RESUMEN

OBJECTIVES: Non-Small Cell Lung Cancer (NSCLC) has attracted much attention on account of the high incidence and mortality of cancers. Vascular Endothelial Growth Factor Receptor 3 (VEGFR3/FLT4), which is a highly expressed receptor in NSCLC, greatly regulates cancer proliferation and migration. Pseudolaric Acid B (PAB) is a diterpenoid acid with antitumor activity isolated from Pseudolarix kaempferi. This study aimed to explore the inhibitory effect of PAB targeting FLT4 in NSCLC. METHODS: Cell membrane chromatography was used to evaluate the affinity of PAB binding on FLT4. NCIH1299 cells were used in this study, and an MTT assay was performed to determine the anti-proliferation effect of PAB. Cell cycle analysis was conducted to study the cycle arrest of PAB. Wound healing and Transwell assays assessed the rate of cell migration. Western blot analysis evaluated the expression of related proteins. RESULTS: PAB showed strong affinity to FLT4 with a KD value of 3.01 × 10- 6 M. Targeting FLT4 by PAB inactivated downstream P38MAPK and PI3K/AKT pathways, which inhibited the proliferation of NCI-H1299 cells. Meanwhile, PAB promoted G2/M phase arrest by influencing CyclinB1 and CDK1 complex formation to inhibit NCI-H1299 cell growth, but the effect was attenuated by knocking down the FLT4. Besides, PAB regulated MMP9 secretion through the Wnt/ß-catenin signaling pathway to inhibit NCI-H1299 cell migration. However, the ability of PAB to inhibit migration was significantly weakened by FLT4 knockdown in NCI-H1299 cells. CONCLUSION: PAB can inhibit the proliferation and migration of NSCLC cells through targeting FLT4 and is expected to be a promising FLT4 inhibitor for NSCLC treatment.

RESUMEN

Introduction: Endothelial cells have a crucial function in transporting and exchanging various nutrients. O-GlcNAcylation, mediated by O-GlcNAc transferase (OGT), involves the addition of N-acetylglucosamine to proteins and serves as an intracellular nutrient sensing mechanism. However, the role of O-GlcNAcylation in endothelial cells remains poorly understood. Objective: This study investigated the role of O-GlcNAcylation in endothelial cells. Methods: Endothelial-cell-specific Ogt -knockout mice (Ogt-ECKO) were generated by crossing Ogt-floxed mice (Ogt-flox) with VE-Cadherin Cre ERT2 mice. Ogt-ECKO mice and Ogt-flox control mice were subjected to a normal or high-fat diet, and their body weight, glucose metabolism, and lipid metabolism were examined. Results: Ogt-ECKO mice exhibited reduced body weight compared with Ogt-flox control mice under a high-fat diet. Lipid absorption was significantly impaired in Ogt-ECKO mice. Changes in the intercellular junctions of small intestinal lacteal endothelial cells from a button-like to a zipper-like configuration were observed. Furthermore, Ogt-ECKO mice showed decreased expression of VEGFR3. The administration of a nitric oxide donor restored lipid absorption and reversed the morphological alterations in Ogt-ECKO mice. Conclusions: These findings demonstrate the critical role of O-GlcNAcylation in endothelial cells in lipid absorption in the intestine through the modulation of lacteal junction morphology. These results provide novel insight into the metabolic regulatory mechanisms under physiological conditions and have implications for the development of new therapeutic strategies for obesity.

RESUMEN

Lymphangiogenesis at primary tumor and draining lymph nodes plays a pivotal role in tumor metastasis, which has been demonstrated to be regulated by the vascular endothelial growth factor receptor 3 (VEGFR-3) pathway. However, the effect of molecular imaging peptides, which specifically bind VEGFR-3, in tracing tumors remains unclear. We prepared a novel peptide, TMVP1448, with high-affinity to VEGFR-3. The dissociation constant (KD) of TMVP1448 with VEGFR-3 was 7.07 ×10-7 M. In vitro cellular assay showed that TMVP1448 could bind specifically with VEGFR-3. Near infrared imaging results showed that Cy7-TMVP1448 was able to accurately trace primary and metastatic cancers, and PET/CT results showed that [68Ga]Ga-DOTA-TMVP1448 was superior to commonly used radiotracers 18F-FDG. Additionally, no significant negative effect of TMVP1448 was found in mice. Our results suggested that TMVP1448 had great potential for future clinical applications in fluorescence imaging and nuclear imaging of tumors.

