Participação dos neutrófilos durante a implantação embrionária no desenvolvimento da placenta em camundongos / Participation of neutrophils during embryonic implantation in the placental development in mice

A implantação do embrião na parede uterina é um processo complexo que consiste na interação do blastocisto com as células epiteliais do útero, e depende de diferentes tipos celulares do microambiente uterino. Embora a literatura mostre a participação de neutrófilos neste processo, os dados ainda são incipientes para proposição da função exata destas células nos períodos iniciais da gestação. Dados do nosso grupo de pesquisa mostraram que neutrófilos pró-angiogênicos induzem a tolerância gestacional, e que a depleção de neutrófilos durante as fases iniciais da gestação prejudica a implantação do blastocisto e a progressão da gestação. Com base nestes resultados, o presente estudo visou investigar se a depleção de neutrófilos na fase pré-receptiva da janela de implantação do blastocisto altera a morfologia placentária. Para tanto, foi utilizado o modelo de gestação alogênica, onde camundongos fêmeas C57BL/6, após cruzamento com machos Balb/C foram tratadas com anticorpo anti-Ly6G ou isotipo no dia 1,5 da gestação (24 horas após a detecção do plug vaginal) em dose suficiente para manter a depleção de neutrófilos circulantes por 48 horas (200µg/ 500µL; i.p). No final da gestação (dia 18,5), o sangue periférico foi coletado e, em seguida, os animais foram submetidos a laparotomia para retirada da placenta, a qual foi submetida à análise histológica. As análises dos leucócitos circulantes evidenciaram a efetividade do tratamento para depleção de neutrófilos periféricos. A análise histológica mostrou alterações significativas na morfologia da placenta nos animais tratados com anti-Ly6G. Foram detectadas a redução da zona juncional, de células trofoblásticas e de fatores angiogênicos, como fator de crescimento do endotélio vascular (VEGF), e das moléculas de adesão intracelular-1 (ICAM-1) e de plaqueta e endotélio (PECAM-1). Esses dados evidenciam a importância dos neutrófilos nos primeiros dias de gestação para o desenvolvimento da placenta

Blastocyst implantation is a complex process, consisting of the interaction between blastocyst and uterine epithelial cells. Also, it is well known that the implantation site resembles an inflammatory response, with a profusion of recruited immune cells into the endometrial stroma and lumen from the blood. The role of macrophages, natural killers, and dendritic cells have been extensively studied, however, the participation of neutrophils in this process remains unclear. Data from our research group showed that pro-angiogenic neutrophils induced gestation tolerance, also peripheral neutrophils depletion at the time of active placental development led to smaller embryo sizes and abnormal placentation in mice. In this context, the present study aimed to investigate whether pharmacological depletion of neutrophils in mice in the blastocyst implantation phase alters placental morphology. Therefore, C7/BL/6 female mice, after mating with Balb/C males, were treated with an anti-Ly6G antibody or isotype on day 1 of gestation (after detection of the vaginal plug) at a dose sufficient to maintain the depletion of circulating neutrophils for 48 hours (200 µg/500µL; i.p). At the end of the gestational day (day 18), peripheral blood was collected, and then the animals were submitted to laparotomy for the placenta removal and subsequent histological analysis. The analysis of circulating leukocytes from neutrophils depleted mice showed a reduction of peripheral neutrophils up to 48 hours after antibody injection. The histological analysis showed significant alterations in the placenta morphology of the animals treated with anti-Ly6G. The morphometric analyses showed a reduction in the size of neutrophils depleted placenta due to diminished junctional zone and reduction of trophoblast cells. Also, it was observed a reduction of vascular endothelial growth factors (VEGF), reduction of adhesion molecules intracell-1 (ICAM-1), and platelets and endothelium (PECAM-1) positive cells in the junctional zone. In conclusion, these data show the importance of neutrophils on the first days of pregnancy for the development of the placenta

Association between Adverse Maternal Clinical Outcomes and Imbalance of Cytokines and Angiogenic Factors in Preterm Preeclampsia / Associação entre desfechos maternos adversos e desbalanço de citocinas e fatores angiogênicos em pré-eclâmpsia pré-termo

Abstract Objective Preeclampsia (PE) is a pregnancy-specific syndrome characterized by abnormal levels of cytokines and angiogenic factors, playing a role in the disease development. The present study evaluated whether immunological markers are associated with the gestational age and with the disease severity in preeclamptic women. Methods Ninety-five women who developed PE were stratified for gestational age as preterm PE (< 37 weeks) and term PE (≥ 37 weeks of gestation) and compared for disease severity as well as plasma concentration of angiogenic factors and cytokines. The concentrations of placental growth factor (PlGF), vascular endothelial growth factor (VEGF), Fms-like soluble tyrosine kinase (sFlt-1) and soluble endoglin (sEng), as well as the cytokines, tumor necrosis factor-α (TNF-α) and interleukin 10 (IL-10), were determined by enzyme-linked immunosorbent assay (ELISA). Results The comparison between preeclamptic groups showed a higher percentage of severe cases in preterm PE (82.1%) than in term PE (35.9%). Similarly, the concentrations of TNF-α, sFlt-1, and sEng, as well as TNF-α/IL-10 and sFlt-1/PlGF ratios were significantly higher in the preterm PE group. In contrast, concentrations of PlGF, VEGF, and IL-10 were significantly lower in women with preterm PE. Negative correlations between TNF-α and IL-10 (r = 0.5232) and between PlGF and sFlt1 (r = 0.4158) were detected in the preterm PE. Conclusion In pregnant women with preterm PE, there is an imbalance between immunological markers, with the predominance of anti-angiogenic factors and TNF-α, associated with adverse maternal clinical outcomes.

