Expression of interleukin-37, vascular endothelial growth factor A, and transforming growth factor-β1 and their correlation with T cells in children with primary immune thrombocytopenia / 中国当代儿科杂志

OBJECTIVES@#To investigate the expression of interleukin-37 (IL-37), vascular endothelial growth factor A (VEGFA), and transforming growth factor-β1 (TGF-β1) in children with primary immune thrombocytopenia (ITP) and their correlation with T cells.@*METHODS@#A retrospective analysis was conducted on 45 children with ITP (ITP group) who were admitted to Handan Central Hospital from January 2020 to April 2022, and 30 healthy children who underwent physical examination during the same period were included as the healthy control group. The mRNA expression levels of IL-37, VEGFA, and TGF-β1 and the levels of regulatory T cells (Treg) and helper T cells 17 (Th17) were measured before and after treatment, and the correlation between the mRNA expression levels of IL-37, VEGFA, and TGF-β1 and the levels of Treg, Th17, and Treg/Th17 ratio were analyzed.@*RESULTS@#Compared with the healthy control group, the ITP group had a significantly higher mRNA expression level of IL-37 and a significantly higher level of Th17 before and after treatment, as well as significantly lower mRNA expression levels of VEGFA and TGF-β1 and significantly lower levels of Treg and Treg/Th17 ratio (P<0.05). After treatment, the ITP group had significant reductions in the mRNA expression level of IL-37 and the level of Th17 and significant increases in the mRNA expression levels of VEGFA and TGF-β1 and the levels of Treg and Treg/Th17 ratio (P<0.05). Correlation analysis showed that in the ITP group, the mRNA expression levels of IL-37 and TGF-β1 were negatively correlated with the levels of Treg and Treg/Th17 ratio (P<0.05) and were positively correlated with the level of Th17 (P<0.05) before and after treatment; the mRNA expression level of VEGFA was positively correlated with the levels of Treg and Treg/Th17 ratio (P<0.05) and was negatively correlated with the Th17 level (P<0.05) before and after treatment.@*CONCLUSIONS@#Abnormal expression levels of IL-37, VEGFA, and TGF-β1 may be observed in children with ITP, which is significantly associated with the imbalance of Treg/Th17 ratio. It is speculated that the cytokines such as IL-37, VEGFA, and TGF-β1 may be involved in the development and progression of ITP or may become important potential targets for the treatment of children with ITP. Citation:Chinese Journal of Contemporary Pediatrics, 2023, 25(11): 1131-1136.

Bioinformatics analysis and identification to immune-related markers of osteoporosis / 细胞与分子免疫学杂志

Objective To identify immune-related dysregulation mechanisms and potential diagnostic predictive biomarkers in osteoporosis. Methods Gene expression data for both osteoporosis and control populations were retrieved from the GSE35958 and GSE56815 datasets. Immune-related differentially expressed genes (DEGs) were obtained by screening DEGs and were compared with the immunology database and analysis portal (ImmPort) database. Enrichment analysis of these immune-related DEGs was conducted using the Clusterprofiler software package. A protein-protein interaction network was built with the STRING database, which is a search tool for finding interacting genes/proteins, and the top 10 genes with the highest network connectivity were identified as candidate genes. Subsequently, the diagnostic predictive effect of candidate genes was evaluated using receiver operating characteristic (ROC) curves, logistic regression, and column plots. Finally, PCR and Western blot analysis were applied to detect the differential expression of these genes in bone marrow tissue of patients with osteoporosis. Results A total of 138 immune-related DEGs were obtained through intersection analysis. The results of the enrichment analysis indicated that these genes were involved in biological functions such as immune inflammation and signaling pathways including T cell receptors, mitogen activated protein kinase (MAPK), rat sarcoma virus oncogene homologs (Ras), osteoclast differentiation, and B cell receptors. In addition, among the candidate genes, upregulated vascular endothelial growth factor A (VEGFA) and epidermal growth factor receptor (EGFR) and downregulated AKT1, SRC, and JUN in osteoporosis showed the highest connectivity. Among them, VEGFA, EGFR, JUN, and AKT1 demonstrated the best diagnostic predictive value. Conclusion The screening of immune-related DEGs will enhance the understanding of osteoporosis and facilitate the development of immunotherapy targets.

Protective effect of Liujing Toutong Tablets on rats with permanent cerebral ischemia via NF-κB signaling pathway / 中国中药杂志

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1β, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(β-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1β, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and β-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1β, IL-6, CGRP, and NO in rat serum, increased VEGF and β-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and β-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1β. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing β-EP levels.

