Vascular Endothelial Growth Factor Staining Intensity Research Articles

Evaluation of VEGF, BDNF, TRKB expression in oral epithelial dysplasia, oral verrucous carcinoma and oral squamous cell carcinoma and their role as prognostic indicator

ABSTRACT Background: Oral squamous cell carcinoma (OSCC) is a serious health disease that can lead to a reduced quality of life or even death. It ranks sixth in terms of cancer expansion. It is one of India’s primary causes of natural death. In OSCC such potentially malignant Disorders (PMDs) are precancerous lesions with such a high risk of progression. Tumor angiogenesis is a one of the basic biomarkers that may influence the proliferation of a precancerous lesion into the cancerous lesion. Tropomyosin receptor kinase B (TrkB), vascular endothelial growth factor (VEGF), and brain-derived neurotrophic factor (BDNF) also play important roles in carcinogenesis by promoting angiogenesis. The construction of new vessels of blood from existing vasculature is referred as angiogenesis. Aim of the Study: To get deep insights of immunohistochemistry expression of VEGF, BDNF, and TRKB in oral epithelial dysplasia (OED), verrucous carcinoma (VC), and OSCC. Material and Methods: The study included 100 formalin-fixed paraffin-embedded tissue blocks from 20 cases of OED, 20 cases of VC, and 60 cases of OSCC [20 cases of well-differentiated oral squamous cell carcinoma (WD-OSCC), 20 cases of moderately differentiated oral squamous cell carcinoma (MD-OSCC), and 20 cases of poorly differentiated oral squamous cell carcinoma (PD-OSCC). The staining intensity and distribution of VEGF, BNDF, and TrkB were examined and statistically analyzed using analysis of variance (ANOVA), post hoc Bonferroni test, independent t-test, Pearson’s Chi-square test, and Pearson’s correlation coefficient test. Results: The immunoexpression of VEGF, BDNF, and TrkB was found to be elevated in the order of OEDs, VC, and OSCC. The percentage of positive was highest in PD-OSCC, followed by MD-OSCC and WD-OSCC. Conclusion: Based on our findings, angiogenesis plays a significant role in tumor growth and metastasis. A substantial relationship was discovered between VEGF, BDNF, TrkB expression, and increases in vascularity throughout the transition from OEDs to VCs and OSCCs.

Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry.

There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

The impact of deproteinized bovine bone particle size on histological and clinical bone healing outcomes in the augmented sinus: A randomized controlled clinical trial.

The effect of different deproteinized bovine bone mineral (DBBM) particle sizes on bone healing in maxillary sinus floor augmentation remains unclear. This study compared the newly formed tissue and angiogenesis-related bone healing after sinus floor augmentation using large or small DBBM particles. Overall 32 patients were randomly divided into two groups using either large (1-2 mm) or small (0.25-1 mm) DBBM particles for sinus floor augmentation. After 6months, the mineralized tissue volume was calculated using micro-computed tomography (micro-CT) analysis. The newly formed tissue composition was histomorphometrically analyzed. Angiogenesis was also examined by means of vascular endothelial growth factor (VEGF) expression. Implant failure and marginal bone loss were measured at a 1-year follow-up. Statistical analysis was performed using independent samples t-test. Micro-CT analysis demonstrated that grafting with large particles resulted in higher bone volume (6.99 ± 2.72 mm3 , p=0.002) and Bone Volume/Tissue Volume (0.25 ± 0.1, p=0.03) compared with small particles (3.76 ± 1.83 mm3 and 0.14 ± 0.13, respectively). Small particles showed higher non-mineralized tissue volume (26.31 mm3 ) compared with large particle group (17.4 ± 5.34 mm3 ) with p=0.001. The histological data revealed significantly higher area of newly formed bone (32.15% ± 14.04% for the large particle and 15.99% ± 14.12% for the small particle groups, p=0.004). Likewise, non-mineralized tissue was significantly greater in the small particle group (66.48% ± 20.97%) compared with the large particle group (44.36%, p=0.016). Moreover, use of large particles resulted in a significantly higher VEGF staining intensity score and VEFG positive cells. No implant failure was recorded in both groups, while no difference was found in terms of marginal bone loss at the 1-year follow-up. Sinus floor augmentation using large DBBM particles resulted in more angiogenesis expression, higher bone volume, and new bone formation at 6months after sinus augmentation. However, clinical outcomes with regards to implant placement were similar in both groups.