Resumo Objetivo A pré-eclâmpsia (PE) é uma síndrome específica da gravidez caracterizada por níveis anormais de citocinas e fatores angiogênicos, que desempenham um papel no desenvolvimento da doença. Este estudo avaliou se os marcadores imunológicos estão associados à idade gestacional e à gravidade da doença em mulheres com pré-eclâmpsia. Métodos Noventa e cinco mulheres que desenvolveram PE foram estratificadas pela idade gestacional em PE pré-termo (< 37 semanas) e PE a termo (≥ 37 semanas de gestação) e comparadas quanto à gravidade da doença, bem como à concentração plasmática de fatores angiogênicos e citocinas. As concentrações de fator de crescimento placentário (PlGF), fator de crescimento endotelial vascular (VEGF), tirosina quinase solúvel semelhante a Fms (sFlt-1) e endoglina solúvel (sEng), bem como as citocinas, fator de necrose tumoral alfa (TNF- α) e interleucina 10 (IL-10), foram determinados porensaio de imunoabsorção enzimática (ELISA, na sigla em inglês). Resultados A comparação entre os grupos com pré-eclâmpsia mostrou maior porcentagem de casos graves em PE pré-termo (82,1%) do que em PE a termo (35,9%). Da mesma forma, as concentrações de TNF-α, sFlt-1 e sEng, bem como as razões TNF-α/IL-10 e sFlt-1/PlGF foram significativamente maiores no grupo de PE pré-termo. Em contraste, as concentrações de PlGF, VEGF e IL-10 foram significativamente menores em mulheres com PE pré-termo. Correlações negativas entre TNF-α e IL-10 (r = 0.5232) e entre PlGF e sFlt1 (r = 0.4158) foram detectadas no grupo de PE pré-termo. Conclusão Em gestantes com PE pré-termo, ocorre um desequilíbrio entre os marcadores imunológicos, com predomínio de fatores antiangiogênicos e TNF-α, associados a desfechos clínicos maternos adversos.

Evaluation of platelet-rich plasma gel as an angiogenesis-inducing agent in canine advancement skin flaps / Avaliação do gel de plasma rico em plaquetas como agente indutor da angiogênese em flapes cutâneos de avanço em cães

This work aimed to evaluate the effect of platelet-rich plasma (PRP) on advancement skin flaps in dogs regarding improvement of vascularization, with focus on increasing its viable area, since there are reports that it is a potential angiogenesis stimulator. The experimental group was composed of eight adult bitches, in which two advancement skin flaps were made in the ventral abdominal region. No product was applied in the control flap (CF), while PRP was used in the contralateral flap, called treated flap (TF). The areas were clinically evaluated every two days until the 7th postoperative day regarding skin color and presence of necrosis. At 10 days, both flaps were removed and submitted to histological examination and blood vessel morphometry. The vessels counted in each group were statistically analyzed by the F-test at 1% probability. Results showed no significant difference in macroscopic changes in the wound, or CF and TF vascularization, thus suggesting that PRP gel did not improve advancement skin flap angiogenesis in bitches under the experimental conditions in which this research was developed.(AU)

Objetivou-se com o presente artigo avaliar a ação angiogênica do gel de plasma rico em plaquetas (PRP) em flapes cutâneos de avanço em animais da espécie canina, visando aumentar a viabilidade da pele após o procedimento, uma vez que existem relatos de que o produto é um potente estimulador da angiogênese. O grupo experimental foi composto por oito cadelas adultas, onde foram confeccionados dois flapes de avanço (de padrão subdérmico) na região abdominal ventral. Em um dos flapes, considerado controle (FC) não foi aplicado nenhum produto, enquanto que no flape contralateral, denominado tratado (FT), foi usado o gel de PRP. As áreas foram macroscopicamente avaliadas a cada dois dias até o 7º dia de pós-operatório em relação à coloração da pele e presença de área de necrose, e com 10 dias ambos os flapes foram coletados por biópsia e submetidos ao exame histológico e morfometria dos vasos sanguíneos. Os vasos contados em cada grupo foram estatisticamente analisados pelo teste de F ao nível de 1% de probabilidades. Os resultados demonstraram que não houve diferença significativa nas alterações macroscópicas das feridas e na morfometria vascular dos FC e FT, sugerindo dessa maneira que dentro das condições experimentais nas quais a pesquisa foi executada, que o gel de PRP não incrementou a angiogênese de flapes de avanço em cadelas.(AU)

Pterigión recidivante y sus alternativas terapéuticas / Recurrent pterygium and its therapeutic alternatives

RESUMEN El pterigión se trata de un crecimiento fibrovascular de morfología triangular que se extiende desde la conjuntiva hacia la córnea. Está clasificado dentro de las degeneraciones no involutivas o tumoraciones epiteliales benignas corneales y se presenta en el 10,2 por ciento de la población. El tratamiento quirúrgico es el más indicado, y tiene una tasa de recidiva independientemente de la técnica quirúrgica utilizada del 10,7 por ciento; esta tasa de recidiva se evidencia por la neovascularización y el tejido cicatrizal antes de los dos meses después de la cirugía. Lo anterior ha incentivado la investigación de nuevos tratamientos que disminuyan esta complicación, por lo que el objetivo de esta revisión bibliográfica fue la búsqueda de alternativas terapéuticas para el pterigión recidivante. Se realizó una búsqueda automatizada sobre el tema, utilizando la plataforma Infomed, cuya información fue resumida para la elaboración del informe final. Concluimos que existen diferentes tratamientos adyuvantes para disminuir la tasa de recurrencia, y es necesario realizar estudios donde se determine el tiempo y la frecuencia en la aplicación de estos para obtener resultados más efectivos en su uso(AU)

