Expression Of VEGF-D Research Articles

Effects of Valproic Acid and Mitomycin C Combination Therapy in a Rabbit Model of Minimally Invasive Glaucoma Surgery.

PurposeThis study aimed to compare the effectiveness of combination therapy consisting of low-dose mitomycin C (MMC) and valproic acid (VPA) against high-dose MMC for improving the scar phenotype in minimally invasive glaucoma surgery (MIGS).MethodsA rabbit model of MIGS incorporating the PreserFlo MicroShunt was treated with high (0.4 mg/mL) or low (0.1 mg/mL) doses of MMC or with combination therapy consisting of low-dose (0.1 mg/mL) MMC and VPA. Operated eyes were examined by live ocular imaging, histochemical evaluation, multiphoton quantitation of collagen characteristics, and molecular analyses.ResultsAlthough high-dose MMC obliterated the vasculature, combination therapy vastly improved the postoperative tissue morphology by maintaining the vasculature without increased vascularization. Combination therapy also altered collagen morphology and reduced encapsulation of the MicroShunt distal end, which remained at risk with MMC treatment alone. Multiphoton quantitation indicated that the combination therapy significantly reduced collagen density and fiber dimensions compared with monotherapy. At the molecular level, combination therapy significantly reduced Vegfa, Vegfc, and Vegfd expression and inhibited Col1a1 upregulation from baseline levels, all of which low-dose MMC alone was unable to achieve. Notably, COL1A1 protein levels appeared more consistently suppressed by combination therapy compared with high-dose MMC alone.ConclusionsCompared with high-dose MMC, combination therapy was less toxic by sparing the vasculature and potentially more effective in reducing scarring via the regulation of collagen content and organization.Translational RelevanceVPA may be combined with low-dose MMC to replace high-dose MMC to deliver safe and effective anti-scarring outcomes.

Carnitine Palmitoyltransferase 1 Regulates Prostate Cancer Growth under Hypoxia.

Simple SummaryCancer cell survival in hypoxia areas, with low oxygen and food supply as well as abundant waste material, is critical to their aggressiveness and associated with disease relapse and mortality. Therefore, it is vital to understand the molecular regulators of cancer cell survival under these harsh physiological conditions. In the present study, we assessed the role of a mitochondrial protein carnitine palmitoyltransferase (CPT1A) in regulating prostate cancer (PCa) cell survival and proliferation under hypoxic conditions in both cell culture and animal models. The results showed that CPT1A expression in PCa cells is key to their survival and proliferation in the hypoxic tumor microenvironment. These results have high translational significance in improving cancer prognosis and therapy.Hypoxia and hypoxia-related biomarkers are the major determinants of prostate cancer (PCa) aggressiveness. Therefore, a better understanding of molecular players involved in PCa cell survival under hypoxia could offer novel therapeutic targets. We previously reported a central role of mitochondrial protein carnitine palmitoyltransferase (CPT1A) in PCa progression, but its role in regulating PCa survival under hypoxia remains unknown. Here, we employed PCa cells (22Rv1 and MDA-PCa-2b) with knockdown or overexpression of CPT1A and assessed their survival under hypoxia, both in cell culture and in vivo models. The results showed that CPT1A knockdown in PCa cells significantly reduced their viability, clonogenicity, and sphere formation under hypoxia, while its overexpression increased their proliferation, clonogenicity, and sphere formation. In nude mice, 22Rv1 xenografts with CPT1A knockdown grew significantly slower compared to vector control cells (~59% reduction in tumor volume at day 29). On the contrary, CPT1A-overexpressing 22Rv1 xenografts showed higher tumor growth compared to vector control cells (~58% higher tumor volume at day 40). Pathological analyses revealed lesser necrotic areas in CPT1A knockdown tumors and higher necrotic areas in CPT1A overexpressing tumors. Immunofluorescence analysis of tumors showed that CPT1A knockdown strongly compromised the hypoxic areas (pimonidazole+), while CPT1A overexpression resulted in more hypoxia areas with strong expression of proliferation biomarkers (Ki67 and cyclin D1). Finally, IHC analysis of tumors revealed a significant decrease in VEGF or VEGF-D expression but without significant changes in biomarkers associated with microvessel density. These results suggest that CPT1A regulates PCa survival in hypoxic conditions and might contribute to their aggressiveness.