An ethanol extract of Senduduk fruit (Melastoma malabathricum L) inhibits the expression of vascular endothelial growth factor and tumour necrosis factor alpha in HeLa cells

Aim Senduduk fruit (Melastoma malabathricum L) is native to Indomalaya and is believed to possess anticancer activity. This study investigated antiangiogenic and anti-inflammation effects of an ethanolic extract of Senduduk fruit (EESF) on HeLa cells. Methods Cytotoxicity was assayed in HeLa cell cultures exposed to a concentration series of 500-7.8 µg/mL of EESF. IC50 was determined with a methylthiazol-tetrazolium (MTT) cell viability assay. Antiangiogenic and anti-inflammation activity was evaluated by an immunocytochemical assay of vascular endothelial growth factor (VEGF) and tumour necrosis factor alpha (TNF-α) expression in HeLa cells cultured with 1× or 2× IC50 or as a control without EESF. Results IC50 of the EESF was 956 µg/mL. The intensity of VEGF staining indicated moderate expression in HeLa cells in response to IC50, weak expression in response to 2×IC50, and strong expression in the absence of the EESF. While the intensity of TNF-α staining indicated moderate expression in HeLa cells in response to IC50 and to the absence of EESF, and weak expression in response to 2× IC50. Conclusion Senduduk fruit extract inhibited VEGF and TNF-α expression in HeLa cells in a concentration-dependent manner.

The effect on wound healing of pazopanib and bevacizumab compared with corticosteroid in experimental glaucoma filtration surgery.

To compare the effects of bevacizumab and pazopanib with corticosteroids on wound healing after trabeculectomy. In the study, 35 New Zealand white rabbits were randomly divided into five groups. Apart from the first group, limbus-based trabeculectomy was performed for the eyes of rabbits. No postoperative treatment was administered for group I. Topically administered saline, prednisolone acetate (1%), bevacizumab 5 mg/mL, pazopanib 5 mg/mL for group II, III, IV and V respectively were applied for groups 6h daily for 28d. On day 28 of the experiment, eyes were enucleated and histologically and immunohistochemically analyzed. The fibroblast counts of groups IV and V were determined to be lower than those of groups II and III (P<0.05). In the mononuclear cell (MNC) count evaluation, no statistically significant difference was determined between the treatment groups (P>0.05). The immunohistochemical staining intensity of fibroblast growth factor β (FGF-β) and vascular endothelial growth factor (VEGF) was determined to be lower in groups IV and V than in groups II and III (P<0.05). No statistically significant difference was determined between groups IV and V in respect of fibroblast count, MNC count, FGF-β and VEGF staining intensity (P>0.05). The platelet derived growth factor β (PDGF-β) intensity was lower in group V than in groups II, III and IV (P<0.05). While the PDGF-β staining intensity was significantly lower in group IV than in group II, the difference compared with group III was not statistically significant (P>0.05). Bevacizumab and pazopanib might be good alternatives of corticosteroid treatment on delaying wound healing in glaucoma surgery.

Relationships of radiological and surgical variables and vascular endothelial growth factor expression with recurrence in juvenile nasopharyngeal angiofibroma

Juvenile nasopharyngeal angiofibroma (JNA) is a rare fibrovascular tumor that is histologically benign but clinically invasive and it is typified by a high recurrence rate. The recurrence rate is influenced by several characteristics including sociodemographic, clinical, radiological, and surgical variables and vascular endothelial growth factor (VEGF) expression. This observational retrospective cohort study aimed to clarify the relationships of these characteristics with the risk of recurrence in 38 male patients with JNA, including 11 patients with recurrence. JNA commonly develops in the second decade of life with a mean age of occurrence of 15.8 years. Nasal blockage and epistaxis were the most common complaints. Young age, early onset, late-stage disease, and high VEGF expression were linked to higher recurrence rates, albeit without significance. Meanwhile, the age of onset, the presence of an oropharyngeal mass, tumor stage, the use of preoperative embolization, and the intensity of VEGF staining appeared to have clinical significance regarding the risk of recurrence. In this study, preoperative embolization did not decrease the recurrence rate. The risk of recurrence was increased by the presence of residual tumor, suggesting that measures should be taken to ensure complete tumor resection during surgery.