RGD peptide inhibits tumor growth by affecting angiogenesis related signaling pathway of laryngeal cancer stem cells / El péptido RGD inhibe el crecimiento tumoral al afectar la vía de señalización relacionada con la angiogénesis de las células madre del cáncer de laringe

SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.

Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.

Expresión génica de mediadores moleculares para potencial terapia celular en el síndrome gastrointestinal inducido por radiación / Genetic expression of molecular mediators for potential cell therapy in radiation-induced gastrointestinal syndrome

Antecedentes: Las células madres intestinales generan las distintas estirpes celulares a dicho nivel. Estas se regulan por interacciones entre el epitelio y las células del nicho celular anexo. Estas se pueden ver dañadas en tratamientos con radiación, generando el síndrome gastrointestinal inducido por radiación. Se ha visto que células madre mesenquimales (MSC) y macrófagos de médula ósea (BMM) tienen propiedades de regeneración tisular. Objetivos: Evaluar la expresión génica de IL-4, Wnt6, VEGF y bFGF, a partir de cultivos celulares primarios independientes de MSC derivadas de tejido adiposo y BMM de ratones C57BL/6, por medio de PCR en tiempo real (qRT-PCR). Diseño experimental: A partir de un análisis in silico, se confeccionaron primers para evaluar la expresión génica de las moléculas propuestas, en los cultivos primarios por medio de qRT-PCR y electroforesis. Resultados y proyecciones: IL-4 y Wnt6 no son expresadas en las muestras de BMM y MSC. VEGF y bFGF son expresadas por diferentes células, dando expresión diferenciada. A futuro, se deben evaluar las mismas estirpes celulares en un ambiente inflamatorio y su efecto en la expresión génica, en especial VEGF y bFGF. Limitaciones: El número de moléculas en estudio es limitado y la expresión se evalúo solo a nivel genético.

Background: Intestinal stem cell generates diferents cellular types in their niche. They're regulated by interactions between epithelium and niche's cells, and can be damaged by medical radiation treatments causing radiation-induced gastrointestinal syndrome. It has seen that mesenchymal stem cells (MSC) d and bone marrow-derived macrophages (BMM) have propierties of tissular regeneration. Objectives: Determinated genetic expression of IL-4, Wnt6, VEGF and bFGF, in primary cellular cultures of MSC derivated of adipose tissue and BMM of C57BL/6 mice, through real time PCR (qRT-PCR). Methods: By an in silico analysis, we created primers to evaluate the proposed molecules in the primary cellular cultives, with qRT-PCR and electrophoresis. Results and projections: IL-4 and Wnt6 were not expressed in the MSC and BMM samples. VEGF and bFGF were expressed by different cells, giving differential expression. In the future, the same samples should be analyzed in an inflammatory environment, especially VEGF and bFGF. Limitations: The number of molecules are limited and the expression of them is only in a genetic level.

Protective effect of ginsenoside Rg_1 aganist diabetic retinopathy by inhibiting NLRP3 inflammasome in type 2 diabetic mice / 中国中药杂志

Ginsenoside Rg_1, one of the main active components of precious traditional Chinese medicine Ginseng Radix et Rhizoma, has the anti-oxidative stress, anti-inflammation, anti-aging, neuroprotection, and other pharmacological effects. Diabetic retinopathy(DR), the most common complication of diabetes, is also the main cause of impaired vision and blindness in the middle-aged and the elderly. The latest research shows that ginsenoside Rg_1 can protect patients against DR, but the protection and the mechanism are rarely studied. This study mainly explored the protective effect of ginsenoside Rg_1 against DR in type 2 diabetic mice and the mechanism. High fat diet(HFD) and streptozotocin(STZ) were used to induce type 2 diabetes in mice, and hematoxylin-eosin(HE) staining was employed to observe pathological changes in the retina of mice. The immunohistochemistry was applied to study the localization and expression of nucleotide-binding oligomerization domain-like receptors 3(NLRP3) and vascular endothelial growth factor(VEGF) in retina, and Western blot was used to detect the expression of nuclear factor-kappa B(NF-κB), p-NF-κB, NLRP3, caspase-1, interleukin-1β(IL-1β), transient receptor potential channel protein 6(TRPC6), nuclear factor of activated T-cell 2(NFAT2), and VEGF in retina. The results showed that ginsenoside Rg_1 significantly alleviated the pathological injury of retina in type 2 diabetic mice. Immunohistochemistry results demonstrated that ginsenoside Rg_1 significantly decreased the expression of NLRP3 and VEGF in retinal ganglion cells, middle plexiform layer, and outer plexiform layer in type 2 diabetic mice. According to the Western blot results, ginsenoside Rg_1 significantly lowered the expression of p-NF-κB, NLRP3, caspase-1, IL-1β, TRPC6, NFAT2, and VEGF in retina of type 2 diabetic mice. These findings suggest that ginsenoside Rg_1 can significantly alleviate DR in type 2 diabetic mice, which may be related to inhibition of NLRP3 inflammasome and VEGF. This study provides experimental evidence for the clinical application of ginsenoside Rg_1 in the treatment of DR.

