Vascular Endothelial Growth Factor Immunoreactivity Research Articles

Expression of Vascular Endothelial Growth Factor (VEGF) in Colorectal Adenoma and Carcinoma in a Tertiary Care Center.

Colorectal cancer is one of the malignancies in which angiogenesis has been implicated and its importance at different stages of malignant disease. Neovascularization begins when the angiogenic switch is turned on and when angiogenesis activators outweigh angiogenic inhibitors. The process of blood vessel formation is regulated through several growth factor systems. The vascular endothelial growth factor (VEGF) system is one of the important ones and may consequently be a key system in relation to different aspects of colorectal carcinoma treatment.Because of the well-defined steps in its progression (adenoma - Tis - T1 invasive cancer- T2 advanced cancer with metastases), colorectal cancer (CRC) represents a model for investigating the effects of angiogenesis throughout tumor development.The aim of this study is to determine the role of VEGF expression by immunohistochemistry (IHC) in normal epithelium, dysplasia, carcinoma of colorectal specimens and to correlate the same with tumor grade, stage, nodal status, and metastasis. This is a retrospective study on paraffin blocks of 50 colon cancer specimens, 40 adenoma specimens, and 10 normal colonic mucosa specimens. Immunohistochemical stain for VEGF was done on the sections along with controls. Monoclonal antibody detected against VEGF antigen was observed in the cytoplasm of the tumor cells and the intensity ofVEGF expression in individual tumor cells was scored on a scale of 0 (no staining) to 3 (strong intensity), and the percentage of cells with VEGF staining at each intensity was estimated from 0 to 100. Pearson's Chi-squared test and Mann-Whitney test were used to determine significant clinicopathological differences between VEGF expression in positive and negative tumors. In normal epithelium, VEGF immunoreactivity was seen in all 10 cases with high intensity. Among adenomas, VEGF expression was seen in 26 (65%) of the 40 cases. Out of which in tubular adenomas VEGF expression was seen in 13 cases (60%) and negative in eight cases (40%). In tubulo villous adenoma, VEGF expression was seen in nine cases (60%) and negative in six cases (40%). Villous adenomas showed VEGF expression in all four cases (100%). In adenocarcinoma, VEGF expression was seen to be expressed in 42 cases (84%) and negative in eight cases (16%) and expression washigher in low-grade carcinomas (70%) compared to high-grade carcinomas. A significant difference in the expression ofVEGF among adenomas and carcinomas was observedwith higher intensity present in adenoma when compared to carcinoma. Expression ofVEGF could be considered as an early carcinogenic factor incolorectal carcinomas as it is expressed in higher intensity in the precancerous lesion and low-grade and stage 1 adenocarcinoma. Hence, we infer that early colorectal carcinomas are an important model for targeted therapy with antiangiogenic factors for VEGF.

The role of cyclophilin A and VEGF in the pathogenesis and recurrence of pterygium.

Pterygium is defined as overgrowth of abnormal conjunctival tissue on the cornea. Many proinflammatory cytokines and various growth factors have been implicated in the pathogenesis of pterygium. Cyclophilin A (CyPA) is a protein that is used by cyclosporin A (CsA) as the intracellular receptor and is secreted in response to inflammatory stimuli. Vascular endothelial growth factor (VEGF) is the most important angiogenic factor. This study aimed to assessment CyPA and VEGF immunoreactivity in pterygium specimens. In this cross-sectional study, 32 primary pterygium samples, 25 recurrent pterygium samples and 25 normal bulbar conjunctiva samples were included. The histopathological features, CyPA and VEGF immunoreactivity of surgically excised pterygium specimens were compared with control conjunctiva specimens obtained from normal bulbar conjunctiva. CyPA immunoreactivity in vascular endothelial cells, epithelial cells, and stromal cells was remarkably higher in pterygium specimens than control conjunctiva specimens (p = 0.004, p = 0.012, p = 0.001, respectively). Morever, VEGF immunoreactivity in endothelial cells was remarkably higher in pterygium specimens than control conjunctiva specimens (p < 0.001). When recurrent and primary pterygium specimens were compared, CyPA and VEGF immunoreactivity was remarkably higher in recurrent pterygium (p = 0.001, p = 0.001, respectively). Pearson correlation showed that CyPA immunoreactivity correlated with stromal vascularity, stromal inflammation, and mast cell count in pterygium specimens. This study aimed to assess CyPA and VEGF may have a important function in the pathogenesis and recurrence of pterygium.

