. Quando os casos foram analisados separadamente em relação à resposta à quimioterapia, somente a expressão positiva de VEGF foi associada à boa resposta clínica (p=0,0157). Conclusão: a expressão de VEGF mostrou ser isoladamente, um importante marcador de boa resposta ao tratamento quimioterápico neoadjuvante das pacientes com carcinoma avançado de colo uterino.

ABSTRACT Objective: to analyze the expression of Vascular Endothelial Growth Factor (VEGF), its receptor (VEGFR-2), age and histological type of advanced cervical carcinomas with respect to the clinical response to neoadjuvant chemotherapy. Methods: we studied 40 patients with cervical carcinoma (IB2 and IVA) diagnosed by biopsies prior to treatment. All patients underwent neoadjuvant chemotherapy and evaluation for clinical response and expression of VEGF. We considered a tumor regression greater than 50% as a good clinical response. Results: eighteen patients (45%) had good response to chemotherapy, and 22 (55%), poor response. VEGF expression was positive in 16 patients and negative in 24. When analyzed separately for response to chemotherapy, only the positive expression of VEGF was associated with good clinical response (p=0.0157). Conclusion: VEGF expression alone was an important marker of good response to neoadjuvant chemotherapy in patients with advanced carcinoma of the cervix.

Pioglitazone Induces Cardiomyocyte Apoptosis and Inhibits Cardiomyocyte Hypertrophy Via VEGFR-2 Signaling Pathway / Pioglitazona Induz Apoptose e Inibe Hipertrofia de Cardiomiócitos pela Via de Sinalização do VEGFR-2

Abstract Background: Pioglitazone has been widely used as an insulin-sensitizing agent for improving glycemic control in patients with type 2 diabetes mellitus. However, cardiovascular risk and protective effects of pioglitazone remain controversial. Objectives: In this study, we investigated whether pioglitazone affects cardiomyocyte apoptosis and hypertrophy by regulating the VEGFR-2 signaling pathway. Methods: Cardiomyocytes were enzymatically isolated from 1- to 3-day-old Sprague-Dawley rat ventricles. Effects of pioglitazone and the VEGFR-2-selective inhibitor apatinib on cardiomyocyte apoptotic rate was determined using flow cytometry, and hypertrophy was evaluated using [3H]-leucine incorporation. The protein expressions of unphosphorylated and phosphorylated VEGFR-2, Akt, P53, and mTOR were determined by Western-Blotting. Analysis of variance (ANOVA) was used to assess the differences between groups. Results: Pioglitazone and VEGFR-2-selective inhibitor apatinib reduced rat cardiomyocyte viability and cardiomyocyte hypertrophy induced by angiotensin II in vitro. Furthermore, in the same in vitro model, pioglitazone and apatinib significantly increased the expression of Bax and phosphorylated P53 and decreased the expression of phosphorylated VEGFR-2, Akt, and mTOR, which promote cardiomyocyte hypertrophy. Conclusions: These findings indicate that pioglitazone induces cardiomyocyte apoptosis and inhibits cardiomyocyte hypertrophy by modulating the VEGFR-2 signaling pathway.

Resumo Fundamento: A pioglitazona tem sido amplamente utilizada como droga sensibilizadora da insulina para melhorar o controle glicêmico em pacientes com diabetes mellitus tipo 2. No entanto, o risco cardiovascular bem como os efeitos protetores da pioglitazona ainda são controversos. Objetivos: Neste estudo, investigamos se os efeitos da pioglitazona sobre a apoptose e a hipertrofia de cardiomiócitos ocorrem via regulação da via de sinalização do VEGFR-2. Métodos: os cardiomiócitos foram isolados enzimaticamente dos ventrículos de ratos Sprague-Dawley de 1-3 anos de vida. Os efeitos da pioglitazona e do inibidor seletivo de VEGFR-2 apatinib sobre a taxa de apoptose dos cardiomiócitos foram avaliados por citometria de fluxo, e a hipertrofia avaliada por incorporação de leucina-[3H]. As expressões de VEGFR-2, Akt, P53, e mTOR fosforiladas e não fosforiladas foram determinadas por Western Blotting. Análise de variância (ANOVA) foi usada para avaliar diferenças entre os grupos. Resultados: a pioglitazona e o apatinib reduziram a viabilidade e a hipertrofia de cardiomiócitos induzida por angiotensina II in vitro. Além disso, no mesmo modelo in vitro, a pioglitazona e o apatinib aumentaram significativamente a expressão de Bax e P53 fosforilada, e diminuiu a expressão de VEGFR-2, Akt, e mTOR, que promove hipertrofia de cardiomiócitos. Conclusões: Esses resultados indicam que a pioglitazona induz a apoptose e inibe a hipertrofia de cardiomiócitos pela modulação da via de sinalização de VEGFR-2.

