Angiogenesis and retinoblastoma -
Positively stained cells were counted in 10 randomly selected fields under a magnification of × Intensity was graded as negative, weak, moderate, or intense, and the percentage of stained cells was classified as less than or equal to For statistical analysis of the results, we calculated frequency and percentage for nominal and ordinal variables.
Clinicohistopathologic data for the patients in the study are given in Table 1. There were more male than female patients The most common symptoms recorded were leukocoria, strabismus, diminished visual acuity, and orbital mass. A mixed-type pattern of growth was seen in patients, and the endophytic pattern was seen in 13 patients.
Other patterns regression, exophytic, and diffuse were seen infrequently Figure 1 A and B. According to the histopathologic results, poorly differentiated tumors were the most frequent type 81 patients , followed by moderately differentiated 24 patients and well differentiated 16 patients Figure 1 C and D.
Anterior chamber invasion was observed in Compromise of the sclera was seen in Assessment of optic nerve invasion yielded no remarkable differences among the 4 groups.
Most patients The mitotic index was variable among the patients; Evaluating the apoptotic index, The immunoreactivity of VEGF was positive in 43 of 44 patients Table 2 and Figure 3.
Three of the 47 patients studied could not be evaluated because of extensive necrosis. No statistically significant correlation was found between VEGF immunostaining and many established retinoblastoma prognostic factors, such as optic nerve or choroidal invasion, anterior chamber invasion, vitreous seeding, or basophilic staining on the vascular walls.
In our series, the lack of follow-up prevents the possibility of establishing a correlation between VEGF immunostaining and prognosis, even though this variable was not related to the stage of the disease, according to the Grabowski and Abramson classification.
Retinoblastoma is the most common intraocular tumor in childhood, with an annual mean incidence of 1 in 18 live births and a mortality rate that varies widely across the globe.
In our study, patients had a mean age at diagnosis of 29 months. This value is similar to those from other Latin American and Asian series 32, 31, and 26 months in Venezuela, Mexico, and Taiwan, respectively.
Most of our patients had poorly differentiated tumors, similar to the work of Biswas et al. Marback et al 15 measured the relative vascular area of the tumor in retinoblastomas and compared their results with optic nerve and choroidal invasion.
They indicated that the relative vascular area of the tumor seems to be a promising prognostic marker for metastatic disease and should be evaluated in a large sample of eyes with retinoblastoma.
Previous studies 19 have reported that angiogenic potential in retinoblastoma correlates with invasive growth and metastasis and that these 2 factors are associated with poor prognosis. These tumors, however, appear to depend on a heterogeneous vasculature composed of angiogenic neovessels and pericyte-committed mature vasculature, the latter composed of endothelial cells dependent on angiogenic factors such as VEGF.
It is known that VEGF messenger RNA is expressed in retinoblastoma neoplastic cells, but little of such expression occurs in tumor endothelial cells, and that VEGF, which is secreted from neoplastic cells, influences nearby endothelial cells and functions as a paracrine mediator.
Of the 7 patients with well-differentiated retinoblastoma, 2 showed focal and weak VEGF staining, whereas the other 5 had strong and diffuse staining. These results differ from those of Kerimoğglu et al, 23 who measured angiogenesis microvessel density and found higher levels in poorly differentiated retinoblastomas.
We found a statistically significant relation between the interval from the onset of the symptoms to the enucleation and VEGF immunostaining intensity.
This finding indicates that the intensity of the immunostaining depends on the time between the onset of symptoms and enucleation. This could suggest that tumors with delayed diagnosis and treatment could have more angiogenic potential and may be more prone to dissemination.
High mitotic and apoptotic indexes reflect high proliferative activity in any tumor. Kerimoğglu et al 23 have suggested that the apoptotic index could be an important metastatic predictor for retinoblastoma. Most of our patients had high mitotic and apoptotic indexes, and we found a statistically significant correlation between VEGF immunostaining intensity and the apoptotic index Figure 4.
Moreover, a relation between VEGF immunostaining intensity and percentage of staining with the mitotic index was also found. With respect to the apoptotic index, it has been demonstrated that deletion of the retinoblastoma gene produces apoptosis rather than tumor formation because the loss of the retinoblastoma gene triggers a pmediated apoptotic response.