Asunto(s)

Péptidos , Tomografía Computarizada por Tomografía de Emisión de Positrones , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Animales , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Humanos , Ratones , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Péptidos/farmacología , Péptidos/química , Línea Celular Tumoral , Ratones Endogámicos BALB C , Radiofármacos , Femenino , Metástasis de la Neoplasia , Neoplasias/patología , Neoplasias/tratamiento farmacológico , Neoplasias/diagnóstico por imagen , Radioisótopos de Galio , Ratones Desnudos , Fluorodesoxiglucosa F18

RESUMEN

PURPOSE: Vascular endothelial growth factor receptor 3 (VEGFR-3) plays a critical role in tumor lymphangiogenesis and metastasis, holding promise as a promising therapeutic target for solid tumors. TMVP1 (LARGR) is a 5-amino acid peptide previously identified in our laboratory from bacterial peptide display system that specifically targets VEGFR-3. Radiolabeled TMVP1 can be used for non-invasive imaging of VEGFR-3 expressing tumors. Homodimeric peptides have better targeting ability than monomeric peptides, and it is worth exploring whether homodimers of TMVP1 ((TMVP1)2) can achieve better imaging effects. This study aimed to explore the peptide properties and tumor assessment value of [68Ga]Ga-labeled (TMVP1)2. METHODS: In this study, we developed a TMVP1 homodimer that was conjugated with 1,4,7-triazacyclononane-N, N', N″-triacetic acid (NOTA) via tetraethyleneglycol (PEG4) and triglyicine (Gly3) spacer, and labeled with 68Ga, to construct [68Ga]Ga-NOTA-(TMVP1)2. Binding of VEGFR-3 by TMVP1 and (TMVP1)2, respectively, was modeled by molecular docking. The affinity of [68Ga]Ga-NOTA-(TMVP1)2 for VEGFR-3 and its ability to bind to cells were evaluated. MicroPET imaging and biodistribution studies of [68Ga]Ga-NOTA-(TMVP1)2 were performed in subcutaneous C33A cervical cancer xenografts. Five healthy volunteers and eight patients with cervical cancer underwent whole-body PET/CT acquisition 30-45 min after intravenous injection of [68Ga]Ga-NOTA-(TMVP1)2. RESULTS: Both molecular docking and cellular experiments showed that homodimeric TMVP1 had a higher affinity for VEGFR-3 than monomeric TMVP1. [68Ga]Ga-NOTA-(TMVP1)2 was excreted mainly through the renal route and partly through the liver route. In mice bearing C33A xenografts, [68Ga]Ga-NOTA-(TMVP1)2 specifically localized in the tumor (2.32 ± 0.10% ID/g). Pretreatment of C33A xenograft mice with the unlabeled peptide NOTA-(TMVP1)2 reduced the enrichment of [68Ga]Ga-NOTA-(TMVP1)2 in tumors (0.58 ± 0.01% ID/g). [68Ga]Ga-NOTA-(TMVP1)2 proved to be safe in all healthy volunteers and recruited patients, with no side effects or allergies noted. In cervical cancer patients, a majority of the [18F]-FDG identified lesions (18/22, 81.8%) showed moderate to high signal intensity on [68Ga]Ga-NOTA-(TMVP1)2. SUVmax and SUVmean were 2.32 ± 0.77 and 1.61 ± 0.48, respectively. With normal muscle (gluteus maximus) as background, tumor-to-background ratios were 3.49 ± 1.32 and 3.95 ± 1.64 based on SUVmax and SUVmean, respectively. CONCLUSION: The favorable characterizations of [68Ga]Ga-NOTA-(TMVP1)2 such as convenient synthesis, high specific activity, and high tumor uptake enable the evaluation of VEGFR-3 in cervical cancer patients and warrant further clinical studies. TRIAL REGISTRATION: ChiCTR-DOD-17012458. Registered August 23, 2017 (retrospectively registered).

Asunto(s)

Radioisótopos de Galio , Compuestos Heterocíclicos con 1 Anillo , Neoplasias del Cuello Uterino , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Neoplasias del Cuello Uterino/diagnóstico por imagen , Neoplasias del Cuello Uterino/metabolismo , Humanos , Femenino , Animales , Ratones , Compuestos Heterocíclicos con 1 Anillo/química , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/química , Radioisótopos de Galio/química , Línea Celular Tumoral , Compuestos Heterocíclicos/química , Distribución Tisular , Péptidos/química , Péptidos/farmacocinética , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radiofármacos/farmacocinética , Radiofármacos/química , Persona de Mediana Edad , Multimerización de Proteína , Trazadores Radiactivos

RESUMEN

IMPORTANCE: Viruses are able to mimic the physiological or pathological mechanism of the host to favor their infection and replication. Virus-mock basement membrane (VMBM) is a Megalocytivirus-induced extracellular structure formed on the surface of infected cells and structurally and functionally mimics the basement membrane of the host. VMBM provides specific support for lymphatic endothelial cells (LECs) rather than blood endothelial cells to adhere to the surface of infected cells, which constitutes a unique phenomenon of Megalocytivirus infection. Here, the structure of VMBM and the interactions between VMBM components and LECs have been analyzed at the molecular level. The regulatory effect of VMBM components on the proliferation and migration of LECs has also been explored. This study helps to understand the mechanism of LEC-specific attachment to VMBM and to address the issue of where the LECs come from in the context of Megalocytivirus infection.

