Sub-zeptomole Detection of Biomarker Proteins Using a Microfluidic Immunoarray with Nanostructured Sensors.

We report here a low-cost electrochemical immunoarray with unprecedented sensitivity in the sub-zeptomole range with up to 5 log-decades dynamic range for accurate, multiplexed protein determinations. The microfluidic array features eight carbon sensors coated with a dense layer of 5 nm gold-nanoparticles derivatized with primary antibodies. Analyte proteins are captured by secondary antibody-poly-HPR (horseradish peroxidase) bioconjugates containing 400 HRP enzyme labels, with amplified amperometric peaks developed using H2O2 activator and hydroquinone mediator. Prostate cancer biomarkers prostate specific antigen (PSA), vascular endothelial growth factor-D (VEGF-D), ETS-related gene protein (ERG), and insulin-like growth factor-1 (IGF-1) were measured simultaneously with sub-fg/mL LODs (0.08-0.22 zmol). These proteins were determined in serum of postprostatectomy cancer patients which had much lower levels than prostate cancer patients without surgery. This immunoassay protocol makes thousands of low-abundance proteins accessible to quantitative measurements down to zeptomole levels.

Vascular Endothelial Growth Factor-D (VEGF-D) is Elevated in Bronchoalveolar Lavage Fluid of Patients with Lung Squamous Carcinoma.

BACKGROUND: Previous studies have found that vascular endothelial growth factor (VEGF) is associated with lung cancer, yet little is known about vascular endothelial growth factor-D (VEGF-D) in bronchoalveolar lavage fluid (BALF) of lung cancer patients. In this study, we aim to investigate the expression and evaluation of VEGF-D in BALF for lung cancer diagnosis. METHODS: BALF samples were acquired from 81 patients: 40 with benign diseases and 41 with lung cancer. The expression of VEGF-D in BALF was measured using sandwich enzyme-linked immune sorbent assays (ELISA), and the evaluation of VEGF-D in BALF for lung cancer diagnosis was also investigated. RESULTS: In the BALF samples, the levels of VEGF-D in the lung cancer group were higher than in the benign disease group; however, there was no statistical significance between the two groups (p > 0.05). In the pathological classification of lung cancer, the levels of VEGF-D in the BALF differed significantly between the lung squamous carcinoma group and the benign disease group (p < 0.05). The diagnostic accuracies of VEGF-D in BALF for discrimination between patients with squamous cell carcinoma and benign disease were reasonable based on receiver operating characteristic (ROC curve) analysis, with a corresponding sensitivity of 64.7% and specificity of 60%, respectively. CONCLUSIONS: This study demonstrated that the detection of VEGF-D levels in BALF is a valuable diagnostic tool for lung squamous carcinoma.

Vascular endothelial growth factor receptor-3 is a novel target to improve net ultrafiltration in methylglyoxal-induced peritoneal injury.

Appropriate fluid balance is important for good clinical outcomes and survival in patients on peritoneal dialysis. We recently reported that lymphangiogenesis associated with fibrosis developed in the peritoneal cavity via the transforming growth factor-ß1-vascular endothelial growth factor-C (VEGF-C) pathway. We investigated whether VEGF receptor-3 (VEGFR-3), the receptor for VEGF-C and -D, might be a new target to improve net ultrafiltration by using adenovirus-expressing soluble VEGFR-3 (Adeno-sVEGFR-3) in rodent models of peritoneal injury induced by methylglyoxal (MGO). We demonstrated that lymphangiogenesis developed in these MGO models, especially in the diaphragm, indicating that lymphangiogenesis is a common feature in the peritoneal cavity with inflammation and fibrosis. In MGO models, VEGF-D was significantly increased in the diaphragm; however, VEGF-C was not significantly upregulated. Adeno-sVEGFR-3, which was detected on day 50 after administration via tail vein injections, successfully suppressed lymphangiogenesis in the diaphragm and parietal peritoneum in mouse MGO models without significant effects on fibrosis, inflammation, or neoangiogenesis. Drained volume in the peritoneal equilibration test using a 7.5% icodextrin peritoneal dialysis solution (the 7.5% icodextrin peritoneal equilibration test) was improved by Adeno-sVEGFR-3 on day 22 (P<0.05) and day 50 after reduction of inflammation (P<0.01), indicating that the 7.5% icodextrin peritoneal equilibration test identifies changes in lymphangiogenesis. The solute transport rate was not affected by suppression of lymphangiogenesis. In human peritoneal dialysis patients, the dialysate to plasma ratio of creatinine positively correlated with the dialysate VEGF-D concentration (P<0.001). VEGF-D mRNA was significantly higher in the peritoneal membranes of patients with ultrafiltration failure, indicating that VEGF-D is involved in the development of lymphangiogenesis in peritoneal dialysis patients. These results indicate that VEGFR-3 is a new target to improve net ultrafiltration by suppressing lymphatic absorption and that the 7.5% icodextrin peritoneal equilibration test is useful for estimation of lymphatic absorption.