The therapeutic effects of coenzyme Q10 on surgically induced endometriosis in Sprague Dawley rats

The aim of this study was to evaluate the effects of coenzyme Q10 in the treatment of endometriosis rat models. Twenty seven Sprague Dawley rats were divided into four groups; Control Group (n = 7; Endometriosis group), Reference Group (n = 6; Endometriosis + Buserelin acetate, 20 mg/kg), CoQ10 Group-I (n = 7; Endometriosis + CoQ10, 50 mg/kg) and CoQ10 Group-II (n = 7; Endometriosis + CoQ10, 100 mg/kg). At the end of the experiment, all the rats were sacrificed, and the volume and histoarchitecture of endometrial implants were evaluated. The mast cells were determined by Toluidine blue and collagen fiber density was analysed by Masson’s Trichrome staining. Tumour necrosis factor and vascular endothelial growth factor (VEGF) levels were analysed by enzyme-linked immunosorbent assay in peritoneal fluid and VEGF and matrix metalloproteinase-9 (MMP-9) were evaluated by immunohistochemistry. Terminal deoxynucleotidil transferase-mediated dUTP Nick end labelling (TUNEL) was also used for the detection of apoptotic cells. The CoQ10 treatment significantly decreased the volume of endometriotic implants, VEGF, and MMP-9 immunoreactivity and increased TUNEL-positive cells. The findings of the study suggest that CoQ10 can be used in endometriosis treatment by suppressing the endometriotic implants. IMPACT STATEMENT What is already known on this subject? Endometriosis is a gynaecological disorder and previous studies have shown that different treatments with antioxidants cause significant regression in the endometriotic implants. What the results of this study add? In this study, CoQ10 reduced intra-abdominal adhesion scores and volume of the endometriotic implants. In addition, CoQ10 treatment affected mast cell, TNF-α, VEGF, and MMP-9. What of these findings for clinical practice and/or further research? CoQ10 treatments may be possible to apply, it can contribute to science in terms of a new therapeutic treatment for endometriosis. Further studies are required to evaluate the Coenzyme Q10’s effects on pain and subfertility in endometriosis.

Effects of neferine on retinal tissue in experimental diabetic rat model.

To investigate vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) immunoreactivities, as well as apoptosis and oxidative stress levels in Streptozotocin (STZ)-induced diabetic rats, and determine how neferine affected these parameters. Thirty-five male Sprague Dawley rats were divided into five groups of seven. Fasting blood glucose was measured 72h after diabetes mellitus (DM) induction in 21 rats using 60mg/kg STZ dissolved in 0.4ml (0.1M) sodium-citrate buffer (pH:4.5), with values > 250mg/dl considered diabetic. Group 1 received no treatment. Group 3 (healthy rats) received daily intraperitoneal (IP) 4mg/kg neferine. Following DM induction: Group 2 (sham) received daily IP 0.25ml/kg 0.9% normal saline; Group 4 received single IP 0.01mL (2.5mg/kg) bevacizumab, followed by daily IP 0.25mL/kg 0.9% normal saline; and Group 5 received daily IP 4mg/kg neferine. Total antioxidant capacity (TAC) and total oxidative stress (TOS) levels in serum and ocular tissue homogenates were evaluated using ELISA. TUNEL method was used for determining apoptosis and immuno-histochemical staining for PCNA and VEGF immunoreactivities. Group 5 had significantly higher TAC and lower TOS in serum and ocular tissue homogenates than Group 4 (p < 0.05). Despite significantly lower VEGF levels and apoptosis (p < 0.05), there was no significant change in PCNA immunoreactivity in Group 5 (p > 0.05). DM was associated with lower TAC, higher TOS and apoptotic cells, as well as VEGF and PCNA immunoreactivities in the retina. Neferine altered parameters other than PCNA in the opposite direction, demonstrating reductive effects on DM.

Brain Activity after Intermittent Hypoxic Brain Condition in Rats.