Isomangiferin, a Novel Potent Vascular Endothelial Growth Factor Receptor 2 Kinase Inhibitor, Suppresses Breast Cancer Growth, Metastasis and Angiogenesis / 한국유방암학회지

PURPOSE: Vascular endothelial growth factor (VEGF) signal transduction mainly depends on its binding to VEGF receptor 2 (VEGFR-2). VEGF downstream signaling proteins mediate several of its effects in cancer progression, including those on tumor growth, metastasis, and blood vessel formation. The activation of VEGFR-2 signaling is a hallmark of and is considered a therapeutic target for breast cancer. Here, we report a study of the regulation of the VEGFR-2 signaling pathway by a small molecule, isomangiferin. METHODS: A human breast cancer xenograft mouse model was used to investigate the efficacy of isomangiferin in vivo. The inhibitory effect of isomangiferin on breast cancer cells and the underlying mechanism were examined in vitro. RESULTS: Isomangiferin suppressed tumor growth in xenografts. In vitro, isomangiferin treatment inhibited cancer cell proliferation, migration, invasion, and adhesion. The effect of isomangiferin on breast cancer growth was well coordinated with its suppression of angiogenesis. A rat aortic ring assay revealed that isomangiferin significantly inhibited blood vessel formation during VEGF-induced microvessel sprouting. Furthermore, isomangiferin treatment inhibited VEGF-induced proliferation of human umbilical vein endothelial cells and the formation of capillary-like structures. Mechanistically, isomangiferin induced caspase-dependent apoptosis of breast cancer cells. Furthermore, VEGF-induced activation of the VEGFR-2 kinase pathway was down-regulated by isomangiferin. CONCLUSION: Our findings demonstrate that isomangiferin exerts anti-breast cancer effects via the functional inhibition of VEGFR-2. Pharmaceutically targeting VEGFR-2 by isomangiferin could be an effective therapeutic strategy for breast cancer.

6-Shogaol reduces progression of experimental endometriosis in vivo and in vitro via regulation of VGEF and inhibition of COX-2 and PGE2-mediated inflammatory responses

Endometriosis (EM) is one of the most common gynaecological disorder affecting women in their reproductive age. Mechanisms involved in the pathogenesis of EM remains poorly understood, however inflammatory responses have been reported to be significantly involved. The efficacy of 6-shogaol on proliferation of endometriotic lesions and inflammatory pathways in experimentally-induced EM model was explored in this study. EM was stimulated in Sprague-Dawley rats by implantation of autologous endometrium onto the peritoneum abdominal wall. Separate groups were treated with 6-shogaol (50, 100 or 150 mg/kg b.wt/day) via oral gavage for one month period. Gestrinone (GTN) group received GTN (0.5 mg/kg/day) as positive control. Five weeks after implantation, the spherical volume of ecto-uterine tissues was determined. Treatment with 6-shogaol significantly reduced the implant size. Histological analysis reported atrophy and regression of the lesions. 6-shogaol administration effectively down-regulated NF-κB signaling, VEGF and VEGFR-2 (Flk-1) expression in the endometriotic lesions. Excess production of IL-1β and IL-6 (pro-inflammatory cytokines), PGE2 and nitric oxide (NO) were reduced. Overall, the results of the study reveal the efficacy of 6-shogaol against endometriosis via effectively suppressing proliferation of the lesions and modulating angiogenesis and COX-2/NF-κB-mediated inflammatory cascades.