ABSTRACT Pterygium is a fibrovascular growth of triangular shape that extends from the conjunctiva to the cornea. It has been classified as a noninvolutionary degeneration or benign corneal epithelial tumor which affects 10.2 percent of the population. The treatment most commonly indicated is surgery, which has a recurrence rate of 10.7 percent, irrespective of the surgical technique used. Recurrence takes the form of neovascularization and scar tissue within two months after surgery. This has fostered research into new treatments to reduce this complication. The objective of the present bibliographic review was precisely to search for therapeutic alternatives for recurrent pterygium. An automated search was conducted about the topic on the platform Infomed. Data were summarized to write the final report. We concluded that there are several adjuvant treatments to reduce recurrence, and it is necessary to carry out studies determining the time and frequency of their application to obtain more effective results(AU)

A functional interaction between the CCR5 and CD34 molecules expressed in hematopoietic cells can support (or even promote) the development of cancer

Abstract Inflammation and angiogenesis are linked to the development of cancer since both can support the establishment of a tumor-prone microenvironment. The CCR5 is a major regulatory molecule involved in inflammation. The CD34 molecule is commonly described as a hematopoietic stem cell marker, and CD34+ cells are involved in the regulation of distinct physiological processes, including angiogenesis. CCR5 participates in the development of various types of cancer, and recently, a reduced CCR5 expression was associated with low CD34+ cell counts in human cord blood. A naturally occurring genetic variant of the CCR5 gene, the so-called CCR5Δ32 polymorphism, consists of a 32 base-pair deletion in the DNA, interfering in the CCR5 protein levels on the cell surface. When in homozygosis, this variant leads to a total absence of CCR5 expression on the cell surface. In heterozygous individuals, CCR5 surface levels are reduced. Based on these key findings, we hypothesize that a functional interaction can connect CCR5 and CD34 molecules (giving rise to a "CCR5-CD34 axis"). According to this, a CCR5-CD34 interaction can potentially support the development of different types of cancer. Consequently, the lack of CCR5 in association with reduced CD34+ cell counts could indicate a protective factor against the development of cancer. It is required to characterize in detail the functional relationship between CCR5 and CD34 proteins, as well as the real influence of both molecules on the susceptibility and development of cancer at population level. If our hypothesis is confirmed, the CCR5-CD34 axis may be a potential target in the development of anti-cancer therapies.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Análise do efeito do tratamento com nanopartículas de prata (AGNO3 ) revestidas com pectina sobre a angiogênese induzida pelas células do Tumor de Ehrlich em modelo da membrana corioalantoidea / Analysis of the effect of treatment of silver nanoparticles (AgNO3 ) with pectin-coated on ehrlich tumor cell-induced angiogenes in the chorioalantoid membrane model

Avaliou-se a interferência das nanopartículas de prata sobre a angiogênese relacionada ao crescimento tumoral. A pesquisa científica foi realizada através da incubação de 42 ovos embrionados de galinhas. Após 24 horas de incubação, esses mesmos ovos foram separados em seis grupos contendo sete ovos cada, para os tratamentos com: Grupo 1: Soro fisiológico; Grupo 2: Tumor de Ehrlich (TE); Grupo 3: Nanopartículas de prata; Grupo 4: Prednisolona; Grupo 5: Nanopartículas de prata e Tumor de Ehrlich; Grupo 6: Prednisolona e Tumor de Ehrlich. Após o tempo total de incubação, as membranas corioalantoideas (MCAs) foram removidas, e analisadas através do microscópio de luz e fotografadas. O grupo 1 apresentou um padrão normal de crescimento e foi utilizado como controle negativo; O grupo 2 apresentou um aumento na quantidade de vasos sanguíneos; o grupo 3 apresentou baixa interferência na angiogenese embrionária e não contribuiu para o desenvolvimento do tumor; O grupo 4 demonstrou diminuição no desenvolvimento de vasos sanguíneos; O grupo 5 indicou que as nanopartículas de prata, quando associadas ao TE, não favorece o desenvolvimento tumoral e o grupo 6 demonstrou que o fármaco prednisolona associado ao TE, se comporta como um excelente inibidor de neoangiogese tumoral. Considera-se através da técnica realizada a possibilidade de utilizar nanopartículas de prata para o tratamento de células tumorais de Ehrlich, porém devem ser realizados testes confirmatórios para estudar a relação da substância descrita às células tumorais empregadas.

The interference of silver nanoparticles on angiogenesis related to tumor growth was evaluated. Scientific research was carried out by incubating 42 embryonated chicken eggs. After 24 hours of incubation, these same eggs were separated into six groups containing seven eggs each, for treatments with: Group 1: Saline; Group 2: Ehrlich's tumor (ET); Group 3: Silver nanoparticles; Group 4: Prednisolone; Group 5: Silver nanoparticles and Ehrlich's Tumor; Group 6: Prednisolone and Ehrlich's Tumor. After the total incubation time, the chorioallantoid membranes (MCAs) were removed, and analyzed using a light microscope and photographed. Group 1 showed a normal growth pattern and was used as a negative control; Group 2 showed an increase in the amount of blood vessels; group 3 showed low interference in embryonic angiogenesis and did not contribute to the development of the tumor; Group 4 demonstrated a decrease in the development of blood vessels; Group 5 indicated that silver nanoparticles, when associated with TE, do not favor tumor development and group 6 demonstrated that the drug prednisolone associated with TE, behaves as an excellent inhibitor of tumor neoangiogenesis. Through the technique performed, the possibility of using silver nanoparticles for the treatment of Ehrlich tumor cells is considered, however, confirmatory tests should be performed to study the relationship of the substance described to the tumor cells employed.

Terapia gênica de isquemia de membro é uma realidade? / Is gene therapy for limb ischemia a reality?