Prognostic significance of vascular endothelial growth factor expression in esophageal carcinoma: a meta-analysis.

PURPOSE: The purpose of this study was to comprehensively and quantitatively review eligible published studies to explore the prognostic significance of vascular endothelial growth factor (VEGF) expression in patients with esophageal carcinoma (EC). METHODS: PubMed and Embase databases were searched until September 11, 2011. A meta-analysis was performed to demonstrate any relationship between VEGF and 5-year overall survival (OS) in EC patients. RESULTS: The final analysis included 1453 patients from 19 studies. The studies were grouped by patient source, histology, VEGF isoform and cutoff value. The estimated risk of death suggested that VEGF positivity had negative impact on prognosis of patients with EC, esophageal squamous cell carcinoma (ESCC) and Asian patients. The risk ratios (RR) and 95% confidence interval (95% CI) were 1.26 (1.16-1.37) in EC patients, 1.28 (1.16-1.40) in ESCC patients and 1.35 (1.24-1.48) in Asian patients. Furthermore, when the cutoff value was set at 10% in 6 studies, the RR (95% CI) was 1.48 in the VEGF positive group (1.27-1.73). In addition, VEGFC was also correlated with patient poor prognosis with a RR (95% CI) of 1.30 (1.15-1.48). However, EC patients from non-Asian countries and cutoff value at 30% showed no significant correlation with survival. Data were not sufficient to determine the prognostic value of VEGF expression in esophageal adenocarcinoma (EA) patients and VEGFD expression. CONCLUSIONS: VEGF positivity indicated poor prognosis in patients with EC, ESCC and of Asian origin. Cutoff value at 10% may be a more appropriate standard to define VEGF positivity. VEGFC also correlated with poor prognosis in EC patients.

Potential role for vascular endothelial growth factor-D as an autocrine factor for human gastric carcinoma cells.

Vascular endothelial growth factor (VEGF)-D induces lymphangiogenesis by activating VEGF receptor (VEGFR)-3, which is expressed mainly by lymphatic endothelial cells. VEGFR-3 has also been detected in several types of malignant cells, but the significance of VEGFR-3 expression by malignant cells remains unclear. We examined the expression and function of VEGF-D/VEGFR-3 in human gastric carcinoma cells. Expression of VEGF-D and VEGFR-3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF-D on the expression of genes associated with disease progression in VEGFR-3-expressing KKLS cells. VEGF-D-transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF-D and VEGFR-3. In vitro treatment of KKLS cells with exogenous VEGF-D increased expression of cyclin D1 and Bcl-2 and stimulated cell proliferation. VEGF-D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF-D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms.

Different significance between intratumoral and peritumoral lymphatic vessel density in gastric cancer: a retrospective study of 123 cases.