Vascular endothelial growth factor induces inflammatory injury of pancreatic tissue by activating autophagy in hyperlipidemic acute pancreatitis rats / 生理学报

This study was to investigate the changes of autophagy in pancreatic tissue cells from hyperlipidemic acute pancreatitis (HLAP) rats and the molecular mechanism of autophagy to induce inflammatory injury in pancreatic tissue cells. Male Sprague Dawley (SD) rats were intraperitoneally injected with caerulein to establish acute pancreatitis (AP) model and then given a high fat diet to further prepare HLAP model. The HLAP rats were treated with autophagy inducer rapamycin or inhibitor 3-methyladenine. Pancreatic acinar (AR42J) cells were treated with caerulein to establish HLAP cell model. The HLAP cell model were treated with rapamycin or transfected with vascular endothelial growth factor (VEGF) siRNA. The inflammatory factors in serum and cell culture supernatant were detected by ELISA method. The histopathological changes of pancreatic tissue were observed by HE staining. The changes of ultrastructure and autophagy in pancreatic tissue were observed by electron microscopy. The expression levels of Beclin-1, microtubule- associated protein light chain 3-II (LC3-II), mammalian target of rapamycin complex 1 (mTORC1), and VEGF were measured by immunohistochemistry and Western blot. The results showed that, compared with control group, the autophagy levels and inflammatory injury of pancreatic tissue cells from HLAP model rats were obviously increased, and these changes were aggravated by rapamycin treatment, but alleviated by 3-methyladenine treatment. In HLAP cell model, rapamycin aggravated the autophagy levels and inflammatory injury, whereas VEGF siRNA transfection increased mTORC1 protein expression, thus alleviating the autophagy and inflammatory injury of HLAP cell model. These results suggest that VEGF-induced autophagy plays a key role in HLAP pancreatic tissue cell injury, and interference with VEGF-mTORC1 pathway can reduce the autophagy levels and alleviate the inflammatory injury. The present study provides a new target for prevention and treatment of HLAP.

Genetic markers for preeclampsia in Peruvian women / Marcadores genéticos de preeclampsia en mujeres peruanas

Abstract Background: Preeclampsia is a multiorgan disorder associated with maternal and perinatal morbi-mortality. In Peru, incidence is 10% and accounts for 22% of maternal deaths. Genome and genetic epidemiological studies have found an association between preeclampsia and genetic polymorphisms. Objective: To determine the association of the vascular endothelial growth factor (VEGF) +936 C/T and +405 G/C, interleukine-6 (IL-6) -174 G/C, IL-1β-511 C/T, Apo A-1-75 G/A, Apo B-100 2488 C/T (Xbal) polymorphisms with preeclampsia in pregnant Peruvian women. Methods: Were included preeclamptic and healthy (control) pregnant women. Maternal blood samples were subjected to DNA extraction, and molecular genetic analysis was conducted using the PCR-RFLP technique and following a specific protocol for each gene. Allele and genotypic frequencies in the cases and controls were compared. Results: No association was found between the VEGF+936C/T and VEGF+405 polymorphisms and preeclampsia. The frequencies of the GG genotypes and the G allele of the -174 G/C polymorphism in the IL6 gene in preeclamptic and controls showed significant differences, with higher frequencies in cases. For the -511 C/T polymorphism of the IL-1β gene, no significant differences were found in the frequencies of TT genotypes compared with CT+CC. The genotypes and alleles of the Apo-A1-75 G/A and Apo-B100 Xbal variants showed no significant differences between cases and controls. Conclusion: No association was found between the studied genetic markers and preeclampsia. However, in the -174G/C polymorphism of the IL-6 gene, significant differences were found mainly in the GG genotype and G allele.