Fibroblast CEBPD/SDF4 axis in response to chemotherapy-induced angiogenesis through CXCR4

Cancer-associated fibroblasts (CAFs) play an essential role in supporting cancer progression. However, the details and consequent effects in response to the communication between CAFs and angiogenesis remain largely uninvestigated, especially in anticancer drug treatments. We found that cisplatin and 5-fluorouracil could induce fibroblast differentiation toward myofibroblasts via CCAAT/enhancer-binding protein delta (CEBPD) and consequently promote proliferation, migration, and in vitro tube formation of vascular endothelial cells and angiogenesis in vivo. Stromal-cell-derived factor 4 (SDF4) is responsive to anticancer drugs via CEBPD activation in CAFs and contributes to create a permissive environment for tumor cell angiogenesis and promotion of distant metastasis. Importantly, we demonstrated that SDF4 interacts with CXCR4 to trigger VEGFD expression through the activation of the ERK1/2 and p38 pathways in endothelial cells. Taken together, our novel findings support that SDF4 can be a therapeutic target in inhibition of angiogenesis for chemotherapy drug-administrated cancer patients.

Dysregulation of Amphiregulin stimulates the pathogenesis of cystic lymphangioma

Along with blood vessels, lymphatic vessels play an important role in the circulation of body fluid and recruitment of immune cells. Postnatal lymphangiogenesis commonly occurs from preexisting lymphatic vessels by sprouting, which is induced by lymphangiogenic factors such as vascular endothelial growth factor C (VEGF-C). However, the key signals and cell types that stimulate pathological lymphangiogenesis, such as human cystic lymphangioma, are less well known. Here, we found that mouse dermal fibroblasts that infiltrate to sponges subcutaneously implanted express VEGF-D and sushi, Von Willebrand factor type A, EGF, and pentraxin domain containing 1 (SVEP1) in response to PDGFRβ signal. In vitro, Pdgfrb knockout (β-KO) fibroblasts had reduced expression of VEGF-D and SVEP1 and overproduced Amphiregulin. Dysregulation of these three factors was involved in the cyst-like and uneven distribution of lymphatic vessels observed in the β-KO mice. Similarly, in human cystic lymphangioma, which is one of the intractable diseases and mostly occurs in childhood, fibroblasts surrounding cystic lymphatics highly expressed Amphiregulin. Moreover, fibroblast-derived Amphiregulin could induce the expression of Amphiregulin in lymphatic endothelial cells. The dual source of Amphiregulin activated EGFR expressed on the lymphatic endothelial cells. This exacerbation cascade induced proliferation of lymphatic endothelial cells to form cystic lymphangioma. Ultimately, excessive Amphiregulin produced by fibroblasts surrounding lymphatics and by lymphatic endothelial cells per se results in pathogenesis of cystic lymphangioma and will be a fascinating therapeutic target of cystic lymphangioma.

Novel COL4A1-VEGFD gene fusion in myofibroma.

Myofibroma is a benign pericytic tumour affecting young children. The presence of multicentric myofibromas defines infantile myofibromatosis (IMF), which is a life‐threatening condition when associated with visceral involvement. The disease pathophysiology remains poorly characterized. In this study, we performed deep RNA sequencing on eight myofibroma samples, including two from patients with IMF. We identified five different in‐frame gene fusions in six patients, including three previously described fusion transcripts, SRF‐CITED1, SRF‐ICA1L and MTCH2‐FNBP4, and a fusion of unknown significance, FN1‐TIMP1. We found a novel COL4A1‐VEGFD gene fusion in two cases, one of which also carried a PDGFRB mutation. We observed a robust expression of VEGFD by immunofluorescence on the corresponding tumour sections. Finally, we showed that the COL4A1‐VEGFD chimeric protein was processed to mature VEGFD growth factor by proteases, such as the FURIN proprotein convertase. In conclusion, our results unravel a new recurrent gene fusion that leads to VEGFD production under the control of the COL4A1 gene promoter in myofibroma. This fusion is highly reminiscent of the COL1A1‐PDGFB oncogene associated with dermatofibrosarcoma protuberans. This work has implications for the diagnosis and, possibly, the treatment of a subset of myofibromas.

