Molecular mechanisms of miR-1236 in the assessment of tumor lymphangiogenesis in human ovarian cancer patients.

BACKGROUND: Tumor lymphangiogenesis is a critical component in the progression of cancers and specific microRNAs have been reported to be implicated in this process. Recent studies revealed the involvement of miR-1236 in lymphangiogenic signaling by targeting vascular endothelial growth factor receptor 3 (VEGFR3). However, the prognostic importance of miR-1236 and its clinical relevance for lymphangiogenesis in ovarian cancer (OC) remains unclear. METHODS: The study included 52 ovarian tumors and 28 normal ovarian tissues. Quantitative real-time PCR was utilized to analyze the VEGFR3, VEGF-C, LYVE-1 and PROX1 mRNA expression as well as miR-1236. VEGFR3 protein expression was measured by immunohistochemistry staining. Immunohistochemistry for the podoplanin marker (D2-40) was performed to measure lymphatic vessel density (LVD). In addition, diagnostic evaluation based on the receiver-operating characteristic (ROC) curve was performed. The influence of miR-1236 on overall survival was evaluated by Kaplan-Meier method. RESULTS: Here, we show that miR-1236 expression was significantly decreased in ovarian tumors compared with control tissues (p < 0.001) and correlated with advanced clinical stage, lymph node metastasis, distant metastasis and patient survival (All P < 0.05). Moreover, in ovarian tumors, LVD as well as the gene expression of VEGFR3, VEGF-C and LYVE-1, but not PROX1, were found to be remarkably higher compared with control tissues. We also detected a more robust positive staining for VEGFR3 in OC tissues than in control tissues. Furthermore, our results demonstrated an inverse association of miR-1236 expression with LVD, VEGFR3, LYVE-1 and PROX1 expression in OC tissues. The ROC curve analysis indicated that miR-1236 expression has the potential to be used as a diagnostic and prognostic biomarker in OC. Survival analysis further verified a lowered overall survival rate in patients with low miR-1236 expression than in those with high expression. CONCLUSIONS: Our results provide evidence for the translational involvement of miR-1236 in the lymphangiogenesis of OC by regulating lymphangiogenesis-related factors and support the clinical importance of miR-1236 as a new diagnostic and prognostic biomarker for OC.

Presence of Donor Lymph Nodes Within Vascularized Composite Allotransplantation Ameliorates VEGF-C-mediated Lymphangiogenesis and Delays the Onset of Acute Rejection.

BACKGROUND: The lymphatic system plays an active role in modulating inflammation in autoimmune diseases and organ rejection. In this work, we hypothesized that the transfer of donor lymph node (LN) might be used to promote lymphangiogenesis and influence rejection in vascularized composite allotransplantation (VCA). METHODS: Hindlimb transplantations were performed in which (1) recipient rats received VCA containing donor LN (D:LN+), (2) recipient rats received VCA depleted of all donor LN (D:LN-), and (3) D:LN+ transplantations were followed by lymphangiogenesis inhibition using a vascular endothelial growth factor receptor-3 (VEGFR3) blocker. RESULTS: Our data show that graft rejection started significantly later in D:LN+ transplanted rats as compared to the D:LN- group. Moreover, we observed a higher level of VEGF-C and a quicker and more efficient lymphangiogenesis in the D:LN+ group as compared to the D:LN- group. The presence of donor LN within the graft was associated with reduced immunoactivation in the draining LN and increased frequency of circulating and skin-resident donor T regulatory cells. Blocking of the VEGF-C pathway using a VEGFR3 blocker disrupts the lymphangiogenesis process, accelerates rejection onset, and interferes with donor T-cell migration. CONCLUSIONS: This study demonstrates that VCA LNs play a pivotal role in the regulation of graft rejection and underlines the potential of specifically targeting the LN component of a VCA to control graft rejection.