Resumen Antecedentes: La preeclampsia es un trastorno multiorgánico asociado con la morbi-mortalidad materna y perinatal. En el Perú, su incidencia es del 10% y causa el 22% de las muertes maternas. Se encontró una asociación entre la preeclampsia y ciertos polimorfismos. Objetivo: Determinar asociación entre los polimorfismos genéticos del factor de crecimiento endotelial vascular (VEGF) +936 C/T y +405 G/C, interleucina-6 (IL-6) -174G/C, IL-1β -511 C/T, Apo A-1 -75 G/A, Apo B-100 2488 C/T (Xbal), y preeclampsia en gestantes peruanas. Métodos: Se incluyeron gestantes preeclámpticas y sanas (controles). Las muestras de sangre fueron procesadas para extracción del ADN, y el análisis se realizó con la técnica PCR-RFLP con protocolos específicos para cada gen y confirmación con secuenciamiento Sanger. Se compararon las frecuencias alélicas y genotípicas en los casos (preeclampsia) y los controles. Resultados: No se halló asociación entre los polimorfismos VEGF+936-C/T y VEGF+405 y la preeclampsia. Las frecuencias de los genotipos GG y el alelo G del polimorfismo -174-G/C en el gen IL6 en preeclámpticas y controles, mostraron diferencias significativas, con frecuencias más altas en los casos. Para el polimorfismo -511-C/T del gen IL-1β, no se encontraron diferencias significativas en las frecuencias de genotipos TT comparados con CT+CC. Los genotipos y alelos de las variantes Apo-A1-75-G/A y Apo-B100 Xbal no mostraron diferencias significativas entre los grupos Conclusión: No se encontró asociación entre los marcadores genéticos estudiados y la preeclampsia. Sin embargo, el polimorfismo -174-G/C en el gen IL6 mostró diferencias significativas principalmente en el genotipo GG y el alelo G.

Methylene tetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF) polymorphisms in Brazilian patients with Hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC)

OBJECTIVE: The folate pathway is involved in hepatic carcinogenesis and angiogenesis. Polymorphisms in genes related to such processes, including methylene tetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF)] may play an important role in the development of hepatocellular carcinoma (HCC). The objective of this study was to evaluate MTHFR and VEGF polymorphisms in Brazilian patients with hepatitis C virus (HCV)-related HCC. METHODS: A total of 119 patients diagnosed with confirmed HCC and HCV were included in the study. SNP genotyping assays were performed using real-time PCR. VEGFA (rs2010963, rs3025039, and rs833061) and MTHFRC677T (rs1801133, rs1801131) polymorphisms were evaluated. RESULTS: The C alleles of MTHFR (rs1801131) and VEGF (rs2010963) were associated with protection against the development of multinodular HCC, while the T allele of MTHFR (rs1801133) was associated with a higher risk of multinodular presentation [p=0.04 OR 1.835 CI (1.022-3.297)]. Multivariate analysis revealed that the GG/GC genotypes of VEGF rs2010963 were independently associated with multinodular tumors at diagnosis (p=0.013; OR 4.78 CI (1.38-16.67)]. CONCLUSION: Our results suggest that these polymorphisms may increase the risk of rapid tumor progression in patients with HCV infection. This subgroup of patients with HCC and who present polymorphism is more likely to be diagnosed with multinodular disease and not be amenable to receiving curative treatments. These data must be validated in larger cohorts, and the screening intervals can be customized based on genetic history.

LncRNA AFAP1-AS1/miR-27b-3p/VEGF-C axis modulates stemness characteristics in cervical cancer cells / 中华医学杂志(英文版)

BACKGROUND@#Long non-coding RNA (lncRNA) actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) functions as a competing endogenous RNA to regulate target genes expression by sponging microRNAs (miRs) to play cancer-promoting roles in cancer stem cells. However, the regulatory mechanism of AFAP1-AS1 in cervical cancer (CC) stem cells is unknown. The present study aimed to provide a new therapeutic target for the clinical treatment of CC.@*METHODS@#Hyaluronic acid receptor cluster of differentiation 44 variant exon 6 (CD44v6)(+) CC cells were isolated by flow cytometry (FCM). Small interfering RNAs of AFAP1-AS1 (siAFAP1-AS1) were transfected into the (CD44v6)(+) cells. The levels of AFAP1-AS1 were measured by quantitative real-time PCR (qRT-PCR). Sphere formation assay, cell cycle analysis, and Western blotting were used to detect the effect of siAFAP1-AS1. RNA pull-down and luciferase reporter assay were used to verify the relationship between miR-27b-3p and AFAP1-AS1 or vascular endothelial growth factor (VEGF)-C.@*RESULTS@#CD44v6(+) CC cells had remarkable stemness and a high level of AFAP1-AS1. However, AFAP1-AS1 knockdown with siAFAP1-AS1 suppressed the cell cycle transition of G(1)/S phase and inhibited self-renewal of CD44v6(+) CC cells, the levels of the stemness markers octamer-binding transcription factor 4 (OCT4), osteopontin (OPN), and cluster of differentiation 133 (CD133), and the epithelial-mesenchymal transition (EMT)-related proteins Twist1, matrix metalloprotease (MMP)-9, and VEGF-C. In the mechanism study, miR-27b-3p/VEGF-C signaling was demonstrated to be a key downstream of AFAP1-AS1 in the CD44v6(+) CC cells.@*CONCLUSIONS@#LncRNA AFAP1-AS1 knockdown inhibits the CC cell stemness by upregulating miR-27b-3p to suppress VEGF-C.

Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris / 生物工程学报

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

VEGF gene rs35569394 polymorphism in patients with Polycystic Ovary Syndrome

SUMMARY OBJECTIVE: The relationship between the clinicopathological and sociodemographics characteristics of acral melanomas diagnosed at BACKGROUND: This study aimed to investigate the frequency of VEGF gene insertion (I) / deletion (D) polymorphism (rs35569394) in patients with Polycystic Ovarian Syndrome (PCOS) and to compare with a control population to verify its association with the pathology. METHODS: 206 women participated in this study, 103 with PCOS (group of patients) and 103 without the disease (control group). After extraction of genomic DNA from the samples, molecular analysis was performed by Polymerase Chain Reaction (PCR) and electrophoresis in polycrylamide. Descriptive analysis, univariate analysis and logistic regression model were used. Results were presented in odds ratio (OR) and 95% confidence interval (95% CI), considering the significance of p <0.05. RESULTS: There were no statistical differences between patients and controls for allele frequencies (χ2 = 1.16, p = 0.56). The genotypic frequency distribution was in Hardy Weinberg equilibrium for the patients (χ2 = 2.42; p <0.05), but not for the control group (χ2 = 7.26; p <0.05). Regarding risk factors for the syndrome, a history of familial PCOS is more frequent among women with the syndrome. CONCLUSIONS: In the present study, there is no association between VEGF gene I / D polymorphism and PCOS.

RESUMO OBJETIVO: Este estudo teve como objetivo investigar a frequência do polimorfismo de inserção (I)/ deleção (D) do gene VEGF (rs35569394) em pacientes com Síndrome dos Ovários Policísticos (SOP) e comparar com uma população controle para verificar sua associação com a patologia. MÉTODOS: Participaram desse estudo 206 mulheres sendo 103 com SOP (grupo de pacientes) e 103 sem a doença (grupo controle). Após extração do DNA genômico das amostras, a análise molecular foi realizada por Reação em Cadeia da Polimerase e eletroforese em gel de poliacrilamida. Utilizou-se análise descritiva, análise univariada e modelo de regressão logística. Os resultados foram apresentados em odds ratio (OR) e intervalo de confiança de 95% (IC-95%), considerando a significância de p < 0,05. RESULTADOS: Não houve diferenças estatísticas entre as pacientes e controles para as frequências alélicas (χ2 = 1,16, p = 0,56). A distribuição da frequência genotípica estava em equilíbrio de Hardy Weinberg para as pacientes (χ2= 2,42; p<0,12), mas não para o grupo controle (χ2= 7,26; p<0,05). Em relação aos fatores de risco para a síndrome, a história de SOP familiar é mais frequente entre as mulheres com a síndrome. CONCLUSÕES: Na casuística estudada, não há associação entre o polimorfismo I/D do gene da VEGF e a SOP.

Polimorfismo +405G> C del gen del factor de crecimiento de endotelio vascular en población cubana / Vascular Endothelial Growth Factor gene +405G> C polymorphism in the Cuban population