Significance of vascular endothelial growth factor and CD31 and morphometric analysis of microvessel density by CD31 receptor expression as an adjuvant tool in diagnosis of psoriatic lesions of skin.

Pathogenesis of psoriasis is a debated issue. Several mechanisms have been proposed to identify the etiology and pathogenesis so that specific treatments can be given to patients with psoriasis. (1) To compare pattern and distribution of vascular endothelial growth factor (VEGF) and CD31 in patients with psoriasis and other psoriasiform lesions of skin. (2) To study the correlation between VEGF and CD31 expression, clinical severity, and histopathology of psoriasiform lesions of skin. (3) Evaluation of microvessel density (MVD) by using computer-assisted quantitative image analysis in psoriatic skin lesions. This study was conducted on eighty cases, out of which forty were diagnosed cases of psoriasis and forty cases of clinically suspected psoriasiform lesions, submitted in the Department of Pathology, Pt. B.D. Sharma, University of Health Sciences, Rohtak, for histopathological examination. Histopathological sections were stained by routine hematoxylin and eosin staining, and these biopsies were further subjected to immunohistochemical staining with VEGF and CD31 as per standard technique. Assessment of various histopathological features revealed strong correlation between epidermal hyperplasia, suprapapillary thinning, and elongation of rete ridges. Suprabasilar keratinocytes in psoriatic lesions stained intensely for VEGF. The difference for number of microvessels and MVD in psoriasis and psoriasiform lesions was statistically significant. Correlation between intensity of VEGF staining by suprabasilar keratinocytes and MVD was found to be highly significant in psoriatic lesions. The present study concluded that psoriatic lesions exhibit potent angiogenic activity. Early lesions show increased MVD along with other histomorphological parameters such as hypogranulosis, parakeratosis and Munro's microabscesses. Overexpression of VEGF by suprabasilar keratinocytes correlated with increased MVD in papillary dermis.

Histopathological effects of mesenchymal stem cells in rats with bladder and posterior urethral injuries

Background/aim: To evaluate the effects of mesenchymal stem cell (MSC) therapy in an experimental bladder and posterior urethral injury model. Materials and methods: The study subjects consisted of 40 male Wistar albino rats that were divided into four groups: control group (n = 10) (the bladder was only surgically opened and closed), sham group (n = 10) (surgical procedure), IVMSC group (n= 10) (surgical procedure and intravenous MSC treatment), and LMSC group (n = 10) (surgical procedure and local MSC treatment). Histopathological evaluation was performed for the degree of fibrosis and inflammation and the extent and intensity of staining of vascular endothelial growth factor (VEGF) and endoglin (CD105). Results: There were no significant differences between the control and LMSC groups with respect to fibrosis (P = 0.070) or inflammation (P = 0.048). Fibrosis and inflammation were significantly lower in the IVMSC (P = 0.034 for fibrosis, P = 0.080 for inflammation) and LMSC (P = 0.01 for fibrosis, P = 0.013 for inflammation) groups when compared with the sham group. No significant differences regarding fibrosis and inflammation were observed between the IVMSC and LMSC groups (P = 0.198 for fibrosis, P = 0.248 for inflammation). A significant difference was noted between the sham and LMSC groups concerning VEGF staining intensity (P = 0.017). However, no significant difference was found among the groups with regard to the extent or intensity of CD105 staining (P > 0.05). Conclusion: MSC treatment significantly decreased the development of fibrosis in a uroepithelial injury model.