Asunto(s)

Membrana Basal , Células Endoteliales , Iridoviridae , Vasos Linfáticos , Membrana Basal/metabolismo , Membrana Basal/virología , Células Endoteliales/citología , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Iridoviridae/fisiología , Vasos Linfáticos/citología , Proliferación Celular , Movimiento Celular , Vasos Sanguíneos/citología , Interacciones Microbiota-Huesped

RESUMEN

Vascular endothelial growth factor receptor-3 (VEGFR-3) is known to participate in tumorigenesis and lymphangiogenesis, and as such, has the potential to serve as a molecular target for cancer therapy. SAR131675 is a highly selective VEGFR-3 antagonist that has an inhibitive effect on lymphatic cell growth. However, the anticancer effects and underlying mechanisms of SAR131675 in ovarian cancer remain poorly understood. In this study, we investigated the pathological role of VEGFR-3, and the effects of SAR131675 on proliferation, cell cycle, migration, and apoptosis in ovarian cancer cells. Our results showed that the mRNA and protein of VEGFR-3 were expressed in OVCAR3 and SKOV3 ovarian cancer cells, and this receptor was activated following stimulation with 50 ng/ml VEGF-C Cys156Ser (VEGF-CS), a selective ligand for VEGFR-3. Enhancing VEGFR-3 phosphorylation by treatment of ovarian cancer cells with VEGF-CS resulted in increased levels of phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2) and AKT. Moreover, our data demonstrated that SAR131675 inhibited VEGF-CS-mediated proliferation, colony formation, and migration of cancer cells in a dose-dependent manner. In addition, inhibition of VEGFR-3 activation with SAR131675 significantly increased cell cycle arrest and promoted apoptosis in both OVCAR3 and SKOV3 cells. Mechanistically, SAR131675 effectively suppressed the VEGF-CS-induced phosphorylation of VEGFR-3 and its downstream effectors including activated ERK1/2 and AKT in ovarian cancer cells. Our results reveal an anticancer activity of SAR131675 on the growth and migration of ovarian cancer cells, which may be through inhibiting VEGFR-3/ERK1/2/AKT pathway. SAR131675 may serve as an effective targeted drug for ovarian cancer.

RESUMEN

Microvascular dysfunction is a key driver of kidney disease. Pathophysiological changes in the kidney vasculature are regulated by vascular endothelial growth factor receptors (VEGFRs), supporting them as potential therapeutic targets. The tyrosine kinase receptor VEGFR-3, encoded by FLT4 and activated by the ligands VEGF-C and VEGF-D, is best known for its role in lymphangiogenesis. Therapeutically targeting VEGFR-3 to modulate lymphangiogenesis has been proposed as a strategy to treat kidney disease. However, outside the lymphatics, VEGFR-3 is also expressed in blood vascular endothelial cells in several tissues including the kidney. Here, we show that Vegfr-3 is expressed in fenestrated microvascular beds within the developing and adult mouse kidney, which include the glomerular capillary loops. We found that expression levels of VEGFR-3 are dynamic during glomerular capillary loop development, with the highest expression observed during endothelial cell migration into the S-shaped glomerular body. We developed a conditional knockout mouse model for Vegfr-3 and found that loss of Vegfr-3 resulted in a striking glomerular phenotype characterized by aneurysmal dilation of capillary loops, absence of mesangial structure, abnormal interendothelial cell junctions, and poor attachment between glomerular endothelial cells and the basement membrane. In addition, we demonstrated that expression of the VEGFR-3 ligand VEGF-C by podocytes and mesangial cells is dispensable for glomerular development. Instead, VEGFR-3 in glomerular endothelial cells attenuates VEGFR-2 phosphorylation. Together, the results of our study support a VEGF-C-independent functional role for VEGFR-3 in the kidney microvasculature outside of lymphatic vessels, which has implications for clinical therapies that target this receptor.NEW & NOTEWORTHY Targeting VEGFR-3 in kidney lymphatics has been proposed as a method to treat kidney disease. However, expression of VEGFR-3 is not lymphatic-specific. We demonstrated developmental expression of VEGFR-3 in glomerular endothelial cells, with loss of Vegfr-3 leading to malformation of glomerular capillary loops. Furthermore, we showed that VEGFR-3 attenuates VEGFR-2 activity in glomerular endothelial cells independent of paracrine VEGF-C signaling. Together, these data provide valuable information for therapeutic development targeting these pathways.