Hypoxic brain injury is accompanied by a decrease in various functions. It is also known that obstructive sleep apnea (OSA) can cause hypoxic brain injury. This study aimed to produce a model of an intermittent hypoxic brain condition in rats and determine the activity of the brain according to the duration of hypoxic exposure. Forty male Sprague–Dawley rats were divided into four groups: the control group (n = 10), the 2 h per day hypoxia exposure group (n = 10), the 4 h per day hypoxia exposure group (n = 10), and the 8 h per day hypoxia exposure group (n = 10). All rats were exposed to a hypoxic chamber containing 10% oxygen for five days. Positron emission tomography–computed tomography (PET-CT) brain images were acquired using a preclinical PET-CT scanner to evaluate the activity of brain metabolism. All the rats were subjected to normal conditions. After five days, PET-CT was performed to evaluate the recovery of brain metabolism. Western blot analysis and immunohistochemistry were performed with vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). The mean SUV was elevated in the 2 h per day and 4 h per day groups, and all brain regions showed increased metabolism except the amygdala on the left side, the auditory cortex on the right side, the frontal association cortex on the right side, the parietal association cortex on the right side, and the somatosensory cortex on the right side immediately after hypoxic exposure. However, there was no difference between 5 days rest after hypoxic exposure and control group. Western blot analysis revealed the most significant immunoreactivity for VEGF in the 2, 4, and 8 h per day groups compared with the control group and quantification of VEGF immunohistochemistry showed more expression in 2 and 4 h per day groups compared with the control group. However, there was no significant difference in immunoreactivity for BDNF among the groups. The duration of exposure to hypoxia may affect the activity of the brain due to angiogenesis after intermittent hypoxic brain conditions in rats.

Inhibition of Hippo Signaling Improves Skin Lesions in a Rosacea-Like Mouse Model

The Hippo signaling pathway plays a key role in regulating organ size and tissue homeostasis. Hippo and two of its main effectors, yes-associated protein (YAP) and WWTR1 (WW domain-containing transcription regulator 1, commonly listed as TAZ), play critical roles in angiogenesis. This study investigated the role of the Hippo signaling pathway in the pathogenesis of rosacea. We performed immunohistochemical analyses to compare the expression levels of YAP and TAZ between rosacea skin and normal skin in humans. Furthermore, we used a rosacea-like BALB/c mouse model induced by LL-37 injections to determine the roles of YAP and TAZ in rosacea in vivo. We found that the expression levels of YAP and TAZ were upregulated in patients with rosacea. In the rosacea-like mouse model, we observed that the clinical features of rosacea, including telangiectasia and erythema, improved after the injection of a YAP/TAZ inhibitor. Additionally, treatment with a YAP/TAZ inhibitor reduced the expression levels of YAP and TAZ and diminished vascular endothelial growth factor (VEGF) immunoreactivity in the rosacea-like mouse model. Our findings suggest that YAP/TAZ inhibitors can attenuate angiogenesis associated with the pathogenesis of rosacea and that both YAP and TAZ are potential therapeutic targets for patients with rosacea.

Effect of Decorin and Bevacizumab on oxygen-induced retinopathy in rat models: A comparative study.

Purpose:The aims of this study were to evaluate the effects of decorin (DCN) in rat oxygen-induced retinopathy (OIR) model and to compare the results with those of bevacizumab.Methods:Twenty-eight newborn Sprague–Dawley rats were randomly divided into four groups. Group I (control): normoxia plus intraperitoneal (ip) normal saline (NS), Group II (sham): OIR plus ip NS, Group III (DCN): OIR plus ip 0.1 mg/kg DCN, and Group IV (bevacizumab): OIR plus ip 2.5 mg/kg bevacizumab. The OIR model was induced by cycling the oxygen concentration between 50% and 10% every 24 h for 14 days following their birth. In all groups, injections were administered on postnatal day (PD) 15. All animals were sacrificed and their right eyes were enucleated on PD 18. The nuclei of neovascular endothelial cells on the vitreal side of the inner limiting membrane were counted, and vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF)-α immunoreactivity were detected in histopathological and immunohistochemical examinations. One-way analysis of variance and post hoc Tukey tests were used for statistical analyses of the data.Results:In Groups II, III, and IV, the mean neovascular cell nuclei counts were 13.14 ± 1.34, 6.57 ± 1.51, and 6.71 ± 1.49, respectively. The mean neovascular cell nuclei count was significantly reduced in treatment groups compared with sham group (P < 0.001). In immunohistochemical staining, the immunoreactivity of VEGF was 0.07 ± 0.02, 0.97 ± 0.21, 0.37 ± 0.12, and 0.23 ± 0.17, respectively. Likewise, immunoreactivity of TNF-α was 0.02 ± 0.02, 1.11 ± 0.36, 0.37 ± 0.13, and 0.62 ± 0.21, respectively. VEGF and TNF-α immunoreactivity increased markedly in the sham group compared with those in the control group (P < 0.001). VEGF and TNF-α immunoreactivity of treatment groups decreased significantly compared to sham group (P < 0.001).Conclusion:The beneficial effects obtained by DCN administration in OIR model were comparable to the effects of bevacizumab.