EFFECTS OF Tempol ON SURVIVAL OF RANDOM PATTERN SKIN FLAPS IN RATS

To study the effects of the new small molecular oxygen free radical scavenger Tempol on the survival and vasculogenesis of the long random pattern skin flap (LRPSF) and its mechanism. Eighty-four male Sprague Dawley rats were randomly divided into control and Tempol groups (42 rats in each group). LRPSF of 9 cm×3 cm in size were prepared on the backs of rats in two groups based on the Mcfarlane flap. Rats were administered with Tempol (100 mg/kg) in the Tempol group and with normal saline in the control group by intraperitoneal injection at 15 minutes before operation and at 1-7 day after operation. The rat and the skin flap survival conditions were observed after operation; the survival rate of skin flap was measured, and the vascular structure, vascular volume, and total length of blood vessels were analyzed with Micro-CT three-dimensional imaging after 7 days; HE staining was used to observe the structure of the skin flaps and inflammation, immumohistochemical staining to observe vascular endothelial growth factor (VEGF) expression; water-soluble tetrazolium-1 method was used to measure the content of superoxide dismutase (SOD) and malondialdehyde (MDA), and ELISA to detect the expressions of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) after 1, 3, and 7 days. All of rats survived after operation, without hemorrhage, edema, and infection. With the extension of time, necrosis occurred in the distal part of the skin flaps in 2 groups, but the necrosis degree of the Tempol group was lower than that of control group; meanwhile, the blood vessel distribution and continuity were better than those of control group. The skin flaps survival rate, vascular volume, and total length of blood vessels of Tempol group were significantly higher than those of control group after 7 days (P<0.05). The clearer skin flaps structure, lighter inflammation reaction and inflammation cell infiltration, and higher VEGF staining intensity were observed in the Tempol group than the control group after 7 days. There was no significant difference in SOD, MDA, and TNF-α, and IL-6 contents between the 2 groups at immediate after operation. SOD significantly increased, but MDA, TNF-α, and IL-6 contents significantly decreased in the Tempol group when compared with control group after 1, 3, and 7 days (P<0.05). Tempol can significantly promote the LRPSF survival rates, its mechanism is closely related to the promotion of vasculogenesis and reduction of oxidative stress and inflammation.

The effect of penile urethral fat graft application on urethral angiogenesis.

Autologous fat grafts are rich in adipose-derived stem cells, providing optimal soft-tissue replacement and significant quantities of angiogenic growth factor. Although fat grafts (FG) are used in several clinical conditions, the use of FG in urethral repairs and the effects of FG to urethral repairs have not yet been reported. An experimental study was performed to evaluate the effect of FG on urethral angiogenesis and tissue growth factor (GF) levels. Sixteen Wistar albino, adult, male rats were allocated into two groups: the control group (CG) (n = 8) and the experiment group (EG) (n = 8). After anesthetization of all rats, 3-mm vertical incisions were made on the urethras, and then sutured with interrupted 5/0 vicryl sutures. The operations were performed under a stereo dissecting microscope under magnification (×20). In the CG, no additional procedure was performed. In the EG after the same surgical procedure, 1 mm(3) FG was removed from the inguinal region by sharp dissection with a knife. The grafts were trimmed to 1 × 1 mm dimensions on millimeter paper. The FGs were placed on the repaired urethras. The skin was then closed. Samples from urethral and penile skin were taken 21 days after surgery in both groups. Density and intensity of staining with vascular-endothelial GF (VEGF), VEGF-receptor, and endothelial-GF receptor (EGFR) in the endothelial and mesenchymal cells of the penile urethral vessels were immunohistochemically evaluated. Data obtained from immunohistochemical evaluations were analyzed with SPSS 15.0. The P-values lower than 0.05 were considered as significant. Density of VEGF staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05). Density of the EGFR staining was significantly decreased in the vascular endothelium of the EG compared to the CG (P < 0.05) (Table). Intensity of VEGF, VEGF-R and EGFR staining was not significantly different between the two groups. There were no significant differences between groups regarding to VEGFR staining and mesenchymal examination. Decreased density was found in the VEGF staining in the vascular endothelium. This could be explained by the day that the tissues were harvested or because autologous fat grafts might cause decreased growth factor levels, which is contrary to the literature data. Fat grafting has an immunohistochemical effect on the growth factor levels that are related to angiogenesis after urethral repair. It is difficult to make a firm conclusion about the role of fat grafting on urethral healing. Therefore, future studies are needed to see if FG can be used as an alternative to other procedures in order to avoid complications.

Expression of vascular endothelial growth factor and transforming growth factor alpha in rat testis during chronic renal failure.