Asunto(s)

Enfermedades Renales , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Ratones , Animales , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Capilares/metabolismo

RESUMEN

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the cardiac function, lymphatic markers, macrophage and inflammatory cytokines in acute myocardial ischemia (AMI) mice, so as to explore its mechanism in improving AMI. METHODS: Male C57BL/6 mice were randomly divided into sham operation, model, EA, inhibitor and inhibitor+EA groups, with 10 mice in each group. AMI model was established by occlusion of left anterior descending coronary artery. For mice in the EA group and inhibitor+EA group, EA (1 mA, 2 Hz/15 Hz) was applied to bilateral "Shenmen"(HT7) and "Tongli"(HT5) for 30 min, once daily for consecutive 3 days. Mice in the inhibitor+EA group were given intraperitoneal injection of vascular endothelial growth factor receptor-3 (VEGFR-3) inhibitor SAR131675 30 min before the EA, while mice in the inhibitor group were given intraperitoneal injection of SAR131675 only. The electrocardiogram (ECG) of the neck-thoracic lead was recorded and analyzed by BL-420F biological function experiment system. Histopathologic changes of myocardial tissue were observed after H.E. staining. The contents of lactate dehydrogenase (LDH), cardiac troponin I (cTnI) in serum and interleukin-18 (IL-18) and interleukin-6 (IL-6) in ischemic myocardium were determined by ELISA. The expressions of hyaluronic acid receptor-1 (LYVE-1) and macrophage mar-ker CD68 (CD68) in the myocardial tissue were detected by immunofluorescence assay. The protein expression levels of vascular endothelial growth factor C (VEGF-C) and VEGFR-3 were detected by Western blot. RESULTS: Compared with the sham operation group, the ECG-ST level, the contents of serum LDH and cTnI, and the contents of IL-18 and IL-6 in the myocardial tissue were significantly increased (P<0.01), the expression of LYVE-1 and the protein expression levels of VEGF-C and VEGFR-3 in the myocardial tissue were significantly decreased (P<0.01), while the number of CD68 positive cells was significantly increased (P<0.01) in the model group. Compared with the model, inhibitor and inhibitor+EA groups, the ECG-ST level, the contents of serum LDH and cTnI, and the contents of IL-18 and IL-6 in the myocardial tissue were decreased (P<0.01), the expression of LYVE-1 and the expression level of VEGFR-3 protein were increased (P<0.01), while the number of CD68 positive cells was significantly decreased (P<0.01) in the EA group. Compared with the model and inhibitor groups, the expression level of VEGF-C was increased (P<0.01) in the EA group. Outcomes of H.E. staining showed that the myocardial fibers were disordered with a large number of inflammatory cell infiltration in the model group, which was milder in the EA group. CONCLUSION: Acupuncture can improve the inflammatory injury of AMI mice, which may be related to activate VEGF-C/VEGFR-3 pathway to promote lymphangiogenesis, reduce macrophage infiltration and inflammatory factors.

Asunto(s)

Electroacupuntura , Isquemia Miocárdica , Animales , Masculino , Ratones , Puntos de Acupuntura , Interleucina-18 , Interleucina-6 , Ratones Endogámicos C57BL , Isquemia Miocárdica/genética , Isquemia Miocárdica/terapia , Factor A de Crecimiento Endotelial Vascular , Factor C de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial Vascular

RESUMEN

Gastric cancer (GC) is one of the most common gastrointestinal malignancies in the world. Growing evidence emphasizes the critical role of long non-coding RNA (lncRNA) in GC tumorigenesis. The aim of the research was to elucidate the effect and mechanism of Babao Dan (BBD) on lymphangiogenesis of GC in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis. The present study investigated BBD significantly decreased the expression of lncRNA-ANRIL and VEGF-C in GC cells (AGS, BGC823, and MGC80-3) by using real-time quantitative polymerasechain reaction (RT-qPCR) and the secretion and expression of VEGF-C by (enzyme linked immunosorbent assay) ELISA and western blot (WB). BBD significantly inhibited the tumor xenograft of GC growth and the expression of lncRNA-ANRIL, VEGF-C, VEGFR-3 and LYVE-1 in vivo. BBD reduced serum VEGF-C level. In vitro, BBD inhibited the tube formation and decreased the cell viability, proliferation and migration of HLECs by using tube formation, MTT, Hoechst and Transwell assays. In addition, WB assay found that BBD decreased the expression levels of VEGF-C, VEGFR-3, matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9), and RT-qPCR assay found that the mRNA expression levels of lncRNA-ANRIL, VEGF-C, VEGFR-3, MMP-2, MMP-9, CDK4, Cyclin D1, and Bcl-2 were down-regulated, and the expression of p21 and Bax were increased. Taken together, these results demonstrated that BBD inhibited lymphangiogenesis of GC in vitro and in vivo via the lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

Asunto(s)