Resumo O conceito de terapia angiogênica surgiu no início da década de 90, o que pode ser feito com genes que codificam fatores de crescimento para promover a formação de novos vasos e o remodelamento de vasos colaterais. Como o procedimento dessa terapia geralmente consiste em apenas injeções locais de vetores, esse processo é pouco invasivo, rápido e de simples realização. Entretanto, desde as primeiras evidências clínicas do efeito de terapia gênica com o fator de crescimento de endotélio vascular (vascular endothelial growth factor, VEGF) vistos nos pacientes com doença arterial obstrutiva periférica até hoje, apenas dois fármacos de terapia angiogênica foram aprovados, um na Rússia e outro no Japão, o que parece um número muito pequeno diante do grande número de investimentos feitos por meio de estudos pré-clínicos e clínicos. Afinal, podemos considerar que a terapia angiogênica já é uma realidade?

Abstract The concept of angiogenic therapy emerged in the early 1990s. The method employs genes that encode growth factors to promote formation of new vessels and remodeling of collateral vessels. Since the procedure involved in this therapy usually only consists of local injections of vectors, the process is minimally invasive, quick, and simple to perform. However, since the first clinical evidence of the effects of gene therapy with vascular endothelial growth factor (VEGF) was observed in patients with peripheral artery disease, to date only two angiogenic therapy drugs have been approved, one in Russia and another in Japan, which seem a very small number, in view of the large volume of investment made in pre-clinical and clinical studies. After all, can we conclude that angiogenic therapy is a reality?

Identificação de Biomarcadores de Angiogênese no Eritema Nodoso Hansênico e na Hanseníase / Identification of Angiogenesis Biomarkers in Erythema Nodosum Leprosy and Leprosy

A hanseníase é uma doença infecciosa de evolução crônica, porém o curso natural da doença pode ser interrompido por episódios reacionais agudos que são caracterizados por uma ativação da resposta inflamatória acelerando o agravamento no dano tecidual e neural. O Eritema Nodoso Hansênico (ENH), um desses episódios reacionais, é uma complicação imunológica grave que acomete os pacientes multibacilares (BL e LL). A presença de neutrófilos na lesão de pele dos pacientes com ENH é considerada uma marca histológica dessa reação, porém ainda não é claro como ocorre o recrutamento desses neutrófilos para o sítio inflamatório e como este tipo celular participa dos mecanismos patológicos. Já foi visto na literatura que a angiogênese está presente de forma aumentada em lesões de pele de pacientes lepromatosos (LL) com e sem ENH sendo associada ao aumento da carga bacilar e progressão da doença. Embora o mecanismo de formação de novos vasos tenha sido bastante estudado na hipóxia tecidual e em outros processos inflamatórios, na hanseníase ainda não está bem descrito. Levando em consideração que a angiogênese está presente nos achados histopatológicos da hanseníase LL e no ENH, esse estudo propõe avaliar os níveis proteicos e a expressão gênica de VEGF-A, VEGFR1, PDGF, HIF1A e ITGB3 em amostras de sangue e pele de pacientes LL com e sem ENH e controles (pacientes BT e doadores sadios). 13 pacientes BT e 28 LL antes de iniciar o tratamento com a poliquimioterapia e 23 pacientes ENH antes do início do tratamento anti-reacional, 13 pacientes ENH 7 dias após o início do tratamento com talidomida e 3 doadores sadios foram recrutados no Ambulatório Souza Araújo, Rio de Janeiro.

Os níveis séricos de VEGF-A e PDGF-BB foram determinados por ELISA. A expressão proteica de VEGFR1 na superfície de neutrófilos circulantes foi determinada por citometria de fluxo multiparamética. A expressão de RNAm de VEGF-A, PDGF-A, HIF1A e ITGB3 na pele foram determinadas por RT-qPCR. Os níveis circulantes de VEGF-A em pacientes LL foram significativamente aumentados quando comparados com pacientes BT. Pacientes LL com e sem ENH apresentaram níveis séricos de VEGF-A comparáveis, porém 7 dias após o início do tratamento com talidomida houve redução. Neutrófilos circulantes de pacientes BT apresentaram maior expressão de VEGFR1 quando comparados com pacientes LL. Os neutrófilos de pacientes LL apresentaram níveis de VEGFR1 similares aos neutrófilos de doadores sadios. A expressão de VEGFR1 em neutrófilos assim com a frequência de neutrófilos VEGFR1+ foi menor em pacientes ENH quando comparados a todos os grupos de pacientes e doadores saudáveis. Os níveis de RNAm de VEGF-A foi significativamente maior na pele de pacientes ENH quando comparados aos pacientes BT e LL não reacionais. A expressão gênica de HIF1A foi maior nas lesões de pele de pacientes ENH quando comparados com LL e BT. Os níveis de PDGFA e ITGB3 nas lesões LL com e sem ENH foram comparáveis. Os nossos resultados sugerem que neutrófilos de pacientes ENH migrem para as lesões de pele através da via VEGF-A/VEGFR1 e que a angiogênese detectada nas lesões de pele de pacientes ENH induzem a expressão de HIF. (AU)

Mecanismo sensor y de adaptación a los niveles de oxígeno y su implicancia en las enfermedades cardiovasculares: a propósito del Premio Nobel de Fisiología-Medicina 2019 / Role of hypoxia-inducible factor-1 (HIF-1) in the adaptacion to oxygen levels: Nobel Prize in physiology-medicine 2019