Vascular endothelial growth factor (VEGF) is known to influence testis function. Transforming growth factor alpha (TGF-α) is expressed in the postnatal testis, and has been demonstrated to stimulate testis development. Systemic diseases such as chronic renal failure (CRF) interfere with hypothalamic-pituitary-gonadal axis, which may cause defective steroidogenesis and gonadal functions. The aim of this study was to investigate the expression and localization of VEGF and TGF-α in testicular tissues of experimental CRF model. Experimental CRF was induced in rats by the resection of more than 85% of renal mass. The expression of VEGF and TGF-α in testicular tissues were assessed by immunohistochemistry on paraffin sections of control, CRF-nondialysed and CRF-dialysed rats. The microscopic evaluation of the testicular structure showed that CRF did not affect testicular histology. Immunohistochemical evaluation showed that VEGF was expressed in the cytoplasm of primary and secondary spermatocyte series as well as the early spermatids. Staining intensity was lower in spermatocytes going through the first meiotic division. TGF-α was expressed in the nuclei of spermatogonia and primary spermatocytes with stronger staining intensity in spermatogonia. The intensity of VEGF staining was similar in control and experimental animals, however, TGF-α expression was lower in the CRF group. The continuous expression of VEGF in spermatocytes and spermatids suggests that the applied model of CRF does not directly disrupt morphology of seminiferous epithelium, thus also spermiogenesis. However, difference between control rats and CRF group in TGF-α immunopositivity, which was localised in spermatogonial mitosis step, may suggest the interference of CRF with early stages of spermatogenesis.

ANNEXIN A2 REGULATES ANGIOGENESIS AND INVASION PHENOTYPES OF MALIGNANT GLIOMA

BACKGROUND: Despite advances in multimodal therapy, patients with malignant glioma have a dismal prognosis. Aberrant angiogenesis and diffuse glioma cell invasion are major obstacles for effective treatment. Therefore, investigation of angiogenesis and invasion is essential for the development of a curative therapy. We established animal models that histologically mimic two invasive and angiogenic phenotypes of glioma, ie, angiogenesis-dependent invasion (J3T-1) and angiogenesis-independent invasion (J3T-2). In our previous study, comparative proteomic analysis showed that annexin A2 was more highly expressed in J3T-1 than in J3T-2. In this study, the function of annexin A2 in malignant glioma in relation to angiogenesis and invasion was investigated using these animal models. METHODS: Annexin A2-downregulated J3T-1 cells (J3T-1shA) and annexin A2-upregulated J3T-2 cells (J3T-2A) were generated by gene transfection. Quantitative reverse transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining of annexin A2 and vascular endothelial growth factor (VEGF) were performed on cultured cells. J3T-1, J3T-1shA, J3T-2, and J3T-2A brain tumors were established in athymic rats. Brain sections were evaluated histopathologically. Human glioblastoma specimens were stained for annexin A2, VEGF, and platelet-derived growth factor (PDGF). RESULTS: QRT-PCR and immunohistochemical staining of cultured cells revealed that expression of VEGF was upregulated in accordance with the expression of annexin A2 in J3T-1 and J3T-2A cells. J3T-1 tumors showed remarkable angiogenic activity and invasion around neovasculature, whereas J3T-1shA tumors, including J3T-2 tumors, showed no angiogenic activity, but diffuse single-cell infiltration of tumor cells into normal parenchyma. J3T-2A tumors were highly angiogenic and showed angiogenesis-dependent invasion. Human glioblastoma specimens revealed that annexin A2 was positive in clusters of tumor cells around dilated vessels (25/30 cases). The intensity of VEGF and PDGF staining corresponded to the expression of annexin A2 (correlation coefficients: annexin A2/VEGF: R = 0.73, P < 0.05; annexin A2/PDGF: R = 0.37, P < 0.05). CONCLUSIONS: Our results revealed that the phenotype of glioma invasion is closely related to angiogenesis, and annexin A2 is one of the factors that regulates angiogenesis and invasion phenotype of malignant glioma. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

Age-related changes in angiogenesis in human dermis

Present research is aimed to examine the number of dermal blood vessels, vascular endothelial growth factor (VEGF), delta-like ligand 4(Dll4) and Jagged-1 (Jag-1) in dermal blood vessels of human from 20weeks of pregnancy to 85years old. Numbers and proliferative activity of dermal fibroblast-like cells were also examined. Blood vessels were viewed with immunohistochemical staining for von Willebrand factor or CD31. VEGF, Dll4, Jag-1, and proliferating cell nuclear antigen (PCNA) were detected immunohistochemically. Results showed that the numbers of fibroblast-like cells, PCNA positive fibroblast-like cells, von Willebrand factor positive or CD31 positive blood vessels in dermis are dramatically decreased with age. The intensity of immunohistochemical staining for VEGF or Jag-1 in blood vessels of dermis is increased from antenatal to deep old period. The degree of immunohistochemical staining of dermal blood vessels for Dll4 has gone up from 20–40weeks of pregnancy to early life period (0–20years), and further decreased below antenatal values. Age-related decrease in the number of dermal blood vessels is suggested to be due to an impairment of VEGF signaling and to be mediated by Dll4 and Jag-1. It may be supposed that diminishing in blood supply of dermis occurring with age is a cause of a decrease in the number and proliferative pool of dermal fibroblasts.