Introducción: El factor de crecimiento endotelial vascular (VEGF) es una proteína involucrada en la proliferación y migración celular del endotelio vascular, en cuyo gen se ha reportado el polimorfismo +405G>C. Se reconoce que no existen reportes genéticos poblacionales de esta variante en Cuba, que permitan caracterizar los perfiles inmunogenéticos a nivel molecular, para su aplicación en estudios de asociación alélica. Objetivo: Describir las frecuencias génicas y genotípicas del polimorfismo VEGF (+405 G>C) en la población cubana. Material y Métodos: Se realizó un estudio observacional, descriptivo, transversal, entre octubre de 2017 y marzo de 2018 en 162 neonatos cubanos, de ambos sexos y sanos, para el pesquisaje neonatal de enfermedades metabólicas, cuyas muestras biológicas se conservaban en el banco de ADN del Centro Nacional de Genética Médica. La caracterización molecular de los genotipos fue realizada mediante un PCR-ARMS. Se utilizó el software GENEPOP 4.4 y el paquete estadístico STATISTICA 8.0 para los cálculos de las frecuencias génicas y genotípicas. Resultados: La población no se ajustó al modelo de equilibrio de Hardy Weinberg para el gen evaluado. Las frecuencias génicas estimadas para el polimorfismo VEGF (+405 G>C) fueron de 0,33 para el alelo G y de 0,67 para el alelo C. El cálculo de las frecuencias genotípicas resultó en 0,14, 0,37 y 0,49, para las variantes GG, GC y CC, respectivamente. Conclusiones: Las frecuencias alélicas VEGF.C fueron superiores a la del alelo VEGF.G, siendo el genotipo VEGF.GG el de menor representación en el conjunto estudiado(AU)

Introduction: The vascular endothelial growth factor (VEGF) is a protein involved in the proliferation and cell migration of the vascular endothelium. In its gene, +405G>C Polymorphism has been reported. There are no population genetic reports of this variant in Cuba that allow the characterization of immunogenetic profiles at a molecular level for its application to allelic association studies. Objectives: To describe the genic and genotypic frequencies of the VEGF (+405 G>C) polymorphism in the Cuban population. Material and Methods: A descriptive cross-sectional observational study was carried out from October 2017 to March 2018 in 162 Cuban healthy newborns of both sexes for the neonatal screening for metabolic diseases, whose biological samples were conserved in the DNA bank of the National Center for Medical Genetics. The molecular characterization of the genotypes was carried out using a PCR-ARMS. The GENEPOP 4.4 software and the statistical software package STATISTICA 8.0 were used for the analysis of genic and genotypic frequencies. Results: The population did not adjust to the Hardy-Weinberg equilibrium model for the gene evaluated. The estimated gene frequencies of VEGF +405 G> C polymorphism were 0.33 for the G allele and 0.67 for the C allele. The calculation of the genotypic frequencies resulted in 0.14, 0.37 and 0.49, for the variants GG, GC and CC, respectively. Conclusions: The allelic frequencies of VEGF.C were higher than the frequencies of the VEGF.G allele, being the VEGF GG the least represented genotype in the group studied(AU)

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?

REC8 suppresses tumor angiogenesis by inhibition of NF-κB-mediated vascular endothelial growth factor expression in gastric cancer cells

BACKGROUND: Tumor angiogenesis is an essential event for tumor growth and metastasis. It has been showed that REC8, a component of the meiotic cohesion complex, played a vital role in Epithelial-Mesenchymal Transition (EMT) in gastric cancer. However, the role of REC8 in gastric cancer angiogenesis remains to be identified. RESULTS: Inhibition of REC8 expression in gastric cancer cells contributed to tumor angiogenesis in the gastric cancer microenvironment. The clinical analysis demonstrated that the loss of REC8 in gastric cancer with enrichment of MVD. Depletion of REC8 expression in gastric cancer cells significantly increased tube formation of human umbilical vein endothelial cells (HUVECs), which is attributed to enhancement of vascular endothelial growth factor (VEGF) secretion caused by REC8 slicing. While addition of neutralizing antibody targeted VEGF into supernatant drastically reversed the effect of REC8 loss in gastric cancer cells on tube formation. Mechanistic analyses indicated that ablation of REC8 promotes nuclear factor-κB (NF-κB) p65 activity and its downstream gene VEGF expression, leading to tube formation. CONCLUSIONS: These results demonstrated a novel REC8 function that suppressed tumor angiogenesis and progression by attenuation of VEGF in gastric cancer microenvironment.

Chinese medicine Buyang Huanwu decoction promotes neurogenesis and angiogenesis in ischemic stroke rats by upregulating miR-199a-5p expression / 浙江大学学报·医学版

OBJECTIVE@#To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats.@*METHODS@#Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting.@*RESULTS@#BYHWD treatment significantly promoted the recovery of neurological function (@*CONCLUSIONS@#Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.