No correlation was found between VEGF immunostaining and tumor staging. This lack of correlation implies that all retinoblastomas, not just those limited to retina and cases with choroidal or optic nerve invasion, have the capacity to produce angiogenic factors and by extension induce angiogenesis.
Marback et al 15 have suggested that retinoblastomas with numerous blood vessels measured with the relative vascular area of the tumor are those at an advanced stage of disease. Although the isolated characterization of VEGF in retinoblastoma should not be taken as a prognostic factor, its association with the apoptotic index suggests a role for this protein in the progression of this disease.
Our data suggest that the use of anti-VEGF therapies in retinoblastoma may be efficacious in targeting immature neovessels within the tumor. This approach, coupled with a strategy to treat pericyte-committed mature tumor vasculature, may be effective in the management of this disease.
Correspondence: Maria E. Orellana, MD, Henry C. Witelson Ocular Pathology Laboratory, McGill University, University St, Room , Montreal, QC H3A 2B4, Canada euorellana gmail.
Submitted for Publication: February 3, ; final revision received September 22, ; accepted October 5, Additional Contributions: Douglas Angulo, MSc, provided valuable statistical analysis. full text icon Full Text. Download PDF Top of Article Abstract Methods Results Comment Article Information References.
Figure 1. View Large Download. Table 1. Clinicohistopathologic Characteristics in Patients With Retinoblastoma a. Vascular Endothelial Growth Factor Staining in 44 Patients With Retinoblastoma.
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Unfortunately, current treatments for Rb have been hampered by an incomplete understanding of the specific pathogenesis of the disease and thus, a lack of a specific molecular target. Earlier studies have focused on blood supply as a potential therapeutic target 3.
The histopathology of Rb has demonstrated the presence of viable tumor cells adjacent to blood vessels surrounded by necrotic cells, which graphically illustrates the exquisite dependence of Rb cells on blood supply 3.
Indeed, the outgrowth of Rb relies on adequate angiogenesis, which is accomplished through expression of proangiogenic factors such as vascular endothelial growth factor A VEGF-A 4. Inhibition of angiogenesis has been shown to effectively eliminate Rb cells 5 , 6 , suggesting that anti-angiogenic therapy may be a promising innovative treatment strategy against this specific vulnerability of the cancer.
However, VEGF-A is a potent proangiogenic factor and plays a substantial role in the homeostasis of various tissues and organs in the human body 7 - 11 and therefore, therapies targeting VEGF-A may cause a multitude of adverse side effects. Thus, an alternative angiogenic target must be sought.
Netrin-1 is a diffusible, laminin-associated protein that acts as a guidance cue during neurogenesis During development, netrin-1 is predominantly expressed in the central nervous system 12 while in adult mammals, netrin-1 and its receptors are expressed in both neural and non-neural tissues Interestingly, netrin-1 has been found to be highly expressed in human metastatic breast tumors 14 and aggressive neuroblastoma 15 and is likely related to tumor-associated angiogenesis Moreover, netrin-1 has been shown to regulate angiogenesis in diabetic kidney disease However, the precise role of netrin-1 in Rb has not been fully examined.
This current study investigated the expression and role of netrin-1 in Rb both in vitro and in vivo. Human Rb cell lines Y79, WERI-Rb-1, NCC-Rbc, NCC-Rbc, and NCC-Rbc-T1 were purchased from American Type Culture Collection ATCC, Rockville, MD, USA and Creative Bioarray Shirley, NY, USA.
Plasmids expressing small interfering si RNA netrin-1 or a scrambled sequence for netrin-1 together with a luciferase reporter to allow in vivo cell tracing by bioluminescent assay under a CMV promoter were purchased from Santa Cruz SC, Beijing, China. Transfections were performed with 1.
All experiments were performed under a project license SH9HA granted by the institutional ethics board of the Shanghai Jiao Tong University, in compliance with institutional guidelines for the care and use of animals.
A protocol was prepared before the study without registration. Male and female week-old nude mice body weight ~22 g; both genders were used to exclude a possible effect of sex on experimental results were purchased from Shanghai Laboratory Animal Center SLAC Shanghai, China.
Both male and female nude mice were used and distributed evenly in each experimental group. Mice were housed under a hour light-dark cycle. Grouping included an allocation concealment method to ensure minimal confounders.
No criteria were used for excluding animals or experimental units during the experiment, and no data were excluded during the analysis. The study did not have humane endpoints.