High expressions of galectin-1 and VEGF are associated with poor prognosis in gastric cancer patients

High expressions of galectin-1 and vascular endothelial growth factor (VEGF) are correlated with biological behavior in some cancers. The aim of this study is to evaluate the expressions of galectin-1 and VEGF in gastric cancer and investigate their relationships with clinicopathological factors and prognostic significance. Immunohistochemical analyses for galectin-1 and VEGF expression were performed on 108 cases of gastric cancer. The relationship between the expression and staining intensity of galectin-1 and VEGF, clinicopathological variables, and survival rates was analyzed. Immunohistochemical staining demonstrated that 68 of 108 gastric cancer samples (63.0%) were positive for galectin-1 and 62 out of 108 gastric cancer samples (57.4%) were positive for VEGF. Galectin-1 expression was associated with tumor size, differentiation grade, TNM stage, lymph node metastases, and VEGF expression. VEGF expression was related to tumor size, TNM stage, and lymph node metastases. Kaplan-Meier survival analysis showed that high galectin-1 and VEGF expressions exhibited significant correlations with poor prognosis for gastric cancer patients. Multivariate analysis revealed that galectin-1 and VEGF expressions were independent prognostic parameters for the overall survival rate of gastric cancer patients. The results of the present study suggest that galectin-1 expression is positively associated with VEGF expression. Both galectin-1 and VEGF can serve as independent prognostic indicators of poor survival for gastric cancer.

Vascular endothelial growth factor expression in hepatitis C virus (HCV)-related advanced hepatocellular carcinoma (HCC) compared with hepatitis B virus (HBV)-related advanced HCC.

4115 Background: HCC with different etiologic factors may result in activation of different signaling pathways. This study aimed to clarify if HBV-related HCC (HBV-HCC) and HCV-related HCC (HCV-HCC) may have difference in the expression of key molecules that are relevant to contemporary molecular targeted therapy. Methods: We enrolled patients diagnosed with advanced HCC from 2001 to 2011 who had tumor tissues obtained upon the diagnosis of advanced HCC at our center. Tumor slides were immunohistochemically stained for phosphorylated extracellular signal-regulated kinases (p-ERK), Raf kinase inhibitory protein (RKIP), and vascular endothelial growth factor (VEGF). The expressions of p-ERK and RKIP were evaluated according to percentages of positive-staining cells and graded as: 0: 0; 1+: 1-10%; 2+: 11-50%; 3+: &gt; 50%. Grades 0 and 1 were considered negative, and grades 2 and 3 positive. VEGF staining was evaluated as strong or weak according to staining intensity. The staining results of VEGF were further recorded as H scores, defined as intensity (0, 1, 2, or 3) × percentages of positive staining. Results: In total, 131 patients were enrolled in this study; 94 (72%) patients had HBV-HCC, and 37 (28%) patients had HCV-HCC. HBV-HCC and HCV-HCC had similar expression of p-ERK (positive: 45% vs. 51%, p = 0.491) and RKIP (positive: 83% vs. 84%, p = 0.912). HCV-HCC was more likely to have strong VEGF staining than HBV-HCC (95% vs. 72%, p = 0.005) and higher H scores for VEGF staining (289.5 vs. 248.8, p&lt; 0.001). In multivariate analysis adjusting for age, sex, macrovascular invasion, extrahepatic metastasis, and α-fetoprotein level, HCV-HCC remained an independent factor associated with strong VEGF staining. VEGF staining intensity was not associated with age, sex, macrovascular invasion, extrahepatic metastasis, and α-fetoprotein level. Conclusions: HCV-HCC has stronger VEGF expression than HBV-HCC. (This study was supported by the grant of NSC101-2314-B-002-141, 100CAP1020-2, and NTUH.101-N1965.)