Relación entre los niveles séricos del factor de crecimiento endotelial-vascular C, la afectación ganglionar axilar y la sobrevida libre de enfermedad en pacientes con cáncer de mama operable sometidas a biopsia selectiva del ganglio centinela / Association between serum levels of vascular-endothelial growth factor-C, axillary node involvement and disease-free survival in patients with operable breast cancer undergoing sentinel node biopsy

Objetivos Investigar la relación entre los niveles séricos del factor de crecimiento endotelial-vascular C (VEGF-C) y la afectación ganglionar axilar. Materiales y métodos Se realizó un estudio prospectivo y subsecuente análisis sobre 174 pacientes con cáncer de mama temprano que habían sido sometidas a la biopsia selectiva de ganglio centinela (BSGC). Resultados El valor medio de la concentración plasmática de VEGF-C fue 6.561,5pg/ml. No se encontró asociación entre el nivel plasmático del VEGF-C y el estado de compromiso ganglionar en la BSGC (p=0,626). En el análisis de regresión logística multivariante los niveles séricos de VEGF-C tampoco fueron predictivos del compromiso ganglionar. El tiempo hasta la recaída o hasta el último seguimiento tuvo una mediana de 51 meses (media: 49 meses), con una máxima de 80 meses (percentil 25: 42 meses; percentil 75: 62 meses) La reducción en 1.000 puntos en los valores séricos de VEGF-C (1k) aumentó el riesgo instantáneo, con un HR 1,357 (IC 95%: 1,004-1,838). Este aumento de riesgo es del 35% por cada 1.000 puntos de disminución de los valores séricos, evidenciable en el test de Kaplan-Meier. Conclusiones Los niveles séricos de VEGF-C no están relacionados con el riesgo de afectación ganglionar axilar en el cáncer de mama temprano y, por tanto, no pueden utilizados para predecir la presencia de afectación ganglionar en la BSGC. Las pacientes con niveles séricos de VEGF-C por encima de la media presentan menor riesgo de recaer en los primeros 4 años de seguimiento que las pacientes con valores por debajo de la media. Esta diferencia fue estadísticamente significativa en el análisis multivariado por lo que merece confirmación en futuros estudios diseñados específicamente para este objetivo. (AU)

Objectives To investigate the association between serum levels of vascular-endothelial growth factor-C (VEGF-C) and axillary node involvement. Material and methods We performed a prospective study and subsequent analysis of 174 patients with early-stage breast cancer who had undergone sentinel node biopsy (SNB). Results The mean VEGF-C plasma concentration was 6561.5 pg/ml. No association was found between VEGF-C plasma concentration and axillary node involvement in SNB (p=0.626). On multivariate logistic regression analysis, serum VEGF-C levels were not predictive of node involvement. The median interval to recurrence or last follow-up was 51 months (mean, 49 months), with a maximum of 80 months (25th percentile: 42 months, 75th percentile, 62 months). A reduction of 1000 points in serum VEGF-C levels (1k) increased instant risk, with HR 1.357 (95%CI: 1.004-1.838). This increased risk was 35% for each 1000-point decrease in serum levels, demonstrated in the Kaplan-Meier analysis. Conclusions Serum VEGF-C levels are not related to risk of node involvement in early-stage breast cancer and, therefore, cannot be used to predict the presence of node involvement in SNB. Patients with serum VEGF-C levels above the mean have a lower risk of recurrence in the first 4 years of follow-up than those with levels below the mean. This difference was statistically significant in the multivariate analysis and consequently deserves confirmation in future studies designed specifically for this aim. (AU)

An origami paper-based nanoformulated immunosensor detects picograms of VEGF-C per milliliter of blood.