ARN Largo no Codificante , Neoplasias Gástricas , Línea Celular Tumoral , Medicamentos Herbarios Chinos , Humanos , Linfangiogénesis/genética , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , ARN Largo no Codificante/genética , ARN Largo no Codificante/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Subsequently to the publication of the above article, the authors have realized that Fig. 4 on p. 145 (showing the results from the colony formation assay) was published containing erroneous images in Fig. 4C. Essentially, incorrect images were selected to represent the negative control (NC) experiments owing to an altered shooting angle and incorrect naming of the files. The corrected version of Fig. 4, now showing the correct data for the NC experiments in Fig. 4C, is shown on the next page. The authors confirm that the error made in the presentation of Fig. 4 did not adversely affect either the results or the conclusions reported in this paper, and they are grateful to the Editor of International Journal of Oncology for granting them this opportunity to publish a Corrigendum. They also apologise to the readership for any inconvenience caused. [International Journal of Oncology 54: 139-151, 2019; DOI: 10.3892/ijo.2018.4623].

RESUMEN

OBJECTIVE: Cerebral cavernous malformations (CCMs) can happen anywhere in the body, although they most commonly produce symptoms in the brain. The role of CCM genes in other vascular beds outside the brain and retina is not well-examined, although the 3 CCM-associated genes (CCM1, CCM2, and CCM3) are ubiquitously expressed in all tissues. We aimed to determine the role of CCM gene in lymphatics. Approach and Results: Mice with an inducible pan-endothelial cell (EC) or lymphatic EC deletion of Ccm3 (Pdcd10ECKO or Pdcd10LECKO) exhibit dilated lymphatic capillaries and collecting vessels with abnormal valve structure. Morphological alterations were correlated with lymphatic dysfunction in Pdcd10LECKO mice as determined by Evans blue dye and fluorescein isothiocyanate(FITC)-dextran transport assays. Pdcd10LECKO lymphatics had increased VEGFR3 (vascular endothelial growth factor receptor-3)-ERK1/2 (extracellular signal-regulated kinase 1/2) signaling with lymphatic hyperplasia. Mechanistic studies suggested that VEGFR3 is primarily regulated at a transcriptional level in Ccm3-deficient lymphatic ECs, in an NF-κB (nuclear factor κB)-dependent manner. CCM3 binds to importin alpha 2/KPNA2 (karyopherin subunit alpha 2), and a CCM3 deletion releases KPNA2 to activate NF-κB P65 by facilitating its nuclear translocation and P65-dependent VEGFR3 transcription. Moreover, increased VEGFR3 in lymphatic EC preferentially activates ERK1/2 signaling, which is critical for lymphatic EC proliferation. Importantly, inhibition of VEGFR3 or ERK1/2 rescued the lymphatic defects in structure and function. CONCLUSIONS: Our data demonstrate that CCM3 deletion augments the VEGFR3-ERK1/2 signaling in lymphatic EC that drives lymphatic hyperplasia and malformation and warrant further investigation on the potential clinical relevance of lymphatic dysfunction in patients with CCM.

Asunto(s)

Endotelio Linfático/fisiopatología , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Sistema de Señalización de MAP Quinasas/fisiología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Células Cultivadas , Células Endoteliales/fisiología , Endotelio Linfático/patología , Femenino , Eliminación de Gen , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Hiperplasia , Masculino , Ratones Endogámicos , Modelos Animales , FN-kappa B/genética , Translocación Genética

RESUMEN

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Asunto(s)

Indoles/toxicidad , Enfermedades Renales/inducido químicamente , Riñón/efectos de los fármacos , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Naftalenos/toxicidad , Inhibidores de Proteínas Quinasas/toxicidad , Receptor 3 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Cisplatino , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Riñón/enzimología , Riñón/patología , Riñón/fisiopatología , Enfermedades Renales/enzimología , Enfermedades Renales/patología , Enfermedades Renales/fisiopatología , Vasos Linfáticos/enzimología , Vasos Linfáticos/patología , Vasos Linfáticos/fisiopatología , Masculino , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fosforilación , Transducción de Señal , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