Roles of Thromboxane Receptor Signaling in Enhancement of Lipopolysaccharide-Induced Lymphangiogenesis and Lymphatic Drainage Function in Diaphragm.

[Figure: see text].

Short-term molecular and cellular effects of ischemia/reperfusion on vascularized lymph node flaps in rats.

Vascularized lymph node (VLN) transfer is an emerging strategy to re-establish lymphatic drainage in chronic lymphedema. However, the biological processes underlying lymph node integration remain elusive. This study introduces an experimental approach facilitating the analysis of short-term molecular and cellular effects of ischemia/reperfusion on VLN flaps. Lymph node flaps were dissected pedicled on the lateral thoracic vessels in 44 Lewis rats. VLN flaps were exposed to 45 or 120 minutes ischemia by in situ clamping of the vascular pedicle with subsequent reperfusion for 24 hours. Flaps not exposed to ischemia/reperfusion served as controls. Lymph nodes and the perinodal adipose tissue were separately analyzed by Western blot for the expression of lymphangiogenic and angiogenic growth factors. Moreover, morphology, microvessel density, proliferation, apoptosis and immune cell infiltration of VLN flaps were further assessed by histology and immunohistochemistry. Ischemia for 120 minutes was associated with a markedly reduced cellularity of lymph nodes but not of the perinodal adipose tissue. In line with this, ischemic lymph nodes exhibited a significantly lower microvessel density and an increased expression of VEGF-D and VEGF-A. However, VEGF-C expression was not upregulated. In contrast, analyses of the perinodal adipose tissue revealed a more subtle decrease of microvessel density, while only the expression of VEGF-D was increased. Moreover, after 120 minutes ischemia, lymph nodes but not the perinodal adipose tissue exhibited significantly higher numbers of proliferating and apoptotic cells as well as infiltrated macrophages and neutrophilic granulocytes compared with non-ischemic flaps. Taken together, lymph nodes of VLN flaps are highly susceptible to ischemia/reperfusion injury. In contrast, the perinodal adipose tissue is less prone to ischemia/reperfusion injury.

Overexpression of VEGF165 is associated with poor prognosis of cervical cancer

Cervical cancer is a major health hazard to Indian women. Human papillomavirus (HPV) infection is an established risk factor for cervical carcinogenesis. However, understanding the cervical cancer biology beyond HPV infection is very crucial to predict aggressive behavior, prognosis, treatment response and survival. In the present study, we explored the role of vascular endothelial growth factor A (VEGFA) isoforms, VEGFC and VEGFD in cervical cancer progression and its association with HPV 16 and 18 infections. A total of 110 cervical cancer tissues and 50 normal cervical tissues were collected for the study. Reverse transcription-polymerase chain reaction was employed to analyze tissue VEGFA isoforms, VEGFC and VEGFD expression. VEGF165 was significantly higher, whereas VEGFC and VEGFD were significantly lower in malignant cervical carcinoma tissues as compared to normal cervix tissues. Expression levels of VEGF121 and VEGFC were significantly associated with type of tumor growth while VEGF165 was significantly associated with lymph node metastasis. VEGF165 transcript levels were significantly higher in patients with squamous cell carcinoma (SCC) and developed recurrence. Most strikingly, higher VEGF165 expression was significantly associated with worst disease-free survival (DFS) specifically in patients with SCC. Association of VEGF165 with lymph node metastasis, disease recurrence and worst DFS indicated that VEGF165 is an important prognostic factor in cervical carcinogenesis.

Investigation of Targeting Relationship between Micro-Rna-22 and Vegfr3 in Lung Squamous Cell Carcinoma.