Lopinavir and curcumin directly alters BAX/BCL2 and VEFG165b mRNA levels to suppress human squamous cervical carcinoma cell growth / Lopinavir y curcumina alteran directamente los niveles de ARNm de BAX/ BCL 2 y VEFG165 para suprimir el crecimiento de células de carcinoma cervical escamoso humano

Following the success of the highly active antiretroviral therapy, the potential of multidrug combination regimen for the management of cancer is intensely researched. The anticancer effects of curcumin on some human cell lines have been documented. Lopinavir is a FDA approved protease inhibitor with known apoptotic activities. Dysregulated apoptosis is important for the initiation of cancer while angiogenesis is required for cancer growth and development, this study therefore investigated the effects of the combination of lopinavir and curcumin on cell viability, apoptosis and the mRNA expression levels of key apoptotic and angiogenic genes; BAX, BCL2 and VEGF165b in two human cervical cell lines; human squamous cell carcinoma cells - uterine cervix (HCS-2) and transformed normal human cervical cells (NCE16IIA). The two human cervical cell lines were treated with physiologically relevant concentrations of the agents for 120 h following which BAX, BCL2 and VEGF165b mRNA expression were determined by Real Time qPCR. The Acridine Orange staining for the morphological evaluation of apoptotic cells was also performed. The combination of lopinavir and curcumin up-regulated pro-apoptotic BAX and antiangiogenic VEGF165b but down-regulated the mRNA levels of anti-apoptotic BCL2 mRNA in the human squamous cell carcinoma (HCS-2) cells only. The fold changes were statistically significant. Micrographs from Acridine Orange staining showed characteristic evidence of apoptosis in the human squamous cell carcinoma (HCS-2) cells only. The findings reported here suggest that the combination of curcumin and the FDA approved drug-lopinavir modulate the apoptotic and angiogenic pathway towards the inhibition of cervical cancer.

Tras el éxito de la terapia antirretroviral altamente activa, se investiga intensamente el potencial del régimen de combinación de múltiples fármacos para el tratamiento del cáncer. Se han documentado los efectos anticancerígenos de la curcumina en algunas líneas celulares humanas. Lopinavir es un inhibidor de proteasa aprobado por la FDA con actividades apoptóticas conocidas. La apoptosis disrregulada es importante para el inicio del cáncer, mientras que la angiogénesis es necesaria para el crecimiento y desarrollo del cáncer. Por lo tanto, este estudio investigó los efectos de la combinación de lopinavir y curcumina sobre la viabilidad celular, la apoptosis y los niveles de expresión del ARNm de genes apoptóticos y angiogénicos clave: BAX, BCL2 y VEGF165b en dos líneas celulares cervicales humanas; células de carcinoma de células escamosas humanas: cérvix uterino (HCS-2) y células cervicales humanas transformadas (NCE16IIA). Las dos líneas celulares cervicales humanas se trataron con concentraciones fisiológicamente relevantes de los agentes durante 120 horas, después de lo cual la expresión de ARNm de BAX, BCL2 y VEGF165b se determinó mediante qPCR en tiempo real. También se realizó la tinción con naranja de acridina para la evaluación morfológica de células apoptóticas. La combinación de lopinavir y curcumina reguló incrementando BAX proapoptósicos y VEGF165b antiangiogénicos, pero reguló a la baja los niveles de ARNm del BCL2 antiapoptótico en células de carcinoma de células escamosas humanas (HCS-2) únicamente. Los cambios en el pliegue fueron estadísticamente significativos. Las micrografías de la tinción con naranja de acridina mostraron evidencia característica de apoptosis solo en las células del carcinoma de células escamosas humanas (HCS-2). Los hallazgos reportados aquí sugieren que la combinación de curcumina y el fármaco aprobado por la FDA lopinavir modulan la vía apoptótica y angiogénica hacia la inhibición del cáncer cervical.

Use of cationic microbubbles targeted to P-selectin to improve ultrasound-mediated gene transfection of hVEGF165 to the ischemic myocardium / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection (TCUMGT) via ultrasound-targeted microbubble destruction (UTMD) to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles (MBs) with pcDNA3.1-human vascular endothelial growth factor 165 (pcDNA3.1-hVEGF165) plasmids targeted to P-selectin (MB+P+VEGFp) were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups: microbubble only (MB), microbubble+P-selectin (MB+P), microbubble+pcDNA3.1-hVEGF165 plasmid (MB+VEGFp), and microbubble+ P-selectin+pcDNA3.1-hVEGF165 plasmid (MB+P+VEGFp). The reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB+P+VEGFp delivery showed greater improvement than MB+VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA3.1-hVEGF165 recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.

