The Expression Of Aquaporins And VEGF In Human Glioma

Objective: Human brain gliomas with peritumoral edemaare clinically common, and more malignant grade, more seriousbrain edema including cytotoxic and vasogenic. Watermetabolism in the pathophysiological course of glioma and itsperitumoral edema'genesis and development must be related toand different from normal to some extent. Recently, it has beenfound that aquaporins (AQP1, AQP4) and vascular endothelialgrowth factor (VEGF), expressed in the human brain, may playan important role in brain water metabolism and abnormalpathophysiological condition, but not clarified. Now, AQP1,AQP4 and VEGF expressions in the normal brain tissue, gliomatumor and peritumoral edema tissue are studied using byimmunohistochemistrical and in situ hybridizational methods,and its actions and relations in the water metabolism of humangliomas are discussed. It provided a molecule level of theoreticalproof to illustrate glioma and its peritumoral edema mechanismand a newly potential path for clinic treatment.Materials and Methods: 66 samples of 43 patientsincluding 37 cases of male and 29 cases of female with glioma,who underwent operation at the Second Affiliated Hospital,Hebei Medical University from 2003 to 2004, were collected.The normal brain samples are 9, peritumoral edema ones 23 andtumor ones 34. The range of patient age was from 9 years old to76 years old, and patients consisted of 16 cases of <20 years old,31 cases of 20-60 years old and 13 cases of >60 years old.Supratentorial were 60, infratentorial 6; supratentorial tumorsamples were 30 (left 11 and right 19); supratentoriallyperitumoral edema samples were 23 (left 7 and right 16);supratentorially normal brain samples were 7 (left 2 and right 5).The range of disease course was from 1 week to 6 months.Chemiotherapy, radiotherapy and immunosuppressive therapywere not performed to all of the patients before operation. Freshsamples obtained in the operations were routinely, timelyprocessed to be fixed by 4% formamint, dehydrated by gradientalcohol, becomed transparent by dimethyl benzene and imbeddedby paraffin. Prepared and pretreated essential regents andinstruments for immunohistochemistrical and in situhybridizational detections; Cut serial sections of 5μm were fromthe blocks, then stained representative sections with H&E inorder to comfirm the histopathological diagnosis based onWHO(2000) CNS tumor classified standards. Tumor samplesconsisted of 8 cases of WHO grade Ⅰ(pilocytic astrocytoma),8 cases of WHO grade Ⅱ(fibrillary astrocytoma 5,protoplasmic astrocytoma 1, gemistocytic astrocytoma 1, andependymoma 1), 10 cases of WHO grade Ⅲ(anaplasticastrocytoma 7, anaplastic ependymoma 1 and malignantpapilloma of choroid plexus 2), and 8 cases of WHO grade Ⅳ(glioblastoma multiforme 6 and medulloblastoma 2). 23peritumoral edema tissue samples included 2 derived from gradeⅠtumor, 7 from grade Ⅱ, 10 from grade Ⅲand 4 from gradeⅣ. All 9 cases of normal brain tissue samples were obtainedfrom the patients with brain cortex-stoma who suffered fromglioma and were being operated and confirmed by preoperationalimaging and postoperational pathological diagnosis.Immunohistochemical stains (SP) were performed toexamine the expression of VEGF,AQP1,AQP4 and in situhybridizational detections to examine the expression ofAQP1mRNA and AQP4mRNA (DAB coloration). Expressingcolor shade was graded into no staining, yellow, buffy and brown;expressing scope was distinguished into no-staining, scattered,limited and widespread. So, combining color shade and scope,expressing strength was graded into four classes presented with"-,+,++,+++".Calculated edema index (EI) by measuring preoperationalimaging.Applying SPSS11.5 statistical package, carry out two-andseveral-samples nonparameter test, linear regression and rankcorrelation analysis on results. P value less than 0.05 wasconsidered statistically significant.Results: Postive expressions were located in plasmamembrane, plasma and/or enation and/or glia, being differentdegree of yellow and buffy.VEGF expressed typically in endothelial cytoplasm,cytoplasm and enations of tumor cells, active astrocytes, somegliocytes. In 9 normal brain tissue samples, there were 3 "-", 5"+", 1 "++"and 0 "+++"; in 23 peritumoral edema tissue samples,1"-", 7 "+", 9 "++"and 6 "+++"; in 34 tumor tissue