BACKGROUND: Patients with gastric cancer in China have worse outcome and poorer prognosis. Tumor-induced lymphangiogenesis plays a crucial role in metastasis and tumor progression. The intratumoral and peritumoral lymphatics were supposed to have different biological effects. Three major growth factors, vascular endothelial growth factor- (VEGF)-A, VEGF-C and VEGF-D, are involved in the activation process via their receptors (VEGFRs). The purpose of current study is to investigate the significant difference between intratumoral and peritumoral lymphatic vessel density (LVD) in gastric cancer and their correlations with lymphangiogenetic growth factors. METHODS: Intratumoral LVD (I-LVD) and peritumoral LVD (P-LVD) of 123 patients with primary gastric cancer were assessed after staining with D2-40, and confirmed by double staining with D2-40/CD34. Proliferative activity of lymphatics endothelium was evaluated by double staining with D2-40/Ki-67. The associations were analyzed between I-LVD/P-LVD and the expression level of VEGF-A, VEGF-C, VEGF-D and the receptor VEGFR-3, which was measured by immunohistochemistry (IHC). The correlations of I-LVD and P-LVD with patient prognosis were also valued. RESULTS: (1) The peritumoral lymphatics (PTLs) were relatively enlarged with dilated lumen compared with the intratumoral lymphatics (ITLs). Increased P-LVD was significantly higher than I-LVD (P < 0.05). (2) P-LVD was found significantly associated with lymph node metastasis (LNM) (P < 0.001), lymphatic vessel invasion (LVI) (P < 0.001), VEGF-C (P = 0.003), VEGF-D expression level (P = 0.005) and VEGFR-3 expression level (P < 0.001) in peritumoral tissues, despite no significant association was found between above variants with I-LVD. However, increased I-LVD was demonstrated to be associated with decreased tumor volume (P < 0.001). Neither I-LVD nor P-LVD was correlated with VEGF-A expression (P > 0.05). (3) Proliferative activity of lymphatics endothelium was observed in PTLs, in spite of ITLs. (4) Increased P-LVD, but not I-LVD, was indicated to be an independent risk factor for lymph node metastasis by multivariate logistic regression analysis, and was related to worse disease-free survival and overall survival. CONCLUSIONS: PTLs play roles in gastric cancer progression. Increased P-LVD, but not I-LVD, was significantly associated with VEGF-C/-D/VEGFR-3 system, and could be an independent risk factor for lymph node metastasis and a prognostic factor in gastric cancer.

Concentrations of vascular endothelial growth factor C and D in amniotic fluid and maternal plasma.

OBJECTIVE: Vascular endothelial growth factor-C (VEGF-C) and VEGF-D promote both endothelial and lymphatic vascularization during embryonic development. We studied their presence in amniotic fluid (AF) and maternal plasma during pregnancy. DESIGN: Descriptive study. Setting. University Central Hospital, Helsinki, Finland. Samples. AF at 14-20 weeks (n=20) and 38-41 weeks' of gestation (n=20), serial plasma samples (n=38) from 16 healthy pregnant women during 8-40 weeks of pregnancy, and plasma from 15 non-pregnant controls. METHODS: Enzyme-linked immunosorbent assay (ELISA). Main outcome measures. Concentrations of VEGF-C and -D in AF and maternal plasma. RESULTS: VEGF-C concentrations in AF decrease as pregnancy advances (p=0.002) and are about 10-fold lower than in plasma at the corresponding gestational age (p=0.001). Plasma VEGF-C concentrations are higher in the first trimester (p=0.014) and the early second trimester (p=0.016) than in non-pregnant women. VEGF-D is not detectable in AF, but in plasma its concentrations become higher at term compared with non-pregnant women (p=0.039). CONCLUSIONS: VEGF-C and -D are present in high concentrations in maternal plasma, while only VEGF-C can be detected in AF.

Expression of vascular endothelial growth factors C and D correlates with lymphangiogenesis and lymph node metastasis in lung adenocarcinoma.

BACKGROUND: Vascular endothelial growth factors (VEGF)-C and -D have been reported to be lymph-specific growth factors. In this study we analyzed the intra- and peritumoral microlymphatic vessel density (MLVD) and the expression of VEGF-C and -D in lung adenocarcinoma. METHODS: Tissue samples were obtained from 48 patients who underwent surgery for lung adenocarcinoma. The expression of VEGF-C, -D and MLVD was examined by immunohistochemistry, and the correlation with lymph node metastasis and clinicopathological features was evaluated. RESULTS: Immunohistochemical analysis revealed the presence of VEGF-C and -D immunoreactivity in 70.8 % (34/48) and 58.3 % (28/48) of the tissue samples, respectively. The group with a high expression of VEGF-C and -D in the tumor margins had a higher incidence of lymph node metastasis. MLVD in both the VEGF-C and the VEGF-D positive groups was higher than that in the negative groups, and the lymph node metastasis was likewise higher. CONCLUSION: VEGF-C and -D may play important roles in lymphangiogenesis and lymph node metastasis in lung adenocarcinoma.

Identification of immunohistochemical prognostic markers for survival after resection of pulmonary metastases from colorectal carcinoma.