RESUMEN: El Premio Nobel 2019 en Fisiología-Medicina se confirió a los Profesores Gregg Semenza, William Kaelin y Sir Peter Ratcliffe por sus investigaciones en la maquinaria molecular que regula la expresión de genes sensibles a los cambios en los niveles de oxígeno. La síntesis de eritropoyetina inducida por la disminución de los niveles sanguíneos de oxígeno condujo al estudio del gen de la eritropoyetina y descubrimiento de los elementos de respuesta a hipoxia (HRE) en la región promotora y posteriormente al factor transcripcional inducible por hipoxia tipo 1 (HIF-1). Este factor consta de dos subunidades: HIF-1α, sensible al oxígeno, y HIF-1β, expresada constitutivamente. HIF1 activa la transcripción de genes que codifican enzimas, transportadores y proteínas mitocondriales que disminuyen la utilización de oxígeno al cambiar el metabolismo oxidativo al metabolismo glicolítico y además aquellos involucrados en la angiogénesis y diferenciación celular. Las investigaciones paralelas en la enfermedad von Hippel-Lindau (VHL), un desorden autosómico dominante, permitieron descubrir el mecanismo de degradación de HIF1 en condiciones de normoxia y como se estabiliza bajo hipoxia. El impacto de HIF en clínica radica en el establecimiento de nuevas dianas terapéuticas para combatir la anemia y diversas enfermedades cardiovasculares. HIF promueve la angiogénesis a través de la expresión del factor de crecimiento vascular endotelial (VEGF), agente cardioprotector con potencial para tratar la isquemia/reperfusión, hipertrofia patológica e insuficiencia cardíaca.

ABSTRACT: The Nobel Prize in Physiology-Medicine was awarded to Drs. Gregg Semenza, William Kaelin and Sir Peter Ratcliffe for their research in the molecular machinery that regulates the expression of genes sensitive to the change in oxygen levels. The synthesis of erythropoietin induced by the decrease levels of oxygen in the blood led to investigate the promoter of the erythropoietin gene where the so-called hypoxia response elements (HRE) were described. Semenza et al. described a protein that binds to HREs and called it hypoxia-inducible transcriptional factor (HIF) that regulates gene expression among those involved in angiogenesis, cell differentiation and glycolytic enzymes. HIF presents two oxygen-sensitive subunits HIF-1α and HIF-1β constitutively expressed. In parallel, Kaelin et al. investigated von Hippel-Lindau disease (VHL), an autosomal dominant disorder, discovering a mutation of this protein generated a behavior similar to hypoxia. The impact of HIF-1α lies in the search for new strategies such as hydrolase inhibitors to combat prevalent diseases, including anemia and cardiovascular diseases These compounds promote the expression of vascular endothelial growth factor (VEGF), a cardioprotective agent with potential use in pre- and post-conditioning therapy, cardiac hypertrophy and heart failure.

Effects of benzalkonium chloride and cyclosporine applied topically to rabbit conjunctiva: a histomorphometric study / Efeitos do uso tópico do conservante cloreto de benzalcônio e ciclosporina na conjuntiva de coelhos: estudo histomorfométrico

ABSTRACT Purpose: Chronic instillation of benzalkonium chloride, a preservative, has inflammatory effects on the ocular surface. However, addition of the anti-inflammatory agent cyclosporine to a therapeutic protocol may mitigate these effects. This study compared the toxic effects of a 0.1% benzalkonium chloride solution and the possible protective effect of 0.05% cyclosporine when applied topically to the rabbit conjunctiva. Methods: Fifteen age- and weight-matched, female New Zealand white rabbits were categorized into three groups and treated for 30 consecutive days. Group 1, 2, and 3 - benzalkonium chloride received 0.1% every 24 h, 0.05% cyclosporine every 6 h, and both treatments, respectively. In each rabbit, the left eye was subjected to treatment and the right eye was a control. The rabbits were euthanized at after the experiment. Goblet cells and blood vessels were then enumerated in conjunctival tissues stained with periodic acid-Schiff and hematoxylin-eosin, respectively. Differences between treated and untreated eyes and between groups were compared using the t-test and analysis of variance. Results: Benzalkonium chloride treatment, with and without cyclosporine, significantly reduced (p≤0.05) in the number of goblet cells in treatment eyes compared with that in respective control eyes. Alternatively, adding cyclosporine to benzalkonium chloride did not prevent the loss of conjunctival goblet cells, and a significant reduction in the number of goblet cells was noted. Benzalkonium chloride-induced significant increase in the number of new blood vessels was mitigated significantly by the addition of cyclosporine. Conclusion: This study demonstrated the magnitude of conjunctival injury caused by chronic instillation of benzalkonium chloride. Although cyclosporine did not mitigate the effects on goblet cells, its addition minimized inflammatory angiogenesis induced by benzalkonium chloride.

RESUMO Objetivo: A instilação crônica de cloreto de benzal­cônio, um conservante, tem efeitos inflamatórios na superfície ocular. No entanto, a adição do agente anti-inflamatório ciclosporina a um protocolo terapêutico pode atenuar esses efeitos. Este estudo comparou os efeitos tóxicos de uma solução de cloreto de benzalcônio a 0,1% e o possível efeito protetor de ciclosporina a 0,05% quando aplicado topicamente à conjuntiva de coelho. Métodos: Quinze coelhos fêmeas brancos da raça Nova Zelândia, pareados por idade e peso, foram categorizados em três grupos e tratados por 30 dias consecutivos. Os grupos 1, 2 e 3 - receberam cloreto de benzalcônio 0,1% a cada 24h, ciclosporina a 0,005% a cada 6h e ambos os tratamentos, respectivamente. Em cada coelho, o olho esquerdo foi submetido a tratamento e o olho direito foi controle. Os coelhos foram submetidos à eutanásia após o experimento. Células caliciformes e vasos sanguíneos foram então enumerados em tecidos conjuntivais corados com ácido periódico-Schiff e hematoxilina-eosina, respectivamente. As diferenças entre os olhos tratados e não tratados e entre os grupos foram comparadas usando o teste t e análise de variância. Resultados: O tratamento com cloreto de benzalcônio, com e sem ciclosporina, reduziu significativamente (p£0,05) o número de células caliciformes nos olhos tratados em comparação com os olhos controle correspondentes. Alternativamente, a adição de ciclosporina ao cloreto de benzalcônio não impediu a perda de células caliciformes conjuntivais, e foi observada uma redução significativa no número de células caliciformes. O aumento significativo induzido pelo cloreto de benzalcônio no número de novos vasos sanguíneos foi significativamente mitigado pela adição da ciclosporina. Conclusão: Este estudo demonstrou a magnitude da lesão conjuntival resultante da instilação crônica de cloreto de benzalcônio. Embora a ciclosporina não tenha atenuado os efeitos nas células caliciformes, sua adição minimizou a angiogênese inflamatória induzida pelo cloreto de benzalcônio.

Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways in vivo

Abstract Purpose To explore the potential role and unclear molecular mechanisms of vaccarin in wound healing. Methods Rats' skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels. Results Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group. Conclusions The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

TNF-alpha increases angiogenic potential in a co-culture system of dental pulp cells and endothelial cells

Abstract We recently demonstrated that a co-culture system of human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs) could enhance angiogenesis ability in vitro. However, whether tumor necrosis factor α (TNF-α) could promote blood vessel formation during pulp regeneration remained unknown. The aim of this study was to investigate the effects of TNF-α on the formation of endothelial tubules and vascular networks in a co-culture system of hDPSCs and HUVECs. hDPSCs were co-cultured with HUVECs at a ratio of 1:5. The Matrigel assay was performed to detect the total tubule branching lengths and numbers of branches, and the Cell-Counting Kit 8 assay was performed to examine the effect of TNF-α on cell proliferation. Real-time polymerase chain reactions and western blot were used to detect vascular endothelial growth factor (VEGF) mRNA and protein expression. The Matrigel assay showed significantly greater total branching lengths and numbers of branches formed in the experimental groups treated with different concentrations of TNF-α compared with the control group. The decomposition times of the tubule structures were also significantly prolonged (P < 0.05). Treatment with 50 ng/ml TNF-α did not significantly change the proliferation of co-cultured cells, but it significantly increased the VEGF mRNA and protein expression levels (p < 0.05). In addition, the migration abilities of HUVECs and hDPSCs increased after co-culture with TNF-α (p < 0.05). TNF-α enhanced angiogenic ability in vitro in the co-culture system of hDPSCs and HUVECs.

Yiqi Tongluo Particles improve Qi-deficiency and blood stasis in multiple cerebral infarction rats by promoting angiogenesis / 中国中药杂志

This research was aimed to evaluate the protective effect and potential mechanism of Yiqi Tongluo Particles(YQTLs).Firstly,an animal model of multiple cerebral infarction(MCI) with Qi deficiency and blood stasis was established.Rats were randomly divided into six groups:SHAM group,Vehicle group,Buyang Huanwu decoction original group(BYHWO),EGb761 group,high and low dose of YQTLs group.Rats underwent sleep deprivation after one week of MCI and the tongues and pulses of rats after six weeks of sleep deprivation were detected,followed by collecting blood to analysis the blood coagulation.Differential expression of angiogenesis associated proteins was examined using proteomic research and verified by immunohistochemical.RESULTS: showed that neurological function score was obviously declined,G and B value of tongue surface was increased significantly and the pulse distension,the activated partial thromboplatin time(APTT) as well as prothrombin time(PT) were recovered following YQTLs 7.56 g·kg-1 treatment.Furthermore,G value of tongue surface,APTT and PT were also improved by YQTLs 3.78 g·kg-1.The results of proteomic technology showed that proteins associated with angiogenesis were reversed compared with Vehicle group.Moreover,the expression of VEGFR2 from immunohistochemical was promoted after YQTLs treatment.The MCI with Qi deficiency and blood stasis was alleviated obviously following YQTLs treatment and the possible mechanism was that YQTLs may enhance angiogenesis during cerebral ischemia.

Induction of Angiogenesis by Malarial Infection through Hypoxia Dependent Manner

Malarial infection induces tissue hypoxia in the host through destruction of red blood cells. Tissue hypoxia in malarial infection may increase the activity of HIF1α through an intracellular oxygen-sensing pathway. Activation of HIF1α may also induce vascular endothelial growth factor (VEGF) to trigger angiogenesis. To investigate whether malarial infection actually generates hypoxia-induced angiogenesis, we analyzed severity of hypoxia, the expression of hypoxia-related angiogenic factors, and numbers of blood vessels in various tissues infected with Plasmodium berghei. Infection in mice was performed by intraperitoneal injection of 2×10⁶ parasitized red blood cells. After infection, we studied parasitemia and survival. We analyzed hypoxia, numbers of blood vessels, and expression of hypoxia-related angiogenic factors including VEGF and HIF1α. We used Western blot, immunofluorescence, and immunohistochemistry to analyze various tissues from Plasmodium berghei-infected mice. In malaria-infected mice, parasitemia was increased over the duration of infection and directly associated with mortality rate. Expression of VEGF and HIF1α increased with the parasitemia in various tissues. Additionally, numbers of blood vessels significantly increased in each tissue type of the malaria-infected group compared to the uninfected control group. These results suggest that malarial infection in mice activates hypoxia-induced angiogenesis by stimulation of HIF1α and VEGF in various tissues.