Detecting vascular endothelial growth factor C (VEGF-C), a kind of tumor biomarker, is of significant clinical importance in evaluating the prognosis of patients with cancer. However, laboratory analyses are usually not suitable for point-of-care testing because they are expensive and time consuming. In response to these challenges, we fabricated an origami paper-based microfluidic electrochemical device. To improve the specificity of VEGF-C detection, nanocomposites, synthesized by new methylene blue (NMB), amino-functional single-walled carbon nanotubes (NH2-SWCNTs), and gold nanoparticles (AuNPs), were used to modify the surface of working electrodes. Results of electrochemical detection showed that the immunosensor had excellent linearity, ranging from 0.01 to 100 ng mL-1 (R2 = 0.988), and the limit of detection was 10 pg mL-1. To confirm the high specificity of the device under real-world conditions, we evaluated the device using clinical serum samples from our hospital. The results demonstrated that the device had an excellent performance and could provide a platform for real-time detection of cancers.

Prognostic value of lymphatic vessel density in oral squamous cell carcinoma.

AIMS: Lymphatic vessel density (LVD) for the evaluation of tumor metastasis and prognosis remains controversial. The aim of this study was to elucidate the association between tumor cells and lymphatic vessels, and evaluate LVD in oral squamous cell carcinoma (OSCC). MAIN METHODS: 128 OSCC cases were used to determine the expression of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and vascular endothelial growth factor C (VEGF-C). Mann-Whitney or Kruskal-Wallis tests were employed to analyze the association between clinicopathological data and intratumoral LVD (ILVD), peritumoral LVD (PLVD), and VEGF-C; comparisons between ILVD and PLVD were made with t-test. Correlations between LVD and VEGF-C were analyzed by Spearman's correlation coefficient. Disease-specific survival curves were obtained with Kaplan-Meier method and compared using the log-rank test. Cox multiple regression was used to clarify the independent effect of clinicopathological data on clinical outcome. KEY FINDINGS: Tumor tissues were positively stained with LYVE-1 and VEGF-C. Both tumor metastasis and recurrence were associated with ILVD. A significant association between ILVD and VEGF-C expression was observed (P < 0.05). A significant association between high ILVD and poor disease-specific survival was observed (P < 0.05). SIGNIFICANCE: This study showed that ILVD was significantly associated with increased lymphatic metastasis, tumor recurrence, and reduced disease-specific survival in patients with OSCC. ILVD could be an indicator to predict the prognosis of OSCC.

Stromal inflammatory cells are associated with poorer prognosis in primary cutaneous melanoma.

We observed that non-tumor-infiltrating inflammatory cells are often present in the stroma of melanoma. The role of these stromal inflammatory cells (SIC) in cancer has not been studied. We evaluated the prognostic significance of SIC in 299 patients with vertical growth phase primary melanomas with at least 10 years of clinical follow-up. Lymphatic density and lymphatic invasion in the areas with SIC was quantified. The prognostic significance of these factors was evaluated using univariable and multivariable Cox models for melanoma-specific death and the time to first recurrence. Of the 299 melanomas, 161 exhibited areas with SIC. Percentages of vertical growth phase tumor-infiltrating lymphocytes and radial growth phase regression were significantly higher in cases with SIC compared to those without SIC (P = .005); lymphatic invasion was also detected more frequently in cases with SIC (P = .001). Lymphatic density in SIC areas was higher than that in other areas of the melanomas. Patients with SIC had poorer clinical outcome. Vascular endothelial growth factor-C (VEGFC) staining in a subset of these melanoma patients showed that VEGFC expression in the stromal macrophages was associated with lymphatic invasion in SIC areas. In conclusion, SIC in melanoma is associated with poorer prognosis, and the prognostic effect is partially mediated through induction of lymphangiogenesis with increased lymphatic invasion.

Prediction of healing in Category I pressure ulcers by skin blotting with plasminogen activator inhibitor 1, interleukin-1α, vascular endothelial growth factor C, and heat shock protein 90α: A pilot study.