Ventilation-induced changes correlate to pulmonary vascular response and VEGF, VEGFR-1/2, and eNOS expression in the rat model of postnatal hypoxia

Neonatal asphyxia occurs due to reduction in oxygen supply to vital organs in the newborn. Rapid restoration of oxygen to the lungs after a long period of asphyxia can cause lung injury and decline of respiratory function, which result from the activity of molecules that induce vascular changes in the lung such as nitric oxide (NO) and vascular endothelial growth factors (VEGF). In this study, we evaluated the pulmonary and vascular morphometry of rats submitted to the model of neonatal asphyxia and mechanical ventilation, their expression of pulmonary VEGF, VEGF receptors (VEGFR-1/VEGFR-2), and endothelial NO synthase (eNOS). Neonate Sprague-Dawley rats (CEUA #043/2011) were divided into four groups (n=8 each): control (C), control submitted to ventilation (CV), hypoxia (H), and hypoxia submitted to ventilation (HV). The fetuses were harvested at 21.5 days of gestation. The morphometric variables measured were body weight (BW), total lung weight (TLW), left lung weight (LLW), and TLW/BW ratio. Pulmonary vascular measurements, VEGFR-1, VEGFR-2, VEGF, and eNOS immunohistochemistry were performed. The morphometric analysis showed decreased TLW and TLW/BW ratio in HV compared to C and H (P<0.005). Immunohistochemistry showed increased VEGFR-2/VEGF and decreased VEGFR-1 expression in H (P<0.05) and lower eNOS expression in H and HV. Median wall thickness was increased in H, and the expression of VEGFR-1, VEGFR-2, VEGF, and eNOS was altered, especially in neonates undergoing H and HV. These data suggested the occurrence of arteriolar wall changes mediated by NO and VEGF signaling in neonatal hypoxia.

Effects of Chronic Exercise on Endothelial Progenitor Cells and Microparticles in Professional Runners / Efeitos de Exercício Crônico Sobre Células Progenitoras Endoteliais e Micropartículas em Corredores Profissionais

Abstract Background: The effects of chronic exposure to exercise training on vascular biomarkers have been poorly explored. Objective: Our study aimed to compare the amounts of endothelial progenitor cells (EPCs), and endothelial (EMP) and platelet (PMP) microparticles between professional runners and healthy controls. Methods: Twenty-five half-marathon runners and 24 age- and gender-matched healthy controls were included in the study. EPCs (CD34+/KDR+, CD133+/KDR+, and CD34+/CD133+), EMP (CD51+) and PMP (CD42+/CD31+) were quantified by flow-cytometry. All blood samples were obtained after 12 h of fasting and the athletes were encouraged to perform their routine exercises on the day before. Results: As compared with controls, the CD34+/KDR+ EPCs (p=0.038) and CD133+/KDR+ EPCs (p=0.018) were increased, whereas CD34+/CD133+ EPCs were not different (p=0.51) in athletes. In addition, there was no difference in MPs levels between the groups. Conclusion: Chronic exposure to exercise in professional runners was associated with higher percentage of EPCs. Taking into account the similar number of MPs in athletes and controls, the study suggests a favorable effect of exercise on these vascular biomarkers.

Resumo Fundamento: Os efeitos da exposição crônica ao exercício sobre biomarcadores vasculares foram pouco estudados. Objetivo: Nosso estudo teve como objetivo comparar as quantidades de células progenitoras endoteliais (CPEs), e de micropartículas endoteliais (MPEs) e plequetárias (MPPs) de corredores profissionais com controles sadios. Métodos: Vinte e cinco corredores de meia maratona e 24 controles pareados quanto à idade e ao sexo foram incluídos no estudo. CPEs (CD34+/KDR+, CD133+/KDR+ e CD34+/CD133+), MPE (CD51+) e MPPs (CD42+/CD31+) foram quantificadas por citometria de fluxo. Todas as amostras de sangue foram obtidas após 12 horas de jejum, e os atletas foram incentivados a realizar seus exercícios de rotina no dia anterior à coleta. Resultados: Em comparação aos controles, CPEs CD34+/KDR+ (p=0,038) e CD133+/KDR+ (p=0,018) estavam aumentados, e CPEs CD34+/CD133+ não foram diferentes (p=0,51) nos atletas. As concentrações de MP não diferiram entre os grupos. Conclusão: A exposição crônica ao exercício em corredores profissionais associou-se a uma maior porcentagem de CPEs. Considerando o número similar de MPs entre atletas e controles, o estudo sugere um efeito favorável do exercício sobre esses biomarcadores vasculares.