Evaluation of BMP-2, VEGF, and Vitamin D Receptor Levels in the Ligamentum Flavum of Patients with Lumbar Spinal Stenosis and Disc Herniation.

To histopathologically evaluate and compare bone morphogenetic protein (BMP)-2, vascular endothelial growth factor (VEGF), and vitamin D receptor (VDR) levels in the ligamentum flavum (LF) of patients with lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). Surgical specimens of the LF in 25 patients who underwent surgery for LDH and 25 patients who underwent surgery for LSS were examined histopathologically. The prevalence and severity of BMP-2, VEGF, and VDR immunoreactivity were evaluated to create histoscores (prevalence × severity), which were compared between groups. The mean BMP-2 histoscore was similar in both groups. In the LSS group, the mean VEGF histoscore was significantly higher and the mean VDR histoscore was significantly lower. Elevated VEGF and decreased VDR levels in the LF in LSS are associated with more intense inflammation and chronic process of the disease. The prominent expression of BMP-2 in the LF in both diseases suggests that BMP-2 might be affected by inflammation regardless of chronic pressure and degeneration.

Poly(ester amide) microspheres are efficient vehicles for long-term intracerebral growth factor delivery and improve functional recovery after stroke

Growth factors promote plasticity in injured brain and improve impaired functions. For clinical application, efficient approaches for growth factor delivery into the brain are necessary. Poly(ester amide) (PEA)-derived microspheres (MS) could serve as vehicles due to their thermal and mechanical properties, biocompatibility and biodegradability. Vascular endothelial growth factor (VEGF) exerts both vascular and neuronal actions, making it suitable to stimulate post-stroke recovery. Here, PEA (composed of adipic acid, L-phenyl-alanine and 1,4-butanediol) MS were loaded with VEGF and injected intracerebrally in mice subjected to cortical stroke. Loaded MS provided sustained release of VEGF in vitro and, after injection, biologically active VEGF was released long-term, as evidenced by high VEGF immunoreactivity, increased VEGF tissue levels, and higher vessel density and more NG2+ cells in injured hemisphere of animals with VEGF-loaded as compared to non-loaded MS. Loaded MS gave rise to more rapid recovery of neurological score. Both loaded and non-loaded MS induced improvement in neurological score and adhesive removal test, probably due to anti-inflammatory action. In summary, grafted PEA MS can act as efficient vehicles, with anti-inflammatory action, for long-term delivery of growth factors into injured brain. Our data suggest PEA MS as a new tool for neurorestorative approaches with therapeutic potential.

Effects of an Aquaporin 4 Inhibitor, TGN-020, on Murine Diabetic Retina.

Purpose: To investigate the effect of a selective aquaporin 4 (AQP4) inhibitor, 2-(nicotinamide)-1,3,4-thiadiazole (TGN-020), on the expression of vascular endothelial growth factor (VEGF) and reactive oxygen species (ROS) production, as well as on the retinal edema in diabetic retina. Methods: Intravitreal injections of bevacizumab, TGN-020, or phosphate-buffered saline (PBS) were performed on streptozotocin-induced diabetic rats. Retinal sections were immunostained for anti-glial fibrillary acidic protein (GFAP), anti-AQP4, and anti-VEGF. Protein levels of VEGF from collected retinas were determined by Western blot analysis. In addition, retinal vascular leakage of Evans Blue was observed in the flat-mounted retina from the diabetic rats in the presence or absence of TGN-020. Volumetric changes of rat retinal Müller cells (TR-MUL5; transgenic rat Müller cells) and intracellular levels of ROS were determined using flow cytometry analysis of ethidium fluorescence in the presence or absence of TGN-020 or bevacizumab under physiological and high glucose conditions. Results: In the diabetic retina, the immunoreactivity and protein levels of VEGF were suppressed by TGN-020. AQP4 immunoreactivity was higher than in the control retinas and the expressions of AQP4 were co-localized with GFAP. Similarly to VEGF, AQP4 and GFAP were also suppressed by TGN-020. In the Evans Blue assay, TGN-020 decreased leakage in the diabetic retinas. In the cultured Müller cells, the increase in cell volumes and intracellular ROS production under high glucose condition were suppressed by exposure to TGN-020 as much as by exposure to bevacizumab. Conclusion: TGN-020 may have an inhibitory effect on diabetic retinal edema.