The present study explored the clinical significance of microRNA-22 (miR-22) expression in lung squamous cell carcinoma and to explore the targeting relationship with vascular endothelial growth factor receptor 3 (VEGFR3). A total of 49 patients with lung squamous cell carcinoma who underwent surgical treatment were selected. The expression of miR-22 was detected by fluorescence quantitative realtime PCR (qPCR), the expression of VEGFR3 was detected by Western blotting assays, and D240 labeled microlymphatic vessels density (MLVD) was detected by immunohistochemistry (IHC). Lung squamous cell carcinoma cell line SK-MES-1 was selected and the targeting relationship between miR-22 and VEGFR3 was analyzed by double luciferase reporter gene assay. Western blotting assays were used to detect the expression of vascular endothelial growth factor-D (VEGFD) and D240 in the blank control group, empty vector transfection group, miR-22 transfection group, miR-22 and VEGFR3 co-transfection group. The expression range of miR-22 in lung squamous cell carcinoma was 0.8-3.5. The expression of miR-22 in lung squamous cell carcinoma was significantly different by tumor maximum diameter, lymph node metastasis, vascular invasion and TNM stage. The expression of miR-22 was linked to survival time. There was a negative correlation between miR-22 and VEGFR3, miR-22 and MLVD. Double luciferase reporter gene assays showed that miR-22 reduced the luciferase activity of pGL3-VEGFR3-WT transfected cells. Compared with the control group, the expression of VEGF-D and D2-40 in the miR-22 transfection group was significantly decreased. However, VEGF-D and D240 in the miR-22 and VEGFR3 co-transfection group reversed the changes. We assumed that the abnormal expression of miR-22 in lung squamous cell carcinoma may be involved in the development and progression of lung squamous cell carcinoma. MiR-22 negatively regulated the target gene VEGFR3 to mediate lymphangiogenesis. The expression of miR-22 may also be linked to the prognosis of the disease.

Increased levels of VEGF-C and macrophage infiltration in lipedema patients without changes in lymphatic vascular morphology

Lipedema is a chronic adipose tissue disorder characterized by the disproportional subcutaneous deposition of fat and is commonly misdiagnosed as lymphedema or obesity. The molecular determinants of the lipedema remain largely unknown and only speculations exist regarding the lymphatic system involvement. The aim of the present study is to characterize the lymphatic vascular involvement in established lipedema. The histological and molecular characterization was conducted on anatomically-matched skin and fat biopsies as well as serum samples from eleven lipedema and ten BMI-matched healthy patients. Increased systemic levels of vascular endothelial growth factor (VEGF)-C (P = 0.02) were identified in the serum of lipedema patients. Surprisingly, despite the increased VEGF-C levels no morphological changes of the lymphatic vessels were observed. Importantly, expression analysis of lymphatic and blood vessel-related genes revealed a marked downregulation of Tie2 (P < 0.0001) and FLT4 (VEGFR-3) (P = 0.02) consistent with an increased macrophage infiltration (P = 0.009), without changes in the expression of other lymphatic markers. Interestingly, a distinct local cytokine milieu, with decreased VEGF-A (P = 0.04) and VEGF-D (P = 0.02) expression was identified. No apparent lymphatic anomaly underlies lipedema, providing evidence for the different disease nature in comparison to lymphedema. The changes in the lymphatic-related cytokine milieu might be related to a modified vascular permeability developed secondarily to lipedema progression.

RAMP1 signaling in immune cells regulates inflammation-associated lymphangiogenesis

AbstractCalcitonin gene-related peptide (CGRP) regulates inflammation via signaling through receptor activity-modifying protein (RAMP) 1. Here, we investigated the role of RAMP1 signaling in growth of lymphatic vessels during inflammation. Lymphangiogenesis in the diaphragm of RAMP1-deficient (−/−) mice or their wild-type (WT) counterparts was induced by repeated intraperitoneal injection of lipopolysaccharide (LPS). Compared with WT mice, LPS-induced lymphangiogenesis in RAMP1−/− mice was suppressed. This was accompanied by the reduced expression of vascular endothelial growth factor (VEGF)-C and VEGF-D. The number of CD4+ cells in diaphragm tissue from WT mice was greater than RAMP1−/− mice. Removing CD4+ cells attenuated lymphangiogenesis and expression of VEGF-C and VEGF-D. CD4+ cells isolated from RAMP1−/− mice exhibited reduced expression of VEGF-C and VEGF-D. The number of CD11b+ cells from RAMP1−/− mice was higher than WT mice and was associated with the upregulated expression of genes related to pro-inflammatory macrophage phenotype and downregulation of reparative macrophage phenotype-related expression. When fluorescein isothiocyanate (FITC)-dextran was injected into the peritoneal cavity, the amount of residual FITC-dextran in WT mice was lower than that in RAMP1−/− mice. The present results suggest that RAMP1 signaling in immune cells plays a critical role in inflammation-related lymphangiogenesis; therefore, it represents a novel target for controlling lymphangiogenesis.