The prevention of progression of Category I pressure ulcers (PUs) to Category II or higher is important, as Category II or higher PUs are open wounds and have a higher infection risk. Prognosis prediction of Category I PUs is necessary to provide successful intensive care for PUs with impaired healing. We focused on skin blotting using plasminogen activator inhibitor 1 (PAI1), interleukin-1α (IL-1α), vascular endothelial growth factor C (VEGF-C), and heat shock protein 90α (HSP90α). This pilot study was conducted at long-term-care and general hospitals to examine the applicability of DESIGN-R and thermography; the feasibility of skin blotting technique; the biomarker candidates, PAI1, IL-1α, VEGF-C, and HSP90α; and sample size for prognosis prediction for Category I PUs. Patients aged >65 years underwent skin blotting, scoring for DESIGN-R, and took thermography images of their Category I PU site. Albumin signals were not detected in one out of three participants. PAI1, IL-1α, VEGF-C, and HSP90α were detected in 19 participants, among whom 11 participants could be followed up after one week. There was no difference in DESIGN-R score and skin surface temperature between normal and impaired healing groups, and the sample size was calculated as 16. In conclusion, the feasibility of skin blotting was confirmed. PAI1, IL-1α, VEGF-C, and HSP90α could be biomarker candidates for prognosis prediction for Category I PU and the combination of VEGF-C and HSP90α could be associated with the prognosis of Category I PU. We need to investigate 842 patients in a future study.

[Changes of lymphatic vessel density in lung adenocarcinoma in situ, minimally invasive adenocarcinoma, and invasive adenocarcinoma and the regulatory factors].

OBJECTIVE: To analyze the changes in tumor lymphatic vessel density (LVD) in patients with lung adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IA) and explore the regulatory factors of LVD. METHODS: Complete clinicopathological data were collected form a total of 301 patients with lung adenocarcinoma, including 28 (9.3%) with AIS, 86 (28.6%) with MIA, and 187 (62.1%) with IA. The LVD of all the adenocarcinomas were calculated after D2-40 immunohistochemical staining, and MT1-MMP and VEGF-C expression levels were also evaluated. The differences in LVD among the groups and the correlations of tumor LVD with the expressions of MT1-MMP and VEGF-C and the clinicopathological factors were analyzed. RESULTS: The LVD differed significantly among AIS, MIA, and IA groups (P= 0.000). The LVDs was significantly correlated with the level of VEGF-C protein expression (r=0.917, P=0.009), tumor size (r= 0.686, P=0.017), lymph node metastasis (r=0.739, P=0.000), and clinical stage (r=0.874, P=0.012) of the patients. CONCLUSIONS: Tumor lymphangiogenesis plays an important role in lung adenocarcinoma progression, and VEGF-C may promote this process.

Podoplanin Expression Correlates with Disease Progression in Mycosis Fungoides.

The aim of this study was to investigate the role of lymphangiogenesis in the clinical progression and outcome of mycosis fungoides. Immunohistochemistry and Western blot techniques were used to assess the expression of podoplanin and vascular endothelial growth factor C in mycosis fungoides. Expression of vascular endothelial growth factor C measured by immunohistochemistry was significantly higher in mycosis fungoides samples in comparison with control cases (chronic benign dermatoses) (p = 0.0012). Increased expression of podoplanin was found in advanced vs. early mycosis fungoides (p < 0.0001), and was positively correlated with cutaneous and nodal involvement (p < 0.001, p < 0.0001; respectively). Higher podoplanin expression was also significantly associated with shorter survival (p < 0.001). Strong positive correlation was observed between expression of podoplanin analysed by immunohistochemistry and Western blot (r = 0.75, p < 0.0001). A similar association was shown regarding expression of vascular endothelial growth factor C (r = 0.68, p = 0.0007). In conclusion, these results suggest that increased expression of podoplanin is associated with poor clinical course, as well as shorter survival, of patients with mycosis fungoides.

Establishment of an Acquired Lymphedema Model in the Mouse Hindlimb: Technical Refinement and Molecular Characteristics.