BACKGROUND: Although aggressive resection of pulmonary metastases prolongs the survival of patients with metastatic colorectal cancer, there is a need for predictive pathologic parameters to understand the key molecular events of metastatic progression. The aim of this study was to verify immunohistochemical markers in addition to established clinical parameters after surgery. METHODS: From our subset of patients undergoing resection of pulmonary metastases from metastatic colorectal carcinoma, we analyzed 39 patients (23 men and 16 women) between 2003 and 2007. Only patients who met the criteria for a potentially curative operation were included. All patients were analyzed with regard to age and sex, primary tumor location, stage of the primary tumor, history of hepatic metastases, number of pulmonary metastases, pre-thoracotomy carcinoembryonic (CEA) serum antigen level, and the presence of thoracic lymph node metastasis. Furthermore, we immunohistochemically investigated the expression of vascular endothelial growth factor (VEGF)-D, FBJ murine osteosarcoma viral oncogene homolog B (FOS-B), and melanoma antigen (MAGE)-A in the surgical specimens of pulmonary metastatic lesions. RESULTS: The overall 3-year survival was 50.6 %. A significantly longer survival was observed with multivariate analysis in patients with a pre-thoracotomy serum carcinoembryonic antigen level of no more than 4.2 ng/mL ( P = 0.001), and Dukes stage A or B primary tumor ( P = 0.001). A significantly longer recurrence-free survival was observed with multivariate analysis in patients without thoracic lymph node involvement compared to patients with pulmonary and/or mediastinal lymph node metastases ( P = 0.006). The stage of the primary tumor remained significant ( P = 0.029), and FOS-B expression in tumor cells showed a trend towards favorable recurrence-free survival after pulmonary metastasectomy ( P = 0.059). No statistically significant difference was found in the overall survival rate or recurrence-free survival rate of patients with expression of VEGF-D or MAGE-A antigen in pulmonary metastatic tumor cells. CONCLUSIONS: Our results suggest that in addition to clinically prognostic factors, FOS-B expression has a debatable impact on patient survival. We conclude that the evaluation of molecular and clinical prognostic parameters at the time of pulmonary metastasectomy offers a greater understanding of the metastatic process and provides important information for patient selection.

Lymphangiogenesis induced by VEGF-C and VEGF-D promotes metastasis and a poor outcome in breast carcinoma: a retrospective study of 61 cases.

PURPOSE: To evaluate lymphangiogenesis in patients with breast carcinoma, explore the underlying mechanism, and study the relationship between lymphangiogenesis and progression of breast carcinoma. METHODS: Sixty-one cases of breast carcinoma with complete clinical and pathological data were analyzed. Using an anti-podoplanin monoclonal antibody, an immunohistochemical study was made of all specimens to detect lymphatic vessel density (LVD) and to investigate its clinicopathological and prognostic value. VEGF-C and VEGF-D were observed by RT-PCR and immunostaining to investigate their clinicopathological and prognostic values and their relationship with lymphangiogenesis. RESULTS: LVD in breast carcinoma (6.28+/-3.73) was significantly higher than in benign mammary lesions (0.50+/-1.27), P<0.01 and was significantly associated with lymphatic metastasis and high TNM stage, P<0.01. The level of VEGF-C and VEGF-D expression was also significantly higher in breast carcinomas than in benign mammary lesions, P<0.01. LVD increased significantly with higher expression of VEGF-C and VEGF-D, P<0.01. Patients with high expression of VEGF-C and VEGF-D were observed to be more likely to have a bad outcome, P<0.05. CONCLUSIONS: Lymphangiogenesis was significantly associated with lymph node metastasis, high TNM, and poor outcome in breast carcinoma. LVD may serve as a predictor of lymph node metastasis and a prognostic factor in breast carcinoma. VEGF-C and VEGF-D play an important role in lymphangiogenesis making the carcinoma more aggressive and leading to a poor prognosis.

Lymphangiogenesis in kidney cancer: expression of VEGF-C, VEGF-D and VEGFR-3 in clear cell and papillary renal cell carcinoma.