CCL25 promotes the migration and invasion of non-small cell lung cancer cells by regulating VEGF and MMPs in a CCR9-dependent manner.

The CC chemokine receptor 9 (CCR9) and its natural secreted ligand CC motif chemokine ligand 25 (CCL25) have been implicated in cancer metastasis. However, their metastatic potential in non-small cell lung cancer (NSCLC) remains unclear. In the present study, immunohistochemistry was used to detect the expression and localization of CCR9, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1 and MMP-7 in lung cancer tissue and adjacent normal tissue. The association between the expression of CCR9 and clinical variables was also examined. Reverse transcription-quantitative PCR and western blotting were conducted to detect the expression of VEGF-C, VEGF-D, MMP-1 and MMP-7 in lung cancer cell lines (A549 and SK-MES-1). Migration and invasion assays were conducted to examine cell migration and invasion. Survival and mutation analysis were conducted using published datasets. The expressions of CCR9, VEGF, MMP-1 and MMP-7 were upregulated in cancer tissue, compared with adjacent normal tissue (all P<0.05). Patients with lower expression of CCR9 or CCL25 had better overall survival (OS) compared with those with higher CCR9 or CCL25 expression (P<0.05 and P=0.05, respectively). Furthermore, the expressions of VEGF-C, VEGF-D, MMP-1 and MMP-7 were higher in the CCL25-treated cell lines (all P<0.05), but MMP-7 protein expression was not affected by CCL25 treatment in SK-MES-1 cells (P>0.05). Following treatment with CCL25, lung cancer cells demonstrated higher migratory and invasive potential, which could be blocked by the CCR9 antibody (P<0.05). Survival analysis demonstrated that low expression levels of both CCR9 and CCL25 mRNA indicated favorable OS in patients with NSCLC. Altogether, these results suggested that CCL25 enhanced the phenotype associated with migration and invasion in NSCLC by regulating the expression of VEGF-C, VEGF-D, MMP-1 and MMP-7.

The immunohistochemical landscape of the VEGF family and its receptors in glioblastomas

BackgroundAngiogenesis is one of the hallmarks of cancer. This complex mechanism of tumor progression provides tumors cells with essential nutrients. There have been a limited number of investigations of markers of angiogenesis in Glioblastomas (GBMs), and most previous studies have focused on VEGF-A. Recent evidence suggests that there is a complex lymphatic system in central nervous system (CNS), which suggests VEGF-C and VEGF–D as interesting biomarker candidates. This study was designed to evaluate the expressions of VEGF-A, −C, −D and their co-receptors, VEGFR-1, VEGFR-2, and VEGFR-3 by immunohistochemistry (IHC) using a series of GBMs. In addition, we evaluate any putative correlations between IHC expression levels of VEGF and clinical data of patients.MethodsTumor samples of 70 GBM patients (64 isocitrate dehydrogenase-1 wildtype (wtIDH-1) and 6 mutant (mutIDH-1)) were assessed by IHC using tissue microarray platforms for VEGF subunits and their co-receptors. The medical records were reviewed for clinical and therapeutic data.ResultsAll VEGF subunits and receptors were highly expressed in GBMs: 57 out of 62 (91.9%), 53 out of 56 (94.6%) and 55 out of 63 cases (87.3%) showed VEGF-A, VEGF-C and -D imunoexpression, respectively. Interestingly, we had found both nuclear and cytoplasmic localization of VEGF-C staining in GBM tumor cells. The frequency of immunoexpression of VEGF receptors was the following: VEGFR-1, 65 out of 66 cases (98.5%); VEGFR-2, 63 out of 64 cases (98.4%); VEGFR-3, 49 out of 50 cases (90.0%). There were no significant differences in the patient overall survival (OS) related to the VEGF staining. A weak and monotonous correlation was observed between VEGF and its cognate receptors. The pattern of VEGF IHC was found to be similar when GBM mutIDH-1 subtypes were compared to wtIDH-1.ConclusionBoth VEGF-C and –D, together with their receptors, were found to be overexpressed in the majority GBMs, and the IHC expression levels did not correlate with OS or IDH status. To understand the significance of the interactions and increased expression of VEGF-C, VEGF-D, VEGFR-2, and VEGFR-3 axis in GBM requires more extensive studies. Also, functional assays using a larger series of GBM is also necessary to better address the biological meaning of nuclear VEGF-C expression in tumor cells.