OBJECTIVES: To compare the effectiveness of two methods of extracorporeal shock-wave therapy (ESWT) in a rat model of forelimb lymphedema, induced by axillary lymph node dissection. METHODS: Sprague-Dawley rats were randomly allocated to a group that received 500 ESWT shocks only in the lymphedematous forelimb (Forelimb/ESWT) and a group that received 300 ESWT shocks in the axilla and 200 shocks in the lymphedematous forelimb (Axilla+Forelimb/ESWT). The circumferences of each limb were then measured. Immunohistochemistry for a pan-endothelial marker (cluster of differentiation [CD]31) and lymphatic vessel endothelial hyaluronan receptor-1, and western blot analysis for vascular endothelial growth factor receptor-3 (VEGFR3) and VEGF-C were performed. RESULTS: The circumferences of the limbs showed significant effects of group and time following surgery. The circumferences at the carpal joint and 2.5 cm above were smallest in the naïve limbs, larger in the Axilla+Forelimb/ESWT group, and the largest in the control group. VEGFR3 tended to be expressed at a higher level in the Axilla+Forelimb/ESWT group (1.96-fold) than in the Forelimb/ESWT group (1.20-fold) versus the opposite non-edematous forelimbs, although this difference was not statistically significant. CONCLUSIONS: These data suggest that ESWT protocols have differential effects on angiogenesis and lymphangiogenesis in lymphedematous limbs.

Asunto(s)

Linfedema , Factor A de Crecimiento Endotelial Vascular , Animales , Linfangiogénesis , Linfedema/terapia , Proyectos Piloto , Ratas , Ratas Sprague-Dawley

RESUMEN

Renal cell carcinoma (RCC) is the most common form of kidney cancer. Vascular endothelial growth factor-C (VEGF-C) and its receptor, VEGFR-3, are involved in lymphangiogenesis. The aim of the present study was to investigate the expression levels of VEGF-C and VEGFR-3 in RCC, and their association with lymphatic vessel density (LVD) and lymph node metastasis. The mRNA expression levels of VEGF-C in 40 RCC tissues and 10 normal renal tissues were determined by reverse transcription-semiquantitative PCR. The differential expression of VEGF-C and VEGFR-3 was examined by immunohistochemistry. Using an anti-D2-40 antibody as a lymphatic marker, the morphology and structure of lymphatic vessels in tissues was examined, and the LVD was calculated. VEGF-C mRNA expression in RCC tissues was higher than that in normal renal tissues, and VEGF-C mRNA expression in the lymph node metastasis group was higher than that in the non-lymph node metastasis group. The positive expression rate of VEGF-C and VEGFR-3 in RCC tissues was significantly higher than that in normal renal tissues. VEGF-C expression in the lymph node metastasis group was significantly higher than that in the non-lymph node metastasis group, and the positive expression of VEGF-C was associated with the clinical staging of RCC. In addition, there was a correlation between VEGF-C and VEGFR-3 expression in tumor cells. The LVD around the tumor was higher than that in the center of the tumor tissues and normal renal tissues, and it was closely associated with lymphatic invasion and lymph node metastasis. Overall, the current findings demonstrated that the VEGF-C/VEGFR-3 signaling pathway promoted lymphangiogenesis around the tumor and provided an approach for tumor lymphatic invasion and lymph node metastasis. Therefore, VEGFC and VEGFR-3 expression may serve an important role in the initiation and development of RCC.

RESUMEN

Recent evidence has shown that the vascular endothelial growth factor (VEGF) system plays a crucial role in several neuropathological processes. We previously reported an upregulation of VEGF-C and its receptor, VEGFR-3, in reactive astrocytes after the onset of status epilepticus (SE). However, it remains unknown, which molecules act as downstream signals following VEGFR-3 upregulation, and are involved in reactive astrogliosis after SE. Therefore, we investigated whether VEGFR-3 upregulation within reactive astrocytes is associated with the activation of mammalian target of rapamycin (mTOR) signaling, which we confirmed by assaying for the phosphorylated form of S6 protein (pS6), and whether VEGFR-3-mediated mTOR activation induces astroglial glutamate transporter-1 (GLT-1) expression in the hippocampus after pilocarpine-induced SE. We found that spatiotemporal expression of pS6 was consistent with VEGFR-3 expression in the hippocampus after SE, and that both pS6 and VEGFR-3 were highly expressed in SE-induced reactive astrocytes. Treatment with the mTOR inhibitor rapamycin decreased astroglial VEGFR-3 expression and GLT-1 expression after SE. Treatment with a selective inhibitor for VEGFR-3 attenuated astroglial pS6 expression as well as suppressed GLT-1 expression and astroglial reactivity in the hippocampus after SE. These findings demonstrate that VEGFR-3-mediated mTOR activation could contribute to the regulation of GLT-1 expression in reactive astrocytes during the subacute phase of epilepsy. In conclusion, the present study suggests that VEGFR-3 upregulation in reactive astrocytes may play a role in preventing hyperexcitability induced by continued seizure activity.