Angiogenesis in primary colorectal cancer and matched metastatic tissues: Biological and clinical implications for anti-angiogenic therapies.

Metastasis remains a notable issue in patients with newly diagnosed colorectal carcinomas (CRC). Although anti-angiogenic therapies target metastatic diseases, hypoxia-inducible factor-1 α (HIF-1α) and vascular endothelial growth factor (VEGF) status are routinely evaluated in primary tumors as metastatic sites are infrequently biopsied. The present study aimed to investigate the expression and significance of HIF-1α, VEGF and microvascular density (MVD) in primary tumors and corresponding metastatic CRC tissues. HIF-1α, VEGF and CD34 status were analyzed via immunohistochemistry analysis in 46 patients who underwent surgical resection of primary CRC (35 colon and 11 rectum) and matched metastases (lymph node and liver metastases) in Shandong Cancer Hospital. The association between selected biomarker status and clinicopathological characteristics was analyzed, and expression levels in primary tumors and corresponding metastases were compared. A total of 46 paired colorectal primary tumor and synchronous metastases samples were acquired for analysis using a standardized HIF-1α, VEGF and CD34 immunohistochemical procedure. The results demonstrated that the positive rates of HIF-1α and VEGF in primary CRC were 70 and 73.9%, respectively. HIF-1α (60.9%) and VEGF (58.7%) expression decreased in the lymph metastatic samples compared with primary CRC. Conversely, the level of MVD in primary tumors was significantly higher compared with metastatic tumors. No significant differences were demonstrated between HIF-1α and VEGF expression and the different clinicopathological features in primary CRC and corresponding metastases. Primary carcinomas and matched metastatic tissues demonstrated a moderate level of consistent immunoreactivity for HIF-1α and VEGF. HIF-1α, VEGF and CD34 were expressed in both primary tumors and corresponding metastases of CRC, suggesting that they may be involved in the development of metastasis. HIF-1α and VEGF expression in primary sites was consistent with that observed in metastases; however, it varied from that exhibited in MVD. The current analysis will improve the current understanding of the metastasis models and provide further evidence for evaluating the response to HIF-1α and VEGF inhibitors.

İskemi/Reperfüzyon Hasarı Sonrası Sıçanların Over ve Uterusunda IL-1 ve VEGF Değişiklikleri

Objective:Ischemia/reperfusion injury causes parenchymal and endothelial cell damage as a result of inflammation. Vascular endothelial growth factor (VEGF) expressed in every kind of tissue in human body has important roles in migration, proliferation, endothelial cell permeability, angiogenesis and vasculogenesis. IL-1 is a one of the cytokine family members, and plays important roles in hematopoiesis, inflammatory reactions and immune system regulation. Furthermore, auto-inflammatory diseases are treated by IL-1 as therapeutic agent. The aim of this study is to observe changes of VEGF and IL-1 immunreactivity in ischemia/reperfused rat uterus and ovary.Method:Rats were separated into two groups. Control group and ischemia/reperfusion group which rats were subjected to 45 min ischemia/45 min reperfusion. Samples from uterus and ovary were fixed with 10% neutral formaldehyde and stained with H&E. VEGF and IL-1 immunohistochemistry was applied.Results:Histopathological results showed severe degeneration of endometrium in uterus and ovarian follicles in ischemia/reperfusion group. VEGF and IL-1 immunoreactivity increased in uteruses and ovaries of ischemia/reperfusion group when compared to control groupConclusionIn consequence, the present results suggest that VEGF and IL-1 may be potential detection marker for ischemia/reperfusion injured uterus and ovary. Moreover, VEGF and IL-1 might be in relation with each other to regenerate uterus and ovary.