Analyses of VEGFC/VEGF-D expressions, density and endothelial lymphatic proliferation in salivary gland neoplasms

ObjectiveThe aim of this study was to assess vascular endothelial growth factor C (VEGF-C) and VEGF-D expressions, tumor lymphatic density (D2–40) and endothelial lymphatic proliferation (D2–40/Ki-67 double labeling) in a series of salivary gland neoplasm cases. Materials and methodsTwenty pleomorphic adenomas (PA), 20 adenoid cystic carcinomas (ACC) and 20 mucoepidermoid carcinomas (MEC) were assessed, as well as 10 normal minor salivary gland (SG) tissues for comparison. ResultsAll cases presented positive VEGF-C expression in the peritumoral and intratumoral regions, and no differences in immunoexpression were detected between groups. However, the ACC group presented a significant difference in VEGF-C immunoexpression according to the predominant histological pattern. Most cases presented poor VEGF-D labeling in the peritumoral and intratumoral regions. Concerning peritumoral, intratumoral and total lymphatic endothelial density, the assessed groups revealed an increasing gradient, with lower values for PA, followed by MEC and ACC. ConclusionNo correlation was detected between VEGF-C and VEGF-D immunoexpression in relation to lymphatic tumor density and endothelial lymphatic proliferation. Western blotting (WB) revealed no difference between VEGF-C and VEGF-D expression among the lesions, corroborating the immunohistochemistry findings.

Intracellular ATP Delivery Causes Rapid Tissue Regeneration via Upregulation of Cytokines, Chemokines, and Stem Cells.

We have reported accelerated wound healing induced by intracellular ATP delivery in rabbits, through early massive accumulation, in situ proliferation, and M2 polarization of macrophages. Granulation tissue started to grow within first 24 h of treatment and continued the growth till the wound cavity is completely covered. However, the mechanisms underlying this macrophage response are totally unclear because no one has ever reported this before. In this study, we performed a preliminary exploration of the possible mechanisms by focusing on the roles of cytokines, growth factors, and stem cells in this process. Among the 33 adult rabbits, 18 were used for cytokine measurements and the remaining were used for histological and immunohistochemical studies. Four wounds were created on the ventral side of each ear. Two wounds on one side were treated with ATP-vesicles (10 mM ATP), and the other two were treated with controls (normal saline or Regranex). Dressing changes were made daily and the rabbits were sacrificed at 5 h, 12 h, and 1, 2, 3, 4, 6, 9, 15, and 26 days after wounding. Tissue samples were analyzed for cytokines and growth factors using real-time PCR and immunohistochemical staining. The control wounds showed an immediate increase in proinflammatory cytokines after wound creation but no further increase after this initial spike. The growth factor levels in the control wounds remained unchanged throughout the study. Conversely, the wounds treated with ATP-vesicles showed significantly higher expression of MCP-1 and stem cell markers (CD44, CD106, CD146, and CD34) at day 1, significantly higher IL-1β and TNF-α expression from day 1–4, and significantly higher VEGF-A, VEGF-D, and VEGFR-2 expression from day 4–6 when compared to the controls. The significant upregulation of these factors corresponded to the very early and rapid macrophage accumulation, in situ proliferation, and M2 polarization, resulting in unprecedented rapid granulation tissue generation due to direct macrophage collagen production and neovascularization.