Clinicopathological and prognostic significance of galectin-1 and vascular endothelial growth factor expression in gastric cancer

To evaluate the expression of galectin-1 and vascular endothelial growth factor (VEGF) in gastric cancer and investigate their relationships with clinicopathologic factors and prognostic significance. Galectin-1 and VEGF were immunohistochemically investigated in tumor samples obtained from 214 gastric cancer patients with all tumor stages. Immunohistochemical analyses for galectin-1 and VEGF expression were performed on formalin-fixed, paraffin-embedded sections of surgical specimens. The relationship between the expression and staining intensity of galectin-1 and VEGF, clinicopathologic variables, and patient survival were analyzed. All patients underwent follow-up until cancer-related death or more than five years after tumor resection. P values < 0.05 were considered statistically significant. Immunohistochemical staining demonstrated that 138 of 214 gastric cancer samples (64.5%) were positive for galectin-1, and 116 out of 214 gastric cancer samples (54.2%) were positive for VEGF. There was a significant association between galectin-1 and VEGF expression; VEGF was detected in 60.1% of galectin-1-positive samples and 43.4% of galectin-1-negative samples (P < 0.05). Galectin-1 expression was associated with tumor size, tumor location, stage, lymph node metastases, and VEGF expression (all P < 0.05). VEGF expression was related to tumor size, stage, and lymph node metastases (all P < 0.05). The 5-year survival rate was 56.6% for galectin-1-positive patients and 69.2% for galectin-1-negative patients, and the prognosis for galectin-1-positive patients was significantly poorer compared with galectin-1-negative patients (χ² = 13.880, P = 0.000). The 5-year survival rates for VEGF-positive and VEGF-negative patients were 53.4% and 70.5%, respectively (χ² = 4.619, P = 0.032). The overall survival rate of patients with both galectin-1 and VEGF overexpression in gastric cancer tissue samples was significantly poorer than other groups (both P < 0.05). Galectin-1 expression was positively associated with VEGF expression. Both galectin-1 and VEGF can serve as independent prognostic indicators of poor survival for gastric cancer after gastrectomy.

Toll-like receptor 9 and vascular endothelial growth factor levels in human kidneys from lupus nephritis patients

In the lupus mouse and systemic lupus erythematosus (SLE) patients, DNA fragments isolated from plasma may mimic microbial DNA and trigger Toll-like receptor 9 (TLR9) signaling with formation of autoantibodies against DNA fragments and nucleosomes. Vascular endothelial growth factor (VEGF) is a tightly regulated angiogenic cytokine in the kidney. The present study investigated glomerular and tubular expression of both TLR9 and VEGF in biopsies from human subjects with lupus nephritis (LN) and normal controls. Kidney biopsies in LN (n=8) and normal controls (n=10) were evaluated for expression of TLR9 and VEGF. The degree of kidney damage was analyzed according to the International Society of Nephrology / Renal Pathology Society classification. Immunohistochemistry was performed, and slides were incubated with antibodies against VEGF and TLR9 monoclonal antibody, stained with hematoxylin and eosin, mounted and microscopically scored at ×10 and ×20. We observed intense staining of glomeruli and tubules for TLR9 up to 3+ from patients with LN. Samples from LN subjects showed 3+ staining of glomeruli but only up to 2+ in tubules for VEGF. There was less significant staining for TLR9 and none for VEGF in controls. There was no correlation observed between LN class severity and intensity of staining for VEGF or TLR9. This is the first study that investigated combined expression of TLR9 and VEGF, which could be an important tool for understanding the role of TLR9 and VEGF in LN, with insights into the early detection and targeted treatment of this disease.

Microvascular density as an independent predictor of clinical outcome in renal cell carcinoma: an automated image analysis study

Tumor microvascular density (MVD) has been shown to correlate with the aggressiveness of several cancers. With the introduction of targeted anti-angiogenic therapy, assessment of MVD has the potential not only as a prognostic but also as a therapeutic marker. The significance of tumor vascularity in clear cell renal cell carcinoma (ccRCC) has been debated, with studies showing contradictory results. Previous studies were limited by manual quantification of MVD within a small area of tumor. Since then, the validity of this method has been questioned. To avoid the inaccuracies of manual quantification, we employed a computerized image analysis, which allowed assessment of large areas of tumor and adjacent normal tissue. The latter was used as an internal reference for normalization. MVD and vascular endothelial growth factor (VEGF) were assessed in 57 cases of ccRCC. Sections were immunostained for CD34 and VEGF. Areas of ccRCC and normal kidney medulla were analyzed within scanned images using software that counted CD34-positive vessels and measured the intensity of VEGF staining. We obtained unadjusted values from tumoral areas and calculated adjusted values as tumor/normal ratios. Unadjusted MVD had no association with clinical outcome. However, similarly to tumor stage, higher adjusted MVD was associated with shorter disease-free survival (log-rank P=0.037, Cox P=0.02). This was significant in univariate and multivariate analyses. MVD did not correlate with tumor stage, pointing to its independent prognostic value. As expected due to the known molecular abnormalities in ccRCC, most tumors showed higher VEGF expression than normal tissue. Higher adjusted VEGF was associated with high tumor grade (P=0.049). The finding of increased MVD as an independent marker of tumor aggressiveness may prove useful in the development of new tests for prognostic and therapeutic guidance. Digital techniques can provide more accurate assessment of immunomarkers and may reveal less obvious associations.