The vascular endothelial growth factors VEGF-C, VEGF-D and its receptor, VEGFR-3, are overexpressed in different malignancies and associated with lymph node metastasis and poor prognosis. We analysed these factors in clear cell (ccRCC) and papillary (pRCC) renal cell carcinoma (RCC). The results were correlated with various clinicopathological parameters (CPP). We constructed a tissue microarray with tumor samples of 135 (81%) ccRCC and 31 (19%) pRCC. After immunohistochemical staining using polyclonal antibodies for VEGF-C, VEGF-D and VEGFR-3, a semiquantitative analysis was performed to determine the levels of expression. The results were compared between the two subgroups and were correlated with CPP. In the two subgroups the expression of VEGF-C was significantly correlated with that of VEGF-D (p<0.001). There was an increased expression of VEGF-C in 11% of ccRCC and 36% of pRCC (p=0.002). VEGF-D expression was positive by means of analysis in 22% of ccRCC and 42% of pRCC (p=0.039). There was no significant difference regarding the expression of VEGFR-3 between the subgroups (44% ccRCC and 61% pRCC, p=0.11). No correlation was found between the expression of the analysed parameters and CPP (TNM, grading, progression-free survival and overall survival) in either the entire group or in the two subgroups. In summary, ccRCC and pRCC show a different expression pattern of the analysed lymphangiogenic factors. Further studies are necessary to confirm these results and to determine whether the VEGF-C/VEGF-D/VEGFR-3-axis can play a role as a prognostic tool or a target for therapeutic intervention in renal cell carcinoma.

Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

BACKGROUND AND AIM: Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. METHODS: Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. RESULTS: In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. CONCLUSIONS: There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

VEGF-D expression correlates with colorectal cancer aggressiveness and is downregulated by cetuximab.

AIM: To gain mechanistic insights into the role played by epidermal growth factor receptor (EGFR) in the regulation of vascular endothelial growth factors (VEGFs) in colorectal cancer (CRC). METHODS: The impact of high-level expression of the growth factor receptors EGFR and VEGF receptor (VEGFR)3 and the VEGFR3 ligands VEGF-C and VEGF-D on disease progression and prognosis in human CRC was investigated in 108 patients using immunohistochemistry. Furthermore, the expression of the lymphangiogenic factors in response to the modulation of EGFR signalling by the EGFR-targeted monoclonal antibody cetuximab was investigated at the mRNA and protein level in human SW480 and SW620 CRC cell lines and a mouse xenograft model. RESULTS: Human CRC specimens and cell lines displayed EGFR, VEGF-C and VEGF-D expression with varying intensities. VEGF-C expression was associated with histological grade. Strong expression of VEGF-D was significantly associated with lymph node metastases and linked to a trend for decreased survival in lymph node-positive patients. EGFR blockade with cetuximab resulted in a significant decrease of VEGF-D expression in vitro and in vivo. CONCLUSION: In conclusion, the expression of VEGF-D in colorectal tumours is significantly associated with lymphatic involvement in CRC patients and such expression might be blocked effectively by cetuximab.

Adenovirus-mediated gene transfer of human vascular endothelial growth factor-d induces transient angiogenic effects in mouse hind limb muscle.

We evaluated the therapeutic potential of adenovirus (Ad)-mediated human vascular endothelial growth factor-D (hVEGF-D) gene delivery in mice. Hind limbs of hypercholesterolemic mice ( n = 120) were injected with AdhVEGF-D, AdhVEGF-A, control AdLacZ (all at 1x10(11)viral particles) or saline. Animals were killed at 4, 7, 14, 28, and 42 days. Newly formed vessels were characterized for their quantity, sprouting, angiogenic versus lymphangiogenic phenotype, and arterial versus venous phenotype by endothelial enzymes markers, pericyte coverage, and electron microscopy. Perfusion was measured by power Doppler ultrasound and edema by magnetic resonance imaging (MRI). AdhVEGF-D induced significant formation of new blood vessels, which featured lumenal enlargement, branching, and sprouting. Branching originated mainly from arterioles. The highest vessel density was present on days 4-7 and the effect lasted up to 28 days. Endothelial marker enzyme activity indicated the predominance of arterial capillaries and arterioles. Forty percent of the neovessels were positive for desmin, indicating that VEGF-D increased pericyte coverage. However, branching vessels were highly positive for smooth muscle actin pericyte marker but negative for desmin. Maximal perfusion was measured during the first week after AdhVEGF-D gene transfer. Ultrastructural analysis showed endothelial cells enriched with vesiculo-vacuolar organelles and cytoplasmic protrusions. Modest lymphangiogenic activity was also detected, which could contribute to the relatively low level of edema detected by MRI. In conclusions, AdhVEGF-D has a strong angiogenic effect and a modest lymphangiogenic effect in mouse skeletal muscle. VEGF-D also increases the presence of pericytes/smooth muscle cells in neovessels. AdhVEGF-D is a potential new agent for the induction of therapeutic vascular growth in skeletal muscle.