samples,4"-", 9 "+", 12 "++"and 9 "+++". AQP1 proteins, and mRNAssimilarly, expressed typically in plasma membrane of endothelialand pericytes, and plasma and enations of tumor cells, activeastrocytes and some peripheral glia cells. In 9 normal brain tissuesamples, there were 2 "-", 7 "+", 7 "++"and 0 "+++"of AQP1proteins, and 2 "-", 4 "+", 3 "++"and 0 "+++"of AQP1 mRNAs;in 23 peritumoral edema tissue samples, 1"-", 8 "+", 12 "++"and2 "+++"of AQP1 proteins, and 0"-", 7 "+", 10 "++"and 6 "+++"of AQP1 mRNAs; in 34 tumor tissue samples, 4"-", 20 "+", 10"++"and 0 "+++"of AQP1 proteins, and 1 "-", 16 "+", 12 "++"and 5 "+++"of AQP1 mRNAs. AQP4 proteins, and mRNAssimilarly, expressed typically in plasma membrane of endothelialand pericytes, and plasma and enations of tumor cells, activeastrocytes and some peripheral glia cells, evidently in glia andrelatively poorly in endothelial cells different from AQP1. In 9normal brain tissue samples, there were 0 "-", 5 "+", 1 "++"and3 "+++"of AQP4 proteins, and 1 "-", 5 "+", 0 "++"and 3 "+++"of AQP4 mRNAs; in 23 peritumoral edema tissue samples, 1"-",10 "+", 8 "++"and 4 "+++"of AQP4 proteins, and 4"-", 11 "+",6 "++"and 2 "+++"of AQP4 mRNAs; in 34 tumor tissuesamples, 2 "-", 11 "+", 14 "++"and 7 "+++"of AQP4 proteins,and 1 "-", 20 "+", 8 "++"and 5 "+++"of AQP4 mRNAs.Two-independent-samples nonparameter test (two-sides)indicated that VEGF expression was different between normaland peritumoral samples, tumor, high grade tumor, grade Ⅱ, Ⅲand Ⅳtumor, peritumoral and low grade tumor, low and highgrade tumor, grade Ⅰtumor and peritumor, grade Ⅱ, ⅢandⅣtumor, low grade tumor and its peritumor, grade Ⅰtumorand its peritumor (homologous P values were 0.004,0.009,0.000,0.002,0.001,0.002,0.037,0.002,0.008,0.002,0.001,0.001,0.027,0.044). AQP1 protein expression wasdifferent between normal and edema, high grade tumor, edemaand tumor, low grade tumor, grade Ⅰtumor, grade Ⅰtumorand grade Ⅲtumor, tumor and edemas of low grade, grade Ⅱand Ⅳ, edemas of high and low grade, grade Ⅱand Ⅲ, gradeⅢand Ⅳ(homologous P values were 0.004,0.046,0.013,0.019,0.002,0.027,0.002,0.040,0.048,0.023,0.002,0.024). AQP1mRNA expression was different between edemaand normal, grade Ⅰtumor, supra-and infra-tentorium of allsamples. AQP4 protein expressions were different only betweenleft and right side of supratentorial edema. AQP4mRNAexpression was not statistically significant.Two-relative-samples nonparameter test (single-side)indicated that the expressions of AQP1 protein and mRNA weredifferent in all samples, all edema, all tumor, grade Ⅰand Ⅳtumor, edemas of high grade tumor and grade Ⅲ, allsupratentorial, supratentorial edema and tumor, grade Ⅰand Ⅲ,supratentorial high and low grade, edemas of high grade andgrade Ⅲ(homologous P values were 0.000,0.036,0.003,0.023,0.032,0.011,0.010,0.000,0.017,0.001,0.023,0.042,0.012,0.018,0.011,0.010). The expressions of AQP4protein and mRNA were different between all samples, all edema,all tumor, grade Ⅲtumor, edema of high grade and grade Ⅲtumor, all supratentorial, all supratentorial edema, supratentorialgrade Ⅲtumor, supratentorial high grade tumor, edema ofsupratentorial high grade tumor and grade Ⅲtumor, allinfrotentorial and infrotentorial tumor (homologous P valueswere 0.001,0.010,0.020,0.023,0.004,0.007,0.002,0.007,0.042,0.048,0.004,0.007,0.048,0.030). The expressions ofVEGF and AQP1 protein were different between all samples, alltumor, grade Ⅲand Ⅳtumor, all supratentorial, supratentorialgrade Ⅲ, Ⅳand high grade tumor (homologous P values were0.001,0.001,0.023,0.012,0.001,0.001,0.019,0.002). Theexpressions of VEGF and AQP4 protein were different betweenall normal samples, supratentorial normal, supratentorial gradeⅢand high grade tumor (homologous P values were 0.019,0.030,0.032,0.015). The expressions of AQP1 and AQP4 proteinwere different between all samples, all normal, all tumor, gradeⅠ, Ⅱand Ⅳtumor, edema of grade Ⅲand Ⅳ, allsupratentorial, supratentorial normal, supratentorial tumor of all,grade Ⅰ, high and low grade, supratentorial edema of grade Ⅲand Ⅳ, and infrasupratentorial tumor (homologous P valueswere 0.001,0.021,0.001,0.030,0.017,0.030,0.030,0.042,0.001,0.032,0.002,0.030,0.042,0.007,0.030,0.042,0.048). The expressions of AQP1 and AQP4 mRNA weredifferent between all samples, all edema, edema of high grade,grade Ⅲand Ⅳ, all supratentorial, supratentorial