BACKGROUND: The pathophysiology of secondary lymphedema remains poorly understood. This study aimed to establish a consistent mouse hindlimb lymphedema model for further investigation of the mechanism and treatment of lymphedema. METHODS: The authors developed a novel postsurgical lymphedema model in the mouse hindlimb by modifying previously described methods. Lymphedema in the hindlimb was created by removing both the inguinal and popliteal lymph nodes together with the surrounding fat pads, followed by silicone splint placement in the inguinal region. Using this modified mouse model, the authors analyzed lymphatic function, histologic changes, and the expression of lymphangiogenic factors including vascular endothelial growth factor C at various time points. RESULTS: The splinted lymphedema model showed a significant increase of edema formation in the hindlimb compared with the sham surgery control animals. Indocyanine green lymphography revealed lymphatic drainage impairment shown by dermal backflow and rerouting of lymph flow in the lymphedema model. Histopathologic and immunohistochemical examinations showed a significant increase of skin thickness and abnormally dilated lymphatics in the lymphedema model. The expression of lymphangiogenic factors in lymphedematous tissues of the splinted lymphedema model was significantly increased compared with controls, depending on the degree of lymphedema. CONCLUSIONS: This splinted lymphedema model closely simulates the volume response, histopathology, and lymphography characteristics of human acquired lymphedema. Given these similarities to human lymphedema, this refinement of a mouse hindlimb model of acquired lymphedema represents a promising platform for the study of lymphatic vascular insufficiency and for the evaluation of new therapeutic modalities.

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.

Evaluation of hypoxia, angiogenesis, and lymphangiogenesis in actinic cheilitis.

BACKGROUND: Actinic cheilitis is a potentially malignant condition caused mainly by chronic sun exposure. Here we aim to evaluate the role of hypoxia, angiogenesis, and lymphatic density in the clinical and morphological progression of a series of cases of actinic cheilitis. MATERIALS AND METHODS: Immunohistochemistry was used to evaluate positivity to hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-C, and D2-40 in 40 cases of actinic cheilitis of the lower lip. RESULTS: The cases studied exhibited variable degrees of positivity to the markers. The median number of lymphatic vessels was 3.2, 2.4, and 3.0 in lesions showing no epithelial dysplasia (NED) and with mild (MED) and moderate (MOED) epithelial dysplasia, respectively. The median VEGF-C positivity index was 82.44% (NED), 92.74% (MED), and 82.83% (MOED), and the median HIF-1α positivity index was 11.57% (NED), 5.26% (MED), and 13.55% (MOED). No significant differences in lymphatic density or median VEGF-C and HIF-1α positivity indices were observed between histological grades or clinical presentations of actinic cheilitis (P > 0.05). CONCLUSIONS: Although representing early events in lip carcinogenesis, the present results suggest that hypoxia, angiogenesis, and lymphangiogenesis do not influence the morphological or clinical progression of actinic cheilitis.

Involvement of syk and VEGF-C in invasion of lung adenocarcinoma A549 cells.

BACKGROUND AND AIMS: Lung cancer has become one of the most dangerous malignant tumors in the world nowadays, whose pathogenesis is complex involving multi-genes and multi-elements. This study aims to investigate the values of spleen tyrosine kinase (Syk) and vascular endothelial growth factor-C (VEGF-C) in lymphangiogenesis and metastasis of lung adenocarcinoma A549 cells. MATERIALS AND METHODS: The pcDNA3.1-VEGF-C and pLNCX-syk were constructed and transfected into A549 cells. After cells with stable expression were sorted, the level of VEGF-C was tested by RT-PCR and immunohistochemistry and the mRNA of syk was tested by RT-PCR. The cell invasion assay was investigated by transwell chamber in vitro. Restriction enzyme digestion and gel electrophoresis demonstrated successful construction of the pcDNA3.1-VEGF-C. RESULTS: RT-PCR and immunohistochemistry revealed higher expression of VEGF-C in VEGFC-construct-transfected A549 cells than that in controls (P < 0.05). Successful construction of the pLNCX-syk was demonstrated by restriction enzyme electrophoresis and sequencing. RT-PCR revealed Syk expression higher in syk-construct-transfected cells than in controls (P < 0.05). CONCLUSIONS: The results indicate a potential link between the upregulation of Syk and VEGF-C expression and lung adenocarcinoma.