Vascular endothelial growth factor G1612A (rs10434) gene polymorphism and neuropsychiatric manifestations in systemic lupus erythematosus patients / Polimorfismo genético do fator de crescimento vascular endotelial G1612A (rs10434) e manifestações neuropsiquiátricas em pacientes com lúpus eritematoso sistêmico

Abstract Aim: To investigate the relation between vascular endothelial growth factor (VEGF) gene polymorphism in systemic lupus erythematosus (SLE) patients and lupus related neuropsychiatric manifestations. Patients and methods: Sixty adult SLE patients recruited from the Rheumatology and Neurology departments of Cairo University hospitals were classified into two groups; Group A: 30 patients with neuropsychiatric manifestations (NPSLE) and Group B: 30 patients without. For both groups the SNP G1612A (rs10434) of the VEGF gene was genotyped by real time polymerase chain reaction (RT-PCR). Results: Statistically significant difference was found in genotype and allele frequencies between both groups (AA [70% vs 13.3%, p < 0.001] and GG [10% vs 66.7%, p < 0.001]). Conclusion: Polymorphism in the gene coding for VEGF may be associated with increased incidence of neuropsychiatric lupus in SLE patients.

Resumo Objetivo: Investigar a relação entre o polimorfismo genético do fator de crescimento vascular endotelial (VEGF) em pacientes com lúpus eritematoso sistêmico (LES) e manifestações neuropsiquiátricas relacionadas com o lúpus. Pacientes e métodos: Foram recrutados 60 pacientes adultos com LES nos departamentos de Reumatologia e Neurologia de hospitais universitários do Cairo e classificados em dois grupos; grupo A: 30 pacientes com manifestações neuropsiquiátricas (LESNP) e grupo B: 30 pacientes sem manifestações neuropsiquiátricas. Genotipou-se o SNP G1612A (rs10434) do gene VEGF em ambos os grupos por reação em cadeia da polimerase em tempo real (RT-PCR). Resultados: Foi encontrada diferença estatisticamente significativa nas frequências genotípicas e alélicas entre os dois grupos (AA [70% vs. 13,3%, p < 0,001] e GG [10% vs. 66,7%, p < 0,001]). Conclusão: O polimorfismo no gene que codifica o VEGF pode estar associado ao aumento na incidência de lúpus neuropsiquiátrico em pacientes com LES.

Pravastatin induces cell cycle arrest and decreased production of VEGF and bFGF in multiple myeloma cell line / Pravastatina induz parada no ciclo celular e diminuição na produção de VEGF e bFGF em linhagem de Mieloma Multiplo

Abstract Multiple myeloma (MM) is a B cell bone marrow neoplasia characterized by inflammation with an intense secretion of growth factors that promote tumor growth, cell survival, migration and invasion. The aim of this study was to evaluate the effects of pravastatin, a drug used to reduce cholesterol, in a MM cell line.Cell cycle and viability were determinate by Trypan Blue and Propidium Iodide. IL6, VEGF, bFGF and TGFβ were quantified by ELISA and qRT-PCR including here de HMG CoA reductase. It was observed reduction of cell viability, increase of cells in G0/G1 phase of the cell cycle and reducing the factors VEGF and bFGF without influence on 3-Methyl-Glutaryl Coenzyme A reductase expression.The results demonstrated that pravastatin induces cell cycle arrest in G0/G1 and decreased production of growth factors in Multiple Myeloma cell line.

Resumo O Mieloma Múltiplo é uma neoplasia de linfócitos B da medula óssea, caracterizada por inflamação com uma intensa secreção de fatores de crescimento que promovem o aumento do volume do tumor, sobrevivência celular, migração e invasão. O objetivo deste estudo foi avaliar os efeitos da pravastatina, uma droga usada para reduzir o colesterol, em um linhagem de MM. O ciclo celular e viabilidade foram determinadas por Trypan Blue e iodeto de propídio. IL6, VEGF, bFGF e TGF foram quantificadas por ELISA e qRT-PCR, incluindo aqui de HMG CoA redutase. Observou-se a redução da viabilidade das células, aumento de células na fase G0/G1 do ciclo celular e redução no VEGF e bFGF, sem influência na expressão da enzima 3-Metil-Glutaril Coenzima A redutase. Os resultados demonstraram que a pravastatina induz parada no ciclo celular em G0/G1 e diminuição da produção de fatores de crescimento em várias linhas de células de Mieloma.