EAF2 loss enhances angiogenic effects of Von Hippel-Lindau heterozygosity on the murine liver and prostate

Von Hippel-Lindau (VHL) disease results from the inactivation of the VHL gene and is characterized by highly vascular tumors. A consequence of VHL loss is the stabilization of hypoxia-inducible factor (HIF) alpha subunits and increased expression of HIF target genes, which include pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF). In mice, homozygous deletion of VHL is embryonic lethal due to vascular abnormalities in the placenta; and, VHL+/− mice develop proliferative vascular lesions in several major organs, most prominently the liver. Loss of ELL-associated factor (EAF2) in murine models has also been shown to induce increased vascular density in the liver as well as the prostate. Previously, EAF2 was determined to be a binding partner of VHL and loss of EAF2 induced a reduction in pVHL levels and an increase in hypoxia induced factor 1α (HIF1α) levels in vitro. Here we characterized the cooperative effects of VHL- and EAF2-deficiency on angiogenesis in the liver and prostate of male mice. VHL deficiency consistently increased the incidence of hepatic vascular lesions across three mouse strains. These vascular lesions where characterized by an increase in microvessel density, and staining intensity of VHL target proteins HIF1α and VEGF. EAF2−/−VHL+/− mice had increased incidence of proliferative hepatic vascular lesions (4/4) compared to VHL+/− (10/18) and EAF2−/− (0/5) mice. Prostates of EAF2−/−VHL+/− mice also displayed an increase in microvessel density, as well as stromal inflammation and prostatic intraepithelial neoplasia. These results suggest that cooperation of VHL and EAF2 may be critical for angiogenic regulation of the liver and prostate, and concurrent loss of these two tumor suppressors may result in a pro-angiogenic phenotype.Electronic supplementary materialThe online version of this article (doi:10.1007/s10456-011-9217-1) contains supplementary material, which is available to authorized users.

Poor prognostic clinicopathologic features correlate with VEGF expression but not with PTEN expression in squamous cell carcinoma of the larynx

BackgroundThe aim of this study was to assess the relationship between expression of vascular endothelial growth factor (VEGF) and phosphatase and tensin homolog deleted in chromosome ten (PTEN), angiogenesis and clinicopathological parameters of squamous cell carcinoma of the larynx.MethodsWe examined immunohistochemical expression of VEGF and PTEN and CD34 for microvessel density (MVD) in sections of formalin-fixed, paraffin embedded tissue blocks of 140 patients with squamous cell carcinoma of the larynx. The intensity of VEGF and PTEN staining and the proportion of cells staining were scored.ResultsThe tumor grade was not significantly related to PTEN expression, but it was to VEGF expression (p = 0.400; p = 0.015, respectively). While there was no significant relationship between PTEN expression and tumor size and cartilage invasion (p = 0.311, p = 0.128), there was a significant relationship between the severity of VEGF expression and tumor size (p = 0.006) and lymph node metastasis (p = 0.048) but not cartilage invasion (p = 0.129). MVD was significantly higher in high-grade tumors (p = 0.003) but had no significant relationship between MVD, lymph node metastasis, and cartilage invasion (p = 0.815, p = 0.204). There was also no significant relationship between PTEN and VEGF expression (p = 0.161) and between PTEN and VEGF expression and the MVD (p = 0.120 and p = 0.175, respectively).ConclusionsIncreased VEGF expression may play an important role in the outcome of squamous cell carcinoma of the larynx. PTEN expression was not related to VEGF expression and clinicopathological features of squamous cell carcinoma of the larynx.