Prognostic significance of VEGF-C immunohistochemical expression in colorectal cancer: A meta-analysis.

BACKGROUND: We sought to comprehensively summarize available evidence for the use of VEGF-C protein to evaluate the clinicopathological and prognostic role of VEGF-C in colorectal cancer. METHODS: Electronic databases from inception to February 2016 were used to search without language restrictions for original articles. A meta-analysis was undertaken to assess the relationship between VEGF-C expression and overall survival (OS) in colorectal cancer. RESULTS: Twenty-seven studies were included in the final meta-analysis. We aggregated 13 trials (n=1.428 patients) that evaluated the correlation between OS and VEGF-C overexpression. Statistics were performed for OS (HR=1.95; 95%CI=1.31-2.92, P=0.007). When the studies were stratified by the pathological variables, including T stage (n=383 patients; OR=1.79; 95%CI=1.14-2.81), lymph node metastasis (n=3212 patients; OR=4.21; 95%CI=3.49-5.08), M stage (n=1106 patients; OR=4.46; 95%CI=2.96-6.70), vascular invasion(n=1471 patients; OR=2.18; 95%CI=1.65-2.88), lymph invasion (n=831 patients; OR=3.95; 95%CI=2.80-5.56), histo-differentiation (n=1695 patients; OR=1.34; 95%CI=1.00-1.79) and Duke's stage(n=778 patients; OR=4.90; 95%CI=3.55-6.75), TNM stage (n=808 patients; OR=1.73; 95%CI=1.18-2.54) provided critical and comprehensive prognostic information. CONCLUSION: Our results demonstrated that VEGF-C overexpression was associated with OS in colorectal cancer.

Src/STAT3 signaling pathways are involved in KAI1-induced downregulation of VEGF-C expression in pancreatic cancer.

A previous study by our group demonstrated that overexpression of KAI1 was associated with lymphatic metastasis in pancreatic cancer. The present study further investigated the signaling pathways involved in KAI1­induced downregulation of vascular endothelial growth factor C (VEGF­C) and lymphatic metastasis in pancreatic cancer. Immunohistochemistry was performed to examine KAI1 and VEGF­C expression in 28 surgically resected pancreatic cancer tissues. MIA PaCa­2 and PCAN1 pancreatic cancer cell lines were transfected with KAI1 overexpression vector. VEGF­C expression as well as phosphorylation of Src and signal transducer and activator of transcription (STAT)3 were assessed by western blot analysis. Furthermore, the signal transduction inhibitors PP2 and AG490 were used to block the Src and STAT3 signaling pathways, respectively. KAI1 was negatively correlated with VEGF­C expression in pancreatic tumor samples. In MIA PaCa­2 cells, VEGF­C expression was more significantly inhibited by restoration of KAI1 than that in PCAN1 cells. In addition, Src and STAT3 phosphorylation was decreased by KAI1 in MIA PaCa­2 cells. Of note, pre­treatment with PP2 efficiently reversed the KAI1-induced enhancement of Src and STAT3 phosphorylation as well as VEGF­C expression. Pre­treatment with AG490 efficiently reversed the KAI1-induced enhancement of STAT3 phosphorylation and VEGF­C expression, but had no effect on the upregulation of Src phosphorylation. The present study identified the involvement of Src/STAT3 signaling pathways in KAI1­induced downregulation of VEGF­C expression and suggested the implication of these pathways in lymphatic metastasis of pancreatic cancer.

Selective Stimulation of Cardiac Lymphangiogenesis Reduces Myocardial Edema and Fibrosis Leading to Improved Cardiac Function Following Myocardial Infarction.