For xenograft procedures and bioluminescence analyses, mice were anesthetized by 2. The xeno-tumor was assessed with a bioluminescent assay IVIS imaging system, Perkin Elmer, Santa Clara, CA, USA 1 month after transplantation.
The mice were assigned into the following three groups, and each group contained 5 mice. Group 1 mice received subcutaneous transplantation of control WERI-Rb-1 cells transfected with a scrambled vector.
The in vitro proliferation potential of Rb cells was assessed with using the Cell Counting Kit-8 CCK-8 assay Roche, Indianapolis, IN, USA , in which the absorbance value OD of the cells was quantified at nm. Immunohistochemistry was performed using primary rabbit anti-netrin-1 Abcam, Cambridge, MA, USA and anti-CD31 antibodies Becton-Dickinson Biosciences, San Jose, CA, USA , followed by a secondary cy3-conjugated anti-rabbit antibody Jackson ImmunoResearch Labs, West Grove, PA, USA.
Five mice were analyzed in each group. ELISA for netrin-1 was performed with a human Netrin-1 ELISA kit ABIN, antibodies-online. The absorption was measured at nm.
The protein concentration was determined by comparing the relative absorbance of the samples with the standards. A SYBR Green PCR Kit Qiagen, Shanghai, China was used for RT-qPCR with the designed primers purchased from Qiagen no sequence information was provided.
The RT-qPCR reactions were performed in triplicate. The gene values were assessed with the 2 -ΔΔCt method and obtained after sequential normalization to β-actin as the experimental control.
Two gene expression profiles GSE and GSE were selected and the GEO2R online analysis tool was used to detect the differentially expressed genes DEGs.
The study was conducted in accordance with the Declaration of Helsinki as revised in The P value, adjusted P value, and log fold change FC were calculated.
org and Kyoto Encyclopedia of Genes and Genomes KEGG All data in the current study were summarized and statistically analyzed with GraphPad Prism 7 GraphPad, Chicago, IL, USA. One-way analysis of variance ANOVA was performed to compare the data from different groups. The values are expressed as mean ± standard deviation SD.
Previous reports have suggested the importance of angiogenesis in the development and progression of Rb. Thus, the angiogenesis status and the levels of netrin-1 were examined in Rb specimens. Data obtained from the GEO database were screened to identify the DEGs between Rb samples and control retina samples.
Analyses of the significantly downregulated and upregulated genes revealed that they were enriched in many pathways including angiogenesis Figure 1A. Moreover, netrin-1 was identified as a significantly upregulated gene in Rb samples Figure 1B. These data suggested the Rb patients showed enhanced angiogenesis and upregulated netrin-1 expression.
Immunohistochemistry revealed significantly higher expression of netrin-1 in Rb specimens compared to normal retina Figure 2A.
Moreover, higher levels of netrin-1 mRNA Figure 2B and protein Figure 2C expression were detected in 5 Rb cell lines, named Y79, WERI-Rb-1, NCC-Rbc, NCC-Rbc, and NCC-Rbc-T1 compared to healthy retina samples. These data are consistent with the analyses of the GEO database samples, demonstrating an upregulation of netrin-1 in Rb.
To assess the functionality of netrin-1 in Rb, plasmids expressing the siRNA for netrin-1 were constructed with a luciferase reporter under a CMV promoter.
Post-workout nutrition for body composition more Increasing thermogenesis naturally about PLOS Subject Areas, click here. Pathological angiogenesis retinoblastoja an essential role in amd aggressiveness and leads to unfavorable prognosis. Angiigenesis aim of Angiobenesis Angiogenesis and retinoblastoma is Angioenesis detect Angiogenesis and retinoblastoma potential retinoblastom of Retinoblastoma binding protein 2 Angiogenesis and retinoblastoma in the tumor angiogenesis of non-small cell lung cancer NSCLC. Immunohistochemical staining was used to detect the expression of RBP2, hypoxia-inducible factor-1α HIF-1αvascular endothelial growth factor VEGF and CD Two pairs of siRNA sequences and pcDNA3-HA-RBP2 were used to down-regulate and up-regulate RBP2 expression in H and SK-MES-1 cells. An endothelial cell tube formation assay, VEGF enzyme-linked immunosorbent assay, real-time PCR and western blotting were performed to detect the potential mechanisms mediated by RBP2 in tumor angiogenesis.
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