Differential Expression of Vascular Endothelial Growth Factor in the Cortex and Hippocampus upon Cerebral Hypoperfusion

Vascular endothelial growth factor (VEGF) provides tolerance against ischemic brain injury, yet, the pattern of VEGF expression in the neurogenic zones following chronic cerebral hypoperfusion has not been studied. Here we evaluated the immunoreactivity of VEGF in a rat model of chronic cerebral hypoperfusion. Chronic hypoperfusion was induced by bilateral common carotid artery ligation in rats. Immunohistochemistry was performed against hypoxia-inducible factor-1α (HIF-1α) and VEGF on brain sections. The density of HIF1α-positive cells in the hypoxia group was increased in the cerebral cortex and hippocampus. Further, the density of VEGF-positive cells was significantly higher in the hypoxia group compared to the control group in the cerebral cortex whereas it was similar in the subventricular zone, and in the dentate gyrus in the hippocampus between the two groups. The pattern of VEGF expression varies in different brain regions following chronic cerebral hypoperfusion.

Immunohistochemical and Ultrastructural Features of the Seasonal Changes in the Epididymal Epithelium of Camel (Camelus dromedarius).

In order to evaluate the influence of reproductive activity on the functional role of the epididymal epithelium in the Egyptian dromedary camel, Connexin-43 (Cx-43), vascular endothelial growth factor (VEGF), and androgen receptor (AR) immunoreactivity in the epididymal epithelium and the fine structure of the principal, dark, basal, apical, and halo cells were investigated. The secretory activity of the principal cells was amplified in the breeding season, while its endocytotic function became more active in the nonbreeding season. This was evidenced by punctate strong immunoreactive signals for Cx-43, which appeared to be more intense in the apical region of these epithelial cells, and the extremely long slender stereocilia (microvilli) with multiple junctional complexes. The nonbreeding principal cells revealed granular immunoreactive signals for VEGF scattered in the apical and basal cytoplasm. Ultrastructurally, both extreme vacuolation and several multivesicular inclusion bodies were observed in their cytoplasm. Dark cell size greatly diminished in the nonbreeding season and their nuclear morphology greatly changed from oval to lobulated shape. The plasma membrane of the apical cells expressed several infoldings (microvilli) in the breeding season. However, it was almost smooth in the nonbreeding season except for a small microvillus that appeared as a bleb-like projection. In some regions, a strong dense immunoreactivity for VEGF could be recognized in the cytoplasm of the apical cells and some basal ones. Halo cells with numerous multivesicular inclusions occupying most of the cytoplasm and a lobulated eccentric nucleus were detected in the nonbreeding season. In conclusion, these findings indicate that the reproductive activity has a significant impact on the immunohistochemical and ultrastructural profiles of the epithelial cells lining the Egyptian dromedary camel epididymis.

Von Hippel-Lindau Gene Mutations and Vascular Endothelial Growth Factor Immunoexpression in Clear Cell Renal Cell Carcinoma

Renal cell carcinoma (RCC) is the most common type of kidney cancer in adults representing about 3% of all newly diagnosed cancers in the United States. Clear cell RCC is the most common subtype (70-80%) of RCC. Clear cell RCC can be familial, but 95% of cases are sporadic resulting from the germline or acquired mutation of Von Hippel-Lindau (VHL) gene. VHL tumor suppressor gene functions as a down regulator of vascular endothelial growth factor (VEGF). Mutations of VHL gene result in overexpression of VEGF, neoangiogenesis and tumor metastasis. Nowadays, anti-VEGF targeted therapy is used for treating metastasis clear cell RCC. However, drug resistance occurs over time. VHL gene targeted therapy combined with anti-VEGF therapy should be considered and detection of VHL gene mutations status becomes essential in these cases. The present study was aimed to detect the VHL gene mutations status and VEGF immunoexpression in 62 clear cell RCC patients by conventional polymerase chain reaction and immunohistochemistry. Three primer pairs were used to detect the mutations of 3 exons in VHL gene. The positive cases for VHL exon 1 mutation, exon 2 mutation and exon 3 mutation were checked by 2% agarose gel electrophoresis. Tumor grading was done by Fuhrman nuclear grading system and staging was done by pathologic TNM staging system. Fifty cases (80.65%) were VHL gene mutation positive and 12 cases (19.35%) were negative. VHL gene mutations were significantly associated with histological grades (p=0.005). Out of 62 cases, 24 cases were weakly positive (1+) and 38 cases were strongly positive (2+) VEGF immuno-reactivity. There was statistically significant association between VEGF immunoexpression and histological grades of clear cell renal cell carcinoma (p=0.00) as well as tumor stage (p=0.01). It was also found that VEGF immunoexpression of clear cell RCC was significantly associated with VHL gene mutation positive tumours (p=0.00). These results can be helpful in further invention of molecular targeted therapy for drug-resistant clear cell RCC patients.