Expression Profile of VEGF-C, VEGF-D, and VEGFR-3 in Different Grades of Endometrial Cancer.

Vascular endothelial growth factor (VEGF)-C, -D, and VEGF receptor-3 are proteins characterized as crucial for tumor lymphangiogenesis. It is accompanied by angiogenesis during wound healing, but also in the neoplastic process. The research studies have shown that the lymphatic system plays a key role in the progression of carcinogenesis. The aim of this study was to evaluate changes in the expression of VEGF-C, VEGF-D and VEGFR-3 in different grades of endometrial cancer (G1-G3). The study included 45 patients diagnosed with endometrial cancer (G1=17; G2=15; G3=13) and 15 patients without neoplastic changes. The expression of VEGF-C, VEGF-D, and VEGFR-3 was assessed using microarray technique and immunohistochemistry. Statistical analysis was performed using the one-way ANOVA and Tukey's post-hoc test. Statistically significant changes in the expression at the transcriptome level were found only in the case of VEGF-C (G1 vs. C, fold change - FC = -1.15; G2 vs. C, FC = -2.33; G3 vs. C, FC = - 1.68). However, VEGF-D and VEGFR-3 were expressed at the protein level. Analysis of VEGF-D expression showed that the optical density of the reaction product in G1 reached 101.7, while the values in G2 and G3 were 142.7 and 184.4, respectively. For VEGF-R3, the optical density of the reaction product reached the following levels: 72 in control, 118.77 in G1, 145.8 in G2, and 170.9 in G3. An increase in VEGF-D and VEGFR-3 levels may indicate that VEGF-D-dependent processes are intensified along with the dedifferentiation of tumor cells. The lack of VEGF-C expression in endometrial cancer samples may suggest that this tumor is characterized by a different mechanism of metastasis than EMT. Our study emphasizes that when analyzing the metastatic potential of cancer, the expression of more than one factor should be taken into account.

Circulating Tumor Biomarkers in Meningiomas Reveal a Signature of Equilibrium Between Tumor Growth and Immune Modulation.

Meningiomas are primary central nervous system (CNS) tumors that originate from the arachnoid cells of the meninges. Recurrence occurs in higher grade meningiomas and a small subset of Grade I meningiomas with benign histology. Currently, there are no established circulating tumor markers which can be used for diagnostic and prognostic purposes in a non-invasive way for meningiomas. Here, we aimed to identify potential biomarkers of meningioma in patient sera. For this purpose, we collected preoperative (n = 30) serum samples from the meningioma patients classified as Grade I (n = 23), Grade II (n = 4), or Grade III (n = 3). We used a high-throughput, multiplex immunoassay cancer panel comprising of 92 cancer-related protein biomarkers to explore the serum protein profiles of meningioma patients. We detected 14 differentially expressed proteins in the sera of the Grade I meningioma patients in comparison to the age- and gender-matched control subjects (n = 12). Compared to the control group, Grade I meningioma patients showed increased serum levels of amphiregulin (AREG), CCL24, CD69, prolactin, EGF, HB-EGF, caspase-3, and decreased levels of VEGFD, TGF-α, E-Selectin, BAFF, IL-12, CCL9, and GH. For validation studies, we utilized an independent set of meningioma tumor tissue samples (Grade I, n = 20; Grade II, n = 10; Grade III, n = 6), and found that the expressions of amphiregulin and Caspase3 are significantly increased in all grades of meningiomas either at the transcriptional or protein level, respectively. In contrast, the gene expression of VEGF-D was significantly lower in Grade I meningioma tissue samples. Taken together, our study identifies a meningioma-specific protein signature in blood circulation of meningioma patients and highlights the importance of equilibrium between tumor-promoting factors and anti-tumor immunity.

Mir526b and Mir655 Promote Tumour Associated Angiogenesis and Lymphangiogenesis in Breast Cancer.