Absence of lymphangiogenesis in ductal breast cancer at the primary tumor site.

Solid evidence for a relationship between lymphangiogenesis and prognosis in human breast cancer is still lacking. Evidence for ongoing lymphangiogenesis in breast cancer is only provided by animal studies. In the present study we investigated lymphatic vessel density as well as the expression level of the lymphangiogenic factors VEGF-C and -D in a series of 121 ductal breast cancer tissues using immunohistochemical stainings. We found that in the primary tumors the lymphatic vessel density, as well as the expression of both VEGF-C and -D, did not relate to grade, tumor stage, progression or patient survival. Furthermore, in tumors in which lymphatic vessels were present, a Ki-67/podoplanin double staining indicated the absence of proliferating lymphatic endothelial cells. In contrast, we did find a correlation between intratumoral lymphatic vessel density inside the lymph node metastases and patient survival. Another parameter that revealed prognostic value was the presence of tumor cells within the lymphatic vessels. This parameter did predict survival in patients with an age below 63 only. Interestingly, expression of VEGF-D was found to be related to the presence of intralymphatic tumor cells.

VEGF-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, NF-kappaB, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits.

Plaque angiogenesis may be associated with the development of unstable and vulnerable plaques. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. Our objective was to determine the effect of diabetes on atherosclerosis and on the expression of angiogenesis-related genes in atherosclerotic lesions. Alloxan was used to induce diabetes in male Watanabe heritable hyperlipidemic (WHHL) rabbits that were sacrificed 2 and 6 months after the induction of diabetes. Nondiabetic WHHL rabbits served as controls. Blood glucose (Glc), serum-free fatty acids (FFA), and serum triglyceride levels were significantly higher in diabetic rabbits. Accelerated atherogenesis was observed in the diabetic WHHL rabbits together with increased intramyocellular lipids (IMCL), as determined by 1H-NMR spectroscopy. Atherosclerotic lesions in the diabetic rabbits had an increased content of macrophages and showed significant increases in immunostainings for vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, RAGE, and NF-kappaB. VEGF-A165 and VEGFR-2 mRNA levels were significantly increased in aortas of the diabetic rabbits, where a trend toward increased plaque vascularization was also observed. These results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, and VEGF receptor-2 expression, and increases NF-kappaB, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.

Quantitative analysis of lymphangiogenic markers in human gastroenteric tumor.

BACKGROUND: Lymphatic spread of gastroenteric tumor cells to regional lymph nodes is one of the early events in metastatic cancers and is often associated with distant metastatic spread and poor prognosis. Expression levels of newly described lymphatic endothelial markers, LYVE-1, VEGF-C, VEGF-D and the VEGF receptors VEGFR-3 were assessed in our study. METHODS: Paired (tumor and corresponding normal tissue) samples were obtained. The expression level of each factor was determined using RT-PCR and quantified by using a real-time quantitative PCR (RT-QPCR) technique, with respective cloned cDNA plasmids as internal standards. RESULTS: The expression of VEGF-C and lymphatic endothelial marker VEGFR-3 was significantly greater in patients with lymph node metastasis than in those without metastasis, but no different expression level of VEGF-D and LYVE-1 was detected in both groups of patients. Lymphatic vessel density (LVD), which was assessed by immunohistochemistry for LYVE-1, was correlated with lymphangiogenesis factors and lymph node metastasis. Expression of VEGF-C and VEGFR-3 was significantly associated with higher peritumoral LVD than normal group, and LVD was found greater in the node-positive group than in the node-negative group. CONCLUSIONS: These results indicate that quantitative analysis of lymphangiogenic marker VEGF-C and VEGFR-3 in gastroenteric specimens may be useful in predicting metastasis of gastroenteric cancer to regional lymph nodes, but the role of LYVE-1 in predicting metastasis of gastroenteric cancer requires further analysis.

Expression level of vascular endothelial growth factor-C and -A in cultured human oral squamous cell carcinoma correlates respectively with lymphatic metastasis and angiogenesis when transplanted into nude mouse oral cavity.