edema,supratentorial tumor of high grade, supratentorial edema of highgrade, grade Ⅲand Ⅳ(homologous P values were 0.042,0.002,0.003,0.017,0.030,0.011,0.002,0.036,0.003,0.017,0.030).Used spearman rank correlation test (two-sides). Statisticalanalysis of all 66-sample results indicated VEGF expression wascorrelated with NPLH, NPT1234, expressions of AQP1proteinand mRNA, expression of AQP4 protein, high or low grade oftissue origin and pathological grade (correlation coefficients were0.332,0.370,0.393,0.502,0.247,0.317,0.437, homologousP values were 0.006,0.004,0.002,0.001,0.000,0.045,0.010,0.000); AQP1mRNA expression was correlated with expressionsof VEGF and AQP1protein, pathological grade of tissue origin;(correlation coefficients were 0.502,0.418,0.268,0.356,homologous P values were 0.000,0.000,0.030,0.003); AQP4protein expression was correlated with expressions of VEGF andAQP4mRNA, left or right side of supratentorial samples(correlation coefficients were 0.247,0.669,0.342, homologous Pvalues were 0.045,0.000,0.007); AQP4mRNA expression wascorrelated with AQP4 protein expression, left or right side ofsupratentorial samples (correlation coefficients were 0.669,0.358,homologous P values were 0.000,0.005); All the expressionalindexes had no correlation with patient sex in the group.Statistical analysis of all 34-tumor-sample results indicated thatVEGF expression had correlation with pathlogical grade andAQP1mRNA expression (correlation coefficients were 0.651,0.504, homologous P values were 0.000,0.002); AQP1 proteinexpression was correlated with AQP4 protein expression(correlation coefficient was 0.373, P value was 0.030);AQP1mRNA expression was correlated with pathological grade,VEGF expression and patient age (correlation coefficients were0.346,0.504,0.475, homologous P values were 0.045,0.002,0.005); AQP4 protein expression was correlated with AQP1protein expression and AQP4mRNA expression (correlationcoefficients were 0.373,0.603, P values were 0.030,0.000);AQP4mRNA expression had correlation with AQP1 proteinexpression and left or right side of supratentorial tumors(correlation coefficients were 0.603,0.376, P value 0.000,0.041);All expressional indexes had no correlation with patient sex.Statistical analysis of all 23-peritumoral-edema-sample resultsshowed that VEGF expression had correlation with AQP4mRNAexpression, AQP1 protein expression with AQP1mRNAexpression, AQP4 protein expression with AQP4mRNAexpression and left or right side of supratentorial edema samples(correlation coefficients were 0.416,0.433,0.631,0.450,homologous P values were 0.048,0.039,0.001,0.031); Allexpressional indexes had no correlation with pathological gradeof tumor, patient sex and age. Statistical analysis of all 19-gliomaedema indexes (EI) showed that EI had positive correlation withpathological grade of tumor, high or low grade, AQP1mRNAexpression and patient age (correlation coefficients were 0.543,0.520,0.503,0.526, homologous P values were 0.016,0.023,0.028,0.021); But EI had no correlation with expression ofVEGF, protein and mRNA of AQP1 and AQP4, patient sex andtumor's side. Statistical analysis of all 13-supratentorial-gliomaEI showed that EI had strongly positive correlation withpathological grade of tumor and high or low grade (correlationcoefficients were 0.925,0.866, P values both were 0.000); But EIhad no correlation with expression of VEGF, protein and mRNAof AQP1 and AQP4, patient age and sex, tumor's side.Conclusions: The expressions of AQP1 protein andmRNA were few in nomal human brain tissue, and more in theperitumoral edema tissue of human glioma and high grade ofhuman glioma; infratentorial tumor poorly than supratentorial.The expressions of AQP4 protein and mRNA were evident innormal human brain tissue, human glioma and peritumoraledema tissue. In contrast with AQP1, AQP4's more extensivedistribution and higher expression were necessary to ensurehigher energy and substance (water) metabolism of brain tissue,which may play roles as stably and inherently basical waterchannel and main water flow regulator. AQP1's more narrowdistribution and lower expression were helpful to sustain andregulate normal water metabolic rate and protect functional areasand intracal volume to some extent; played an important role atspecial site and during pathophisiological process of cytotoxic...