Recombinant Human Erythropoietin Augments Neovascularization Responses in a Neonatal Rat Model of Premature Brain Damage by Phosphatidylinositol 3 Kinase/Akt Pathway / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recombinant human-erythropoietin (rh-EPO) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti-inflammation. In the present study, we found that the rh-EPO was related to the promotion of neovascularization. Our aim was to investigate whether rh-EPO augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway.</p><p><b>METHODS</b>Postnatal day 5 (PD5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. All the rat pups were randomized into five groups as follows: (1) control group; (2) hypoxia-ischemic (HI) group; (3) HI + LY294002 group; (4) HI + rh-EPO group; and (5) HI + rh-EPO + LY294002 group. The phospho-Akt protein was tested 90 min after the whole operation, and CD34, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor (VEGF) were also tested 2 days after the whole operation.</p><p><b>RESULTS</b>In the hypoxic and ischemic zone of the premature rat brain, the rh-EPO induced CD34+ cells to immigrate to the HI brain zone (P < 0.05) and also upregulated the VEGFR2 protein expression (P < 0.05) and VEGF mRNA level (P < 0.05) through the PI3K/Akt (P < 0.05) signaling pathway when compared with other groups.</p><p><b>CONCLUSIONS</b>The rh-EPO treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the PI3K/Akt signaling pathway. Besides, the endogenous EPO may exist in the HI zone of rat brain and also has neovascularization function through the PI3K/Akt signaling pathway.</p>

Effect of Anti-vascular Endothelial Growth Factor Antibody on the Survival of Cultured Retinal Ganglion Cells

PURPOSE: To investigate the effects of anti-vascular endothelial growth factor (VEGF) antibody on the survival of retinal ganglion cell (RGC)-5 cells differentiated with staurosporine under oxidative stress. METHODS: We used real-time polymerase chain reaction and Western blot to confirm the expression of VEGF, VEGF receptor (VEGFR)-1 and VEGFR-2 in RGC-5 cells differentiated with staurosporine for 6 hours. The differentiated RGC-5 cells were treated with 800 µM hydrogen peroxide (H₂O₂) for 24 hours to induce oxidative stress. Then, the survival rate of RGC-5 was confirmed by lactate dehydrogenase assay at each concentration (0, 0.01, 0.1, and 1 mg) using bevacizumab as the anti-VEGF antibody. The expression of VEGF, VEGFR-1, and VEGFR-2 was confirmed using real-time polymerase chain reaction. RESULTS: VEGF, VEGFR-1, and VEGFR-2 were all expressed in differentiated RGC-5 cells. When RGC-5 cells were simultaneously treated with bevacizumab and 800 µM H₂O₂, survival of RGC-5 decreased with bevacizumab concentration. VEGF expression in RGC-5 cells increased with increasing concentration of bevacizumab. Similar patterns were observed for VEGFR-1 and VEGFR-2, but the degree of increase was smaller than that for VEGF. CONCLUSIONS: When bevacizumab was administered to differentiated RGC-5 cells, the cell damage caused by oxidative stress increased. Therefore, given these in vitro study results, caution should be exercised with bevacizumab treatment.

Estrogen deficiency in ovariectomized rats: can resistance training re-establish angiogenesis in visceral adipose tissue?

OBJECTIVE: The purpose of this study was to investigate the effects of resistance training on angiogenesis markers of visceral adipose tissue in ovariectomized rats. METHOD: Adult Sprague-Dawley female rats were divided into four groups (n=6 per group): sham-sedentary, ovariectomized sedentary, sham-resistance training and ovariectomized resistance training. The rats were allowed to climb a 1.1-m vertical ladder with weights attached to their tails and the weights were progressively increased. Sessions were performed three times per week for 10 weeks. Visceral adipose tissue angiogenesis and morphology were analyzed by histology. VEGF-A mRNA and protein levels were analyzed by real-time PCR and ELISA, respectively. RESULTS: Ovariectomy resulted in higher body mass (p=0.0003), adipocyte hypertrophy (p=0.0003), decreased VEGF-A mRNA (p=0.0004) and protein levels (p=0.0009), and decreased micro-vascular density (p=0.0181) in the visceral adipose tissue of the rats. Resistance training for 10 weeks was not able to attenuate the reduced angiogenesis in the visceral adipose tissue of the ovariectomized rats. CONCLUSION: Our findings indicate that the resistance training program used in this study could not ameliorate low angiogenesis in the visceral adipose tissue of ovariectomized rats.