BACKGROUND: The lymphatic system regulates interstitial tissue fluid balance, and lymphatic malfunction causes edema. The heart has an extensive lymphatic network displaying a dynamic range of lymph flow in physiology. Myocardial edema occurs in many cardiovascular diseases, eg, myocardial infarction (MI) and chronic heart failure, suggesting that cardiac lymphatic transport may be insufficient in pathology. Here, we investigate in rats the impact of MI and subsequent chronic heart failure on the cardiac lymphatic network. Further, we evaluate for the first time the functional effects of selective therapeutic stimulation of cardiac lymphangiogenesis post-MI. METHODS AND RESULTS: We investigated cardiac lymphatic structure and function in rats with MI induced by either temporary occlusion (n=160) or permanent ligation (n=100) of the left coronary artery. Although MI induced robust, intramyocardial capillary lymphangiogenesis, adverse remodeling of epicardial precollector and collector lymphatics occurred, leading to reduced cardiac lymphatic transport capacity. Consequently, myocardial edema persisted for several months post-MI, extending from the infarct to noninfarcted myocardium. Intramyocardial-targeted delivery of the vascular endothelial growth factor receptor 3-selective designer protein VEGF-CC152S, using albumin-alginate microparticles, accelerated cardiac lymphangiogenesis in a dose-dependent manner and limited precollector remodeling post-MI. As a result, myocardial fluid balance was improved, and cardiac inflammation, fibrosis, and dysfunction were attenuated. CONCLUSIONS: We show that, despite the endogenous cardiac lymphangiogenic response post-MI, the remodeling and dysfunction of collecting ducts contribute to the development of chronic myocardial edema and inflammation-aggravating cardiac fibrosis and dysfunction. Moreover, our data reveal that therapeutic lymphangiogenesis may be a promising new approach for the treatment of cardiovascular diseases.

The significance of lymphatic space invasion and its association with vascular endothelial growth factor-C expression in ovarian cancer.

The aim of this study is to investigate the significance of lymphatic space invasion (LSI) and tumor VEGF-C expression in the lymphatic spread of ovarian cancer. By performing immunostaining using human ovarian cancer specimens, we first investigated the association between the extent of LSI and tumor VEGF-C expression, tumor lymphangiogenesis, or the lymphatic metastasis. Moreover, by performing in vitro and in vivo experiments, we elucidated the role of VEGF-C in tumor lymphangiogenesis and lymph node metastasis as well as its role as a therapeutic target in ovarian cancer. The presence of LSI was associated with lymph node metastasis in patients with ovarian cancer. VEGF-C overexpression was significantly associated with the increased LSI and LVD in ovarian cancer. VEGF-C stimulated the lymphangiogenesis in vitro, induced the new lymph vessel formation, and increased the lymph node metastasis in mice models of ovarian cancer. The attenuation of VEGF-C expression by the treatment with mTORC1 inhibitor significantly inhibited lymphangiogenesis, and decreased lymph node metastasis in mice models of ovarian cancer. The presence of LSI is an indicator of nodal metastasis and is associated with higher tumor VEGF-C expression and worse clinical outcome of ovarian cancer patients. VEGF-C plays a crucial role in tumor lymphangiogenesis and lymph node metastasis of ovarian cancer.

Resistive-Pulse Measurements with Nanopipettes: Detection of Vascular Endothelial Growth Factor C (VEGF-C) Using Antibody-Decorated Nanoparticles.

Quartz nanopipettes have recently been employed for resistive-pulse sensing of Au nanoparticles (AuNP) and nanoparticles with bound antibodies. The analytical signal in such experiments is the change in ionic current caused by the nanoparticle translocation through the pipette orifice. This paper describes resistive-pulse detection of cancer biomarker (Vascular Endothelial Growth Factor-C, VEGF-C) through the use of antibody-modified AuNPs and nanopipettes. The main challenge was to differentiate between AuNPs with attached antibodies for VEGF-C and antigen-conjugated particles. The zeta-potentials of these types of particles are not very different, and, therefore, carefully chosen pipettes with well-characterized geometry were necessary for selective detection of VEGF-C.