Asunto(s)

Estado Epiléptico , Sistema de Transporte de Aminoácidos X-AG , Astrocitos/metabolismo , Transportador 2 de Aminoácidos Excitadores , Hipocampo/metabolismo , Humanos , Pilocarpina/toxicidad , Estado Epiléptico/inducido químicamente , Serina-Treonina Quinasas TOR/metabolismo , Factor A de Crecimiento Endotelial Vascular , Receptor 3 de Factores de Crecimiento Endotelial Vascular

RESUMEN

OBJECTIVE: To ascertain the expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in placental conducting and exchange villi from normotensive, preeclamptic (PE) and antiretroviral treated pregnant women, using morphometric image analysis. STUDY DESIGN: This study utilizes retrospectively collected, paraffin wax-embedded, placental samples (n = 90) that were immuno-stained for VEGFR-3. During selection of the retrospective study, women with chronic illnesses were filtered out, to exclusively allow for the examination of VEGFR-3 immuno-expression in HIV and preeclamptic women. The study population consisted of normotensive (n = 30) and preeclamptic (n = 60) groups which were further divided on the basis of HIV status (negative - and positive +), and early and late onset preeclampsia (EOPE and LOPE respectively). The resulting groups were as follows; N- (n = 15), N+ (n = 15), EOPE- (n = 15), EOPE+ (n = 15), LOPE- (n = 15) and LOPE+ (n = 15). Microscopic examination and morphometric image analysis were performed on the immuno-stained placental tissue samples. RESULTS: Analysis on HIV status did not yield a significant difference in conducting (p = 0.3015) or exchange (p = 0.4535) villi, regardless of pregnancy type. The N vs. PE analysis showed a reduced immuno-expression of VEGFR-3 in both conducting (p = 0.0107) and exchange (p < 0.0001) villi. Results from a multiple group comparative analysis of N vs. EOPE vs. LOPE VEGFR-3 immuno-expression, showed a significant difference between the N vs. EOPE groups. CONCLUSION: The results presented provide compelling evidence that HIV infection does not significantly alter angiogenesis in placental villi. PE however, has caused angiogenic dysregulation and trophoblast pathology was observed. We report a severe downregulation of VEGFR-3 in placental villi from EOPE woman, regardless of HIV status. Hence we suggest a future investigation into EOPE's aetiology and its downstream effects on pregnancy.

Asunto(s)

Infecciones por VIH , Preeclampsia , Receptor 3 de Factores de Crecimiento Endotelial Vascular , Femenino , Humanos , Placenta , Embarazo , Estudios Retrospectivos , Factor A de Crecimiento Endotelial Vascular

RESUMEN

BACKGROUND: The occurrence and development of gastric cancer is a multi-factor, multi-stage, multi-gene abnormal accumulation process. Both genetic and epigenetic mechanisms play an important role in the molecular mechanism of gastric cancer. DNA methylation is one of the most studied epigenetic expression mechanisms. To study the correlation between gene promoter methylation status and protein expression of vascular endothelial growth factor receptor 3 (VEGFR3), as well as their association with clinicopathological features in early gastric cancer (EGC) cases. METHODS: Immunohistochemical analysis and methylation-specific PCR (MSP) were used to detect the expression of VEGFR3 protein and methylation status of the VEGFR3 promoter in 50 cases of EGC and their paired normal gastric mucosa tissues. The level of DNA methylation of the VEGFR3 promoter, in situ VEGFR3 protein expression, and the clinicopathological characteristics of EGC patients were statistically analyzed. RESULTS: The positive rate of VEGFR3 protein expression in EGC tumor tissue (60%) was significantly higher than that in the normal gastric mucosa (10%). The detectable methylation frequency of VEGFR3 promoter in EGC tumor tissue (48%) was significantly lower than that in the normal gastric mucosa (85%). As anticipated, the methylation level of the VEGFR3 gene promoter was negatively associated with the overexpression of VEGFR3 protein. In addition, methylation status of the VEGFR3 gene promoter was positively correlated with lymph node metastasis in EGC patients (P<0.05), but was not linked to patients' gender, age, tumor size, degree of differentiation, or tumor invasion depth (P>0.05). CONCLUSIONS: Hypomethylation of the VEGFR3 gene promoter is one of the major mechanisms underlying VEGFR3 gene overexpression in EGC tumor tissues and is related to lymph node metastasis in EGC patients. DNA methylation of VEGFR3 is expected to become a molecular diagnostic and prognostic biomarker for EGC.