Chronic Cerebral Hypoperfusion Induces Alterations of Matrix Metalloproteinase-9 and Angiopoietin-2 Levels in the Rat Hippocampus

Angiogenic factors contribute to cerebral angiogenesis following cerebral hypoperfusion, and understanding these temporal changes is essential to developing effective treatments. The present study examined temporal alterations in angiogenesis-related matrix metalloproteinase-9 (MMP-9) and angiopoietin-2 (ANG-2) expression in the hippocampus following bilateral common carotid artery occlusion (BCCAo). Male Wistar rats (12 weeks of age) were randomly assigned to sham-operated control or experimental groups, and expression levels of MMP-9 and ANG-2 were assessed after BCCAo (1 week, 4 weeks, and 8 weeks), using western blotting. Protein expression increased 1 week after BCCAo and returned to control levels at 4 and 8 weeks. In addition, immunofluorescence staining demonstrated that the MMP-9- and ANG-2-positive signals were primarily observed in the NeuN-positive neurons with very little labeling in non-neuronal cells and no labeling in endothelial cells. In addition, these cellular locations of MMP-9- and ANG-2-positive signals were not altered over time following BCCAo. Other angiogenic factors such as vascular endothelial growth factor and hypoxia-inducible factor did not differ from controls at 1 week; however, expression of both factors increased at 4 and 8 weeks in the BCCAo group compared to the control group. Our findings increase understanding of alterations in angiogenic factors during the progression of cerebral angiogenesis and are relevant to developing effective temporally based therapeutic strategies for chronic cerebral hypoperfusion-associated neurological disorders such as vascular dementia.

Fabrication of Microchannels and Evaluation of Guided Vascularization in Biomimetic Hydrogels

BACKGROUND: The fabrication of microchannels in hydrogel can facilitate the perfusion of nutrients and oxygen, which leads to guidance cues for vasculogenesis. Microchannel patterning in biomimetic hydrogels is a challenging issue for tissue regeneration because of the inherent low formability of hydrogels in a complex configuration. We fabricated microchannels using wire network molding and immobilized the angiogenic factors in the hydrogel and evaluated the vasculogenesis in vitro and in vivo. METHODS: Microchannels were fabricated in a hyaluronic acid-based biomimetic hydrogel by using “wire network molding” technology. Substance P was immobilized in acrylated hyaluronic acid for angiogenic cues using Michael type addition reaction. In vitro and in vivo angiogenic activities of hydrogel with microchannels were evaluated. RESULTS: In vitro cell culture experiment shows that cell viability in two experimental biomimetic hydrogels (with microchannels and microchannels + SP) was higher than that of a biomimetic hydrogel without microchannels (bulk group). Evaluation on differentiation of human mesenchymal stem cells (hMSCs) in biomimetic hydrogels with fabricated microchannels shows that the differentiation of hMSC into endothelial cells was significantly increased compared with that of the bulk group. In vivo angiogenesis analysis shows that thin blood vessels of approximately 25–30 µm in diameter were observed in the microchannel group and microchannel + SP group, whereas not seen in the bulk group. CONCLUSION: The strategy of fabricating microchannels in a biomimetic hydrogel and simultaneously providing a chemical cue for angiogenesis is a promising formula for large-scale tissue regeneration.

Comparison of Angiogenic Activities of Three Neuropeptides, Substance P, Secretoneurin, and Neuropeptide Y Using Myocardial Infarction

BACKGROUND: The interplay between neurogenesis and angiogenesis is crucial during the development mediated by neuro-angiogenic morphogens. In particular, the angiogenic activity of neuropeptides and their role in tissue regeneration have long been investigated for better understanding of their biological mechanisms and further applications. However, there have been few studies for direct comparison of angiogenic activities of neuropeptides for in vitro and in vivo models. In this study, we report that direct comparison of the angiogenic activities of neuropeptide Y, secretoneurin, and substance P (SP) immobilized on hydrogels in in vitro and in vivo experiments. METHODS: A hyaluronic acid-based hydrogel is prepared by utilizing acrylated hyaluronic acid and thiolated peptides as a crosslinker and angiogenic factors, respectively. Angiogenic activities of three neuropeptides are evaluated not only by in vitro angiogenic and gene expression assays, but also by an in vivo chronic myocardial infarction model. RESULTS: The comparison of in vitro angiogenic activities of three peptides demonstrates that the SP-immobilized hydrogel shows a higher degree of cell network formation and angiogenic-specific genes than those of the other peptides and the control case. In addition, a three-dimensional angiogenic assay illustrates that more sprouting is observable in the SP group. Evaluation of regenerative activity in the chronic myocardial infarction model reveals that all three peptideimmobilized hydrogels induce increased cardiac function as well as structural regeneration. Among all the cases, the SP group provided the highest regenerative activity both in vitro and in vivo. CONCLUSION: In our comparison study, the SP-immobilized hydrogel shows the highest angiogenic activity and tissue regeneration among the test groups. This result suggests that nerve regeneration factors help angiogenesis in damaged tissues, which also highlights the importance of the neuro-angiogenic peptides as an element of tissue regeneration.

Aberrant Promoter Hypomethylation of Sortilin 1: A Moyamoya Disease Biomarker / 대한뇌졸중학회지

BACKGROUND AND PURPOSE: The pathogenesis of moyamoya disease (MMD) remains poorly understood, and no reliable molecular biomarkers for MMD have been identified to date. The present study aimed to identify epigenetic biomarkers for use in the diagnosis of MMD. METHODS: We performed integrated analyses of gene expression profiles and DNA methylation profiles in endothelial colony forming cells (ECFCs) from three patients with MMD and two healthy individuals. Candidate gene mRNA expression and DNA methylation status were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and pyrosequencing analysis of an expanded ECFC sample set from nine patients with MMD and ten controls. We evaluated the diagnostic accuracy of the potential biomarkers identified here using receiver operating characteristic curve analyses and further measured major angiogenic factor expression levels using a tube formation assay and RT-qPCR. RESULTS: Five candidate genes were selected via integrated analysis; all five were upregulated by hypomethylation of specific promoter CpG sites. After further validation in an expanded sample set, we identified a candidate biomarker gene, sortilin 1 (SORT1). DNA methylation status at a specific SORT1 promoter CpG site in ECFCs readily distinguished patients with MMD from the normal controls with high accuracy (area under the curve 0.98, sensitivity 83.33%, specificity 100%). Furthermore, SORT1 overexpression suppressed endothelial cell tube formation and modulated major angiogenic factor and matrix metalloproteinase-9 expression, implying SORT1 involvement in MMD pathogenesis. CONCLUSIONS: Our findings suggest that DNA methylation status at the SORT1 promoter CpG site may be a potential biomarker for MMD.