Significance of Vascular Endothelial Growth Factor (VEGF)-C and VEGF-D in the Progression of Cutaneous Melanoma.

INTRODUCTION: Induction of tumor lymphangiogenesis by vascular endothelial growth factor (VEGF)-C and VEGF-D promotes metastasis in many human cancers. AIM: The aim of this study was to examine the role of VEGF-C and VEGF-D in lymphangiogenesis and lymph node metastasis in patients with cutaneous melanoma. MATERIALS AND METHODS: Fifty-four melanoma specimens (18 with lymph node metastasis, 36 nonmetastatic) were investigated by immunostaining for VEGF-C, VEGF-D, and for lymphatic endothelial marker D2-40. VEGF-C and VEGF-D expression was assessed as a percentage and intensity of stained tumor cells, tumor-associated macrophages and fibroblasts. The quantification of lymphangiogenesis was conducted by computer-assisted morphometric analysis. RESULTS: The expressions of both VEGF-C and VEGF-D in tumor cells were significantly higher in lymph node metastatic melanomas compared with nonmetastatic melanomas (P = .015 VEGF-C; P = .005 VEGF-D). There was no statistically significant difference between metastatic and nonmetastatic melanomas regarding the expression of VEGF-C and VEGF-D in macrophages and fibroblasts. Metastatic melanomas showed a significantly higher intratumoral and peritumoral lymphatic vessel density (LVD) compared with nonmetastatic melanomas (P = .000 intratumoral, P = .000 peritumoral). Melanomas with VEGF-C positive tumor cells showed a significantly higher intratumoral and peritumoral LVD compared with VEGF-C negative tumor cells group of melanomas (P = .006 intratumoral, P = .010 peritumoral). VEGF-C expression in macrophages, fibroblasts, as well as VEGF-D expression in tumor cells, macrophages, and fibroblasts, showed no correlation with the intratumoral and peritumoral LVD. CONCLUSIONS: Our findings show the significance of VEGF-C in tumor cells in the induction of intratumoral and peritumoral lymphangiogenesis. This study suggests that both VEGF-C and VEGF-D in tumor cells promote lymph node metastasis, and that the immunohistochemical analysis of expression can be a useful tool for predicting clinical behavior of cutaneous melanoma.

Expression of vascular endothelial growth factor-C (VEGF-C) and its receptor (VEGFR-3) in the glial reaction elicited by human mesenchymal stem cell engraftment in the normal rat brain.

To determine whether vascular endothelial growth factor-C (VEGF-C) and its receptor (VEGFR-3) are involved in the glial reaction elicited by transplanted mesenchymal stem cells (MSCs), we examined the cellular localization of VEGF-C and VEGFR-3 proteins in the striatum of adult normal rats that received bone marrow-derived human MSCs. The MSC grafts were infiltrated with activated microglia/macrophages and astrocytes over a 2-week period post-transplantation, which appeared to parallel the loss of transplanted MSCs. VEGF-C/VEGFR-3 was expressed in activated microglia/macrophages recruited to the graft site, where the induction of VEGF-C protein was rather late compared with that of its receptor. VEGF-C protein was absent or very weak on day 3, whereas VEGFR-3 immunoreactivity was evident within the first three days. Furthermore, within three days, VEGF-C could be detected in the brain macrophages localized immediately adjacent to the needle track. At the same time, almost all the brain macrophages in both regions expressed VEGFR-3. Reactive astrocytes at the graft site expressed VEGFR-3, but not VEGF-C. These data demonstrated the characteristic time- and cell-dependent expression patterns for VEGF-C and VEGFR-3 within the engrafted brain tissue, suggesting that they may contribute to neuroinflammation in MSC transplantation, possibly through the recruitment and/or activation of microglia/macrophages and astrogliosis.