RESUMEN

Vascular endothelial growth factor (VEGF)-C and its receptor, vascular endothelial growth factor receptor (VEGFR)-3, are responsible for lymphangiogenesis in both embryos and adults. In epilepsy, the expression of VEGF-C and VEGFR-3 was significantly upregulated in the human brains affected with temporal lobe epilepsy. Moreover, pharmacologic inhibition of VEGF receptors after acute seizures could suppress the generation of spontaneous recurrent seizures, suggesting a critical role of VEGF-related signaling in epilepsy. Therefore, in the present study, the spatiotemporal expression of VEGF-C and VEGFR-3 against pilocarpine-induced status epilepticus (SE) was investigated in C57BL/6N mice using immunohistochemistry. At 1 day after SE, hippocampal astrocytes and microglia were activated. Pyramidal neuronal death was observed at 4 days after SE. In the subpyramidal zone, VEGF-C expression gradually increased and peaked at 7 days after SE, while VEGFR-3 was significantly upregulated at 4 days after SE and began to decrease at 7 days after SE. Most VEGF-C/VEGFR-3-expressing cells were pyramidal neurons, but VEGF-C was also observed in some astrocytes in sham-manipulated animals. However, at 4 days and 7 days after SE, both VEGFR-3 and VEGF-C immunoreactivities were observed mainly in astrocytes and in some microglia of the stratum radiatum and lacunosum-moleculare of the hippocampus, respectively. These data indicate that VEGF-C and VEGFR-3 can be upregulated in hippocampal astrocytes and microglia after pilocarpine-induced SE, providing basic information about VEGF-C and VEGFR-3 expression patterns following acute seizures.

RESUMEN

Lymphangiogenesis is an important biological process associated with cancer metastasis. The development of new drugs that block lymphangiogenesis represents a promising therapeutic strategy. Marine fungus-derived compound phomaketide A, isolated from the fermented broth of Phoma sp. NTOU4195, has been reported to exhibit anti-angiogenic and anti-inflammatory effects. However, its anti-lymphangiogenic activity has not been clarified to date. In this study, we showed that phomaketide A inhibited cell growth, migration, and tube formation of lymphatic endothelial cells (LECs) without an evidence of cytotoxicity. Mechanistic investigations revealed that phomaketide A reduced LECs-induced lymphangiogenesis via vascular endothelial growth factor receptor-3 (VEGFR-3), protein kinase Cδ (PKCδ), and endothelial nitric oxide synthase (eNOS) signalings. Furthermore, human proteome array analysis indicated that phomaketide A significantly enhanced the protein levels of various protease inhibitors, including cystatin A, serpin B6, tissue factor pathway inhibitor (TFPI), and tissue inhibitor matrix metalloproteinase 1 (TIMP-1). Importantly, phomaketide A impeded tumor growth and lymphangiogenesis by decreasing the expression of LYVE-1, a specific marker for lymphatic vessels, in tumor xenograft animal model. These results suggest that phomaketide A may impair lymphangiogenesis by suppressing VEGFR-3, PKCδ, and eNOS signaling cascades, while simultaneously activating protease inhibitors in human LECs. We document for the first time that phomaketide A inhibits lymphangiogenesis both in vitro and in vivo, which suggests that this natural product could potentially treat cancer metastasis.

Asunto(s)

Inhibidores de la Angiogénesis/farmacología , Antinematodos/farmacología , Ascomicetos/química , Linfangiogénesis/efectos de los fármacos , Policétidos/farmacología , Células A549 , Inhibidores de la Angiogénesis/aislamiento & purificación , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antinematodos/aislamiento & purificación , Antinematodos/uso terapéutico , Organismos Acuáticos/química , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Metástasis Linfática , Vasos Linfáticos/citología , Masculino , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Óxido Nítrico Sintasa de Tipo III/metabolismo , Policétidos/aislamiento & purificación , Policétidos/uso terapéutico , Proteína Quinasa C-delta/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

The association between homeobox (HOX)B9 expression and tumor malignancy was identified recently. It was reported that HOXB9 induced tumor angiogenesis, and associated with poor prognosis in patients with breast and colon cancer. On the other hand, regional lymph nodes are the most common site of tumor spread, and lymph node metastasis is a major prognostic factor in gastric cancer. It was hypothesized that HOXB9 promotes tumor lymphangiogenesis and induces tumor progression, invasion and metastasis in gastric cancer. The aim of the present study was to evaluate the correlation between HOXB9 expression, prognosis and clinicopathologic factors in patients with gastric cancer, and to assess the contribution of HOXB9 expression to tumor cell lymphangiogenesis in vitro. HOXB9 expression was evaluated by immunohistochemistry in resected tumor tissues from 58 patients with gastric cancer, and the association between prognosis and clinicopathologic factors was determined. HOXB9 gene was overexpressed in human gastric cancer TMK-1 cells and the effect of HOXB9 overexpression on the expression of vascular endothelial growth factor (VEGF)-C, VEGF-D and VEGF receptor (R)-3 was determined. It was demonstrated that the depth of tumor invasion, the number of node metastases, lymphatic invasion and vascular invasion were significantly associated with HOXB9 expression. Overall survival was decreased in patients with HOXB9 expression. The mRNA expression of VEGF-D but not of VEGF-C and VEGFR-3 was increased in HOXB9-overexpressing TMK-1 cells compared with control cells. In conclusion, HOXB9 expression was positively correlated with gastric cancer progression and lymphangiogenesis marker expression. HOXB9 may be associated with lymphogenic metastasis.