Estudo da imunoexpressão do VEGF165 e do VEGF165B em lesões orais de líquen plano e pênfigo vulgar / Study of VEGF165 and VEGF165B immunoexpression in oral lesions of lichen planus and pemphigus vulgaris

O Líquen Plano Oral (LPO) é uma doença mucocutânea mediada imunologicamente, de etiologia desconhecida, relativamente comum, com prevalências, na população mundial, que variam de 0,22 a 5%. O pênfigo vulgar é uma doença autoimue crônica que pode acometer a mucosa oral sendo o mais comum dos tipos de pênfigo. Entretanto, sua ocorrência é rara, com incidência estimada na população geral de um a cinco casos por milhão de pessoas diagnosticadas a cada ano. O VEGF-A é a proteína angiogênica mais potente tanto na angiogênese normal quanto na patológica. O splicing alternativo do éxon 8 do gene do VEGFA dá origem a duas famílias conhecidas de proteínas isofórmicas, uma desempenhando papel angiogênico, VEGFxxxa, e outra um papel antiangiogênico, VEGFxxxb. Este trabalho se propôs a avaliar a expressão imunoistoquímica do VEGF165 (angiogênico), do VEGF165b (antiangiogênico) em 46 casos de LPO reticular, 23 casos de LPO erosivo e 12 casos de PV, usando como controle 11 casos de hiperplasia fibrosa. Todos os espécimes das lesões e os casos controle foram divididos em e zonas para a análise das marcações, em zona superficial (Z1), média (Z2) e profunda (Z3). Os resultados deste experimento foram submetidos a testes estatístico não-paramétricos com nível de significância de 5%. Comparando apenas as lesões para o marcador anti-VEGF165 foram observadas diferenças significativas apenas nas zonas mais profundas entre as lesões de LPO reticular e PV, e entre as lesões de LPO erosivo e PV. Para o marcador anti-VEGF165b diferenças significativas foram observadas nas zonas médias entre as lesões de LPO reticular e PV; e nas zonas profundas entre LPO erosivo e PV e entre LPO reticular e PV. Avaliando o marcador VEGF165b nos espécimes sem categorizá-los por zonas foram observadas diferenças significativas entre as lesões de LPO reticular e PV. Na análise da correlação entre ambos os marcadores em cada lesão foram observadas correlação positiva fraca e significativa nas zonas média e profunda do LPO reticular e na zona superficial do LPO erosivo. Os resultados do presente estudo sugerem a participação do processo angiogênico na patogênese do LPO e na progressão das lesões de líquen plano oral e pênfigo vulgar, porém outros estudos devem ser realizados a fim de que esses achados, principalmente em relação ao pênfigo vulgar seja fundamentado, uma vez que a presente pesquisa é uma das primeiras que avalia a angiogênese na lesão já estabelecida dessa doença (AU).

Oral Lichen Planus is an immunologically mediated mucocutaneous disease of relatively unknown etiology with prevalences in the world population varying from 0.22 to 5%. Pemphigus vulgaris is a chronic autoimmune disease that may affect the oral mucosa being the most common type of pemphigus. However, its occurrence is rare, with an estimated incidence in the general population of one to five cases per million people diagnosed each year. Angiogenesis plays an important role in tumor growth and in the progression of chronic inflammatory diseases. VEGF-A is the most potent angiogenic protein in both normal and pathological angiogenesis. The alternative splicing of exon 8 VEGF-A gene gives rise to two known families of isoform proteins, one playing angiogenic role, VEGFxxxa, and another an antiangiogenic role, VEGFxxxb. The aim of this study was to evaluate the immunohistochemical expression of VEGF165 (angiogenic), VEGF165b (antiangiogenic) in 46 cases of reticular OLP, 23 cases of erosive OLP and 12 cases of PV, using as control 11 cases of fibrous hyperplasia. All specimens of the lesions and the control cases were divided into zones for the analysis of the immunohistochemical stains, in superficial (Z1), medium (Z2) and deep zones (Z3). The results of this experiment were submitted to non-parametric statistical tests with significance level of 5%. For all immunohistochemical stains the comparison between the lesions with the control group (HF) showed significant differences. Comparing only the lesions to the anti-VEGF165 stains, significant differences were observed only in the deeper zones between the reticular LPO lesions X PV; and between erosive LPO lesions X PV. For the anti-VEGF165b stains, significant differences were observed in medium zones between reticular OLP X PV lesions; and in deep zones between erosive LPO X PV and between reticular LPO and PV. And evaluating VEGF165b stains in specimens without categorizing them by zones was observed a significant difference between reticular LPO and PV lesions. In the analysis of the correlation between both markers in each lesion, a weak and significant positive correlation was observed in medium and deep zones of reticular OLP; and a weak positive correlation in superficial zone of erosive LPO. The present study results suggest angiogenic process participation in the pathogenesis and progression of lesions of oral lichen planus and pemphigus vulgaris. However other studies must be carried out in order that this implication, mainly in relation to pemphigus vulgaris be based once this is one of the first studies to evaluate angiogenesis in the already established lesion of this disease (AU).