MicroRNAs (miRNAs) are small endogenously produced RNAs, which regulate growth and development, and oncogenic miRNA regulate tumor growth and metastasis. Tumour-associated angiogenesis and lymphangiogenesis are processes involving the release of growth factors from tumour cells into the microenvioronemnt to communicate with endothelial cells to induce vascular propagation. Here, we examined the roles of cyclo-oxygenase (COX)-2 induced miR526b and miR655 in tumour-associated angiogenesis and lymphangiogenesis. Ectopic overexpression of miR526b and miR655 in poorly metastatic estrogen receptor (ER) positive MCF7 breast cancer cells resulted in upregulation of angiogenesis and lymphangiogenesis markers vascular endothelial growth factor A (VEGFA); VEGFC; VEGFD; COX-2; lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1); and receptors VEGFR1, VEGFR2, and EP4. Further, miRNA-high cell free conditioned media promoted migration and tube formation by human umbilical vein endothelial cells (HUVECs), and upregulated VEGFR1, VEGFR2, and EP4 expression, showing paracrine stimulation of miRNA in the tumor microenvironment. The miRNA-induced migration and tube formation phenotypes were abrogated with EP4 antagonist or PI3K/Akt inhibitor treatments, confirming the involvement of the EP4 and PI3K/Akt pathway. Tumour supressor gene PTEN was found to be downregulated in miRNA high cells, confirming that it is a target of both miRNAs. PTEN inhibits hypoxia-inducible factor-1 (HIF1α) and the PI3K/Akt pathway, and loss of regulation of these pathways through PTEN results in upregulation of VEGF expression. Moreover, in breast tumors, angiogenesis marker VEGFA and lymphangiogenesis marker VEGFD expression was found to be significantly higher compared with non-adjacent control, and expression of miR526b and miR655 was positively correlated with VEGFA, VEGFC, VEGFD, CD31, and LYVE1 expression in breast tumour samples. These findings further strengthen the role of miRNAs as breast cancer biomarkers and EP4 as a potential therapeutic target to abrogate miRNA-induced angiogenesis and lymphangiogenesis in breast cancer.

Expression profiles of VEGF-A, VEGF-D and VEGFR1 are higher in distant metastases than in matched primary high grade epithelial ovarian cancer

BackgroundIn many malignancies including ovarian cancer, different angiogenic factors have been related to poor prognosis. However, data on their relations to each other or importance as a prognostic factor in ovarian cancer is missing. Therefore, we investigated the expressions of VEGF-A, VEGF-C, and VEGF-D, and the receptors VEGFR1, VEGFR2, and VEGFR3 in patients with malignant epithelial ovarian neoplasms. We further compared expression levels between primary tumors and related distant omental metastases.MethodsThis study included 86 patients with malignant ovarian epithelial tumors and 16 related distant metastases. Angiogenic factor expression was evaluated using immunohistochemistry (n = 102) and qRT-PCR (n = 29).ResultsCompared to primary high grade serous ovarian tumors, the related omental metastases showed higher expressions of VEGF-A (p = 0.022), VEGF-D (p = 0.010), and VEGFR1 (p = 0.046). In univariate survival analysis, low epithelial expression of VEGF-A in primary tumors was associated with poor prognosis (p = 0.024), and short progression-free survival was associated with high VEGF-C (p = 0.034) and low VEGFR3 (p = 0.002). The relative expressions of VEGF-D, VEGFR1, VEGFR2, and VEGFR3 mRNA determined by qRT-PCR analyses were significantly correlated with the immunohistochemically detected levels of these proteins in primary high grade serous ovarian cancer and metastases (p = 0.004, p = 0.009, p = 0.015, and p = 0.018, respectively).ConclusionsThe expressions of VEGF receptors and their ligands significantly differed between malignant ovarian tumors and paired distant metastases. VEGF-A, VEGF-D, and VEGFR1 protein expressions seem to be higher in distant metastases than in the primary high grade serous ovarian cancer lesions.

CCL27/CCL28-CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells.

Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine-chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis. SIGNIFICANCE: The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.