Combining Ellagic Acid with Temozolomide Mediates the Cadherin Switch and Angiogenesis in a Glioblastoma Model

We aimed to evaluate the combined effect of ellagic acid (EA) and temozolomide (TEM) on the cadherin switch and angiogenesis in the C6 glioma cell line. A total of 100 μM EA and 100 μM TEM were applied to rat C6 glioma cells for 24, 48, and 72 hours. Cell proliferation was detected by 5-bromo-2'-deoxyuridine immunohistochemistry. The messenger RNA and protein levels of E-cadherin, N-cadherin, and vascular endothelial growth factor (VEGF) were determined by real-time polymerase chain reaction and their immunohistochemistry, respectively, subsequent to EA treatment combined with TEM. EA in combination with TEM conspicuously reduced the viability of C6 glioma cells at all incubation times (P < 0.001). EA upregulated the expression of E-cadherin at thegene and protein levels in a time-independent manner (P< 0.05 and P < 0.001, respectively). By the presence of TEM,the increase was exaggerated at 24-hour incubation (P< 0.01). Conversely, EA reduced N-cadherin expression and immunoreactivity in a time-independent manner (P < 0.05 and P < 0.001, respectively), and combination with TEM enhanced this effect at the 24th hour (P < 0.001). Combination also downregulated the gene expression (P < 0.001) and immunoreactivity of VEGF only at 72 hours (P < 0.001). A successful therapeutic efficacy of EA combined with TEM is suggested probably by inhibiting the cadherin switch and angiogenesis.

Histological and Immunohistochemical Study on the Possible Effect of Mangosteen and Mesenchymal Stem Cells on Isoproterenol Induced Myocardial Infarction in Adult Male Albino Rats

Background: Myocardial infarction (MI) is one of the causes of cardiovascular diseases that lead to death despite of the great advancements in the medical interventions.Aim of the work: To assess the possible effect of mangosteen and mesenchymal stem cells (MSCs) in Isoproterenol (ISO)-induced MI in adult male albino rats, monitored by histological and immunohistochemical methods.Materials and Methods: Fifty-five adult male albino rats were divided into: control group (GI), group II (ISO), group III (Mangosteen+ISO), group IV (ISO+MSCs), group V (Mangosteen+Isoproterenol+MSCs). ISO was given subcutaneously, twice (250mg/kg/d), mangosteen was given orally (18mg/200gm) and MSCs (5x106 cells in 1mL of PBS) were injected intracardiac. Myocardial sections were stained with H&E, Masson’s trichrome and immunohistochemical stain for caspase-3 and vascular endothelial growth factor (VEGF). The mean area % of collagen fibers and immunoreactivity of caspase-3 and VEGF were measured by the image analyzer. The statistical analysis was applied by using ANOVA.Results: Myocardial sections of G II revealed discontinuity and degeneration of cardiac muscle fibers, pyknotic nuclei, inflammatory cellular infiltration and congested blood vessels. There were increased collagen deposition and significant increase in caspase-3 immunoreactivity. Mangosteen administration (G III) showed partial improvement in cardiac muscle fibers and reduced inflammatory cellular infiltration.MSCs treatment (G IV) resulted in obvious decline in myocardial damage, apoptosis with significant reduction of collagen deposition and significant increase in VEGF immunoreactivity. Better results are obtained when MSCs and mangosteen were combined (G V).Conclusion: Mangosteen exerts a synergistic effect with MSCs against ISO-induced MI.