Angiogenesis is induced by various angiogenic factors including vascular endothelial growth factors (VEGFs), such as VEGF-A, -B, -C and -D, and is involved in tumor progression and metastasis. In an effort to define the expression pattern of VEGFs in oral squamous cell carcinoma (OSCC) and its correlation with clinicopathological factors, we determined the expression levels of VEGFs in OSCC cell lines (HSC-2, HSC-3, HSC-4 and OSC-19) by quantitative RT-PCR and examined their relationship with regional lymph node (LN) and distant metastasis, intratumoral microvessel density (MVD) in tumor transplanted nude mice. We found that HSC-2 and OSC-19 expressed significantly higher levels of VEGF-A and VEGF-C, and caused frequent regional LN metastasis and higher MVD than did the other cell lines. Since VEGF-C is a lymphangiogenic factor, these results suggest that expression of VEGF-C is a useful predictor for LN metastasis.

Precision assessment of heterogeneity of lymphedema phenotype, genotypes and risk prediction.

Lymphedema following breast cancer surgery is considered to be mainly due to the mechanical injury from surgery. Recent research identified that inflammation-infection and obesity may be the important predictors for lymphedema. The purpose of this exploratory research was to prospectively examine phenotype of arm lymphedema defined by limb volume and lymphedema symptoms in relation to inflammatory genes in women treated for breast cancer. A prospective, descriptive and repeated-measure design using candidate gene association method was used to enroll 140 women at pre-surgery and followed at 4-8 weeks and 12 months post-surgery. Arm lymphedema was determined by a perometer measurement of ≥5% limb volume increase from baseline of pre-surgery. Lymphedema symptom phenotype was evaluated using a reliable and valid instrument. Saliva samples were collected for DNA extraction. Genes known for inflammation were evaluated, including lymphatic specific growth factors (VEGF-C & VEGF-D), cytokines (IL1-a, IL-4, IL6, IL8, IL10, & IL13), and tumor necrosis factor-a (TNF-a). No significant associations were found between arm lymphedema phenotype and any inflammatory genetic variations. IL1-a rs17561 was marginally associated with symptom count phenotype of ≥8 symptoms. IL-4 rs2070874 was significantly associated with phenotype of impaired limb mobility and fluid accumulation. Phenotype of fluid accumulation was significantly associated with IL6 rs1800795, IL4 rs2243250 and IL4 rs2070874. Phenotype of discomfort was significantly associated with VEGF-C rs3775203 and IL13 rs1800925. Precision assessment of heterogeneity of lymphedema phenotype and understanding the biological mechanism of each phenotype through the exploration of inherited genetic susceptibility is essential for finding a cure. Further exploration of investigative intervention in the context of genotype and gene expressions would advance our understanding of heterogeneity of lymphedema phenotype.

Bronchial involvement in advanced stage lymphangioleiomyomatosis: histopathologic and molecular analyses.

Lymphangioleiomyomatosis (LAM), a rare progressive disease that almost exclusively affects women, is characterized by pulmonary cysts and neoplastic proliferation of smooth muscle-like cells (LAM cells). Airflow obstruction is a physiologic consequence that is commonly observed in LAM and has been attributed to narrowing of peripheral airways. However, histopathologic examinations of the entire airway have been precluded by the limited availability of such specimens. Here, we used explanted lung tissues from 30 LAM patients for a thorough histologic analysis with a special emphasis on the bronchi. We found bronchial involvement by LAM cells and lymphatics in all patients examined. Furthermore, a moderate to severe degree of chronic inflammation (73%), goblet cell hyperplasia (97%), squamous cell metaplasia (83%) of the epithelium, and thickening of basal lamina (93%) were identified in the bronchi. Because LAM cells are transformed by the functional loss of the TSC genes leading to a hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, we confirmed the expression of phospho-p70S6K, phospho-S6, phospho-4E-BP1, and vascular endothelial growth factor (VEGF)-D in LAM cells from all of the patients examined. In contrast, no protein expression of hypoxia-inducible factor 1α, a downstream molecule indicative of mTORC1 activation and leading to VEGF production, was detected in any patient. Our study indicates that late-stage LAM patients commonly have bronchi involved by the proliferation of both LAM cells and lymphatics and that chronic inflammation complicated their disease. Furthermore, the up-regulation of hypoxia-inducible factor 1α, a common event in mTORC1-driven tumor cells, does not occur in LAM cells and plays no role in VEGF-D expression in LAM cells.