Poly(Ester Amide) Microspheres are Efficient Vehicles for Long-Term Intracerebral Growth Factor Delivery and Improve Functional Recovery after Stroke

Growth factors promote plasticity in injured brain and improve impaired functions. For clinical application, efficient approaches for growth factor delivery into the brain are necessary. Poly(ester amide) (PEA)-derived microspheres (MS) could serve as vehicles due to their thermal and mechanical properties, biocompatibility and biodegradability. Vascular endothelial growth factor (VEGF) exerts both vascular and neuronal actions, making it suitable to stimulate post-stroke recovery. Here, PEA (composed of adipic acid, L-phenyl-alanine and 1,4-butanediol) MS were loaded with VEGF and injected intracerebrally in mice subjected to cortical stroke. Loaded MS provided sustained release of VEGF in vitro and, after injection, biologically active VEGF was released long-term, as evidenced by high VEGF immunoreactivity, increased VEGF tissue levels, and higher vessel density and more NG2+ cells in injured hemisphere of animals with VEGF-loaded as compared to non-loaded MS. Loaded MS gave rise to more rapid recovery of neurological score. Both loaded and non-loaded MS induced improvement in neurological score and adhesive removal test, probably due to anti-inflammatory action. In summary, grafted PEA MS can act as efficient vehicles, with anti-inflammatory action, for long-term delivery of growth factors into injured brain. Our data suggest PEA MS as a new tool for neurorestorative approaches with therapeutic potential.

Immunomodulatory drugs alleviate l-dopa-induced dyskinesia in a rat model of Parkinson's disease.

Thalidomide and closely related analogues are used clinically for their immunomodulatory and antiangiogenic properties mediated by the inhibition of the proinflammatory cytokine tumor necrosis factor α. Neuroinflammation and angiogenesis contribute to classical neuronal mechanisms underpinning the pathophysiology of l-dopa-induced dyskinesia, a motor complication associated with l-dopa therapy in Parkinson's disease. The efficacy of thalidomide and the more potent derivative 3,6'-dithiothalidomide on dyskinesia was tested in the 6-hydroxydopamine Parkinson's disease model. Three weeks after 6-hydroxydopamine infusion, rats received 10 days of treatment with l-dopa plus benserazide (6 mg/kg each) and thalidomide (70 mg/kg) or 3,6'-dithiothalidomide (56 mg/kg), and dyskinesia and contralateral turning were recorded daily. Rats were euthanized 1 hour after the last l-dopa injection, and levels of tumor necrosis factor-α, interleukin-10, OX-42, vimentin, and vascular endothelial growth factor immunoreactivity were measured in their striatum and substantia nigra reticulata to evaluate neuroinflammation and angiogenesis. Striatal levels of GLUR1 were measured as a l-dopa-induced postsynaptic change that is under tumor necrosis factor-α control. Thalidomide and 3,6'-dithiothalidomide significantly attenuated the severity of l-dopa-induced dyskinesia while not affecting contralateral turning. Moreover, both compounds inhibited the l-dopa-induced microgliosis and excessive tumor necrosis factor-α in the striatum and substantia nigra reticulata, while restoring physiological levels of the anti-inflammatory cytokine interleukin-10. l-Dopa-induced angiogenesis was inhibited in both basal ganglia nuclei, and l-dopa-induced GLUR1 overexpression in the dorsolateral striatum was restored to normal levels. These data suggest that decreasing tumor necrosis factor-α levels may be useful to reduce the appearance of dyskinesia, and thalidomide, and more potent derivatives may provide an effective therapeutic approach to dyskinesia. © 2019 International Parkinson and Movement Disorder Society.

Effect of regular swimming exercise to duration-intensity on neurocognitive function in cerebral infarction rat model

ABSTRACTObjective: This study investigated the effect of regular swimming exercise according to the duration-intensity on neurocognitive function in a cerebral infarction rat model.Methods: Forty male Sprague–Dawley 10-week-old rats, weighing 300 ± 50 g, were subjected to photothrombotic cerebral infarction. The remaining 36 rats were randomly divided into four groups (n = 9 per group: non-exercise (group A); swimming exercise of short duration-intensity (5 min/day, group B); swimming exercise of moderate duration-intensity (10 min/day, group C); and swimming exercise of long duration-intensity (20 min/day, group D). Exercise was performed five times a week for 4 weeks, beginning the day after cerebral infarction. Neurocognitive function was evaluated with the Morris water maze test. Immunohistochemistry and western blot analysis examined brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) at 4 weeks postinfarction.Results: At 4 weeks postinfarction, escape latency was found to be shorter in group C than in any of groups A, B, or D. Immunohistochemistry revealed the most significant immunoreactivity for BDNF and VEGF in group C. Western blot analysis demonstrated that BDNF and VEGF proteins were markedly expressed in group C.Conclusions: Regular swimming exercise of moderate duration-intensity may be the most effective exercise protocol for the recovery of neurocognitive function in cerebral infarction rat model.