Vascular Endothelial Growth factor-C Expression Research Articles

ADSCs enhance VEGFR3-mediated lymphangiogenesis via METTL3-mediated VEGF-C m6A modification to improve wound healing of diabetic foot ulcers

BackgroundAdipose-derived mesenchymal stem cells (ADSCs) are an important focus in regenerative medicine. However, the biological function of ADSCs in the wound repair of diabetic foot ulcers (DFUs) remains unclear. This study aimed to determine the underlying mechanisms of ADSCs involved in the wound healing of DFUs.MethodsThe cell surface markers cluster of differentiation 34 (CD34), stromal cell antigen 1 (Stro-1), cluster of differentiation 90 (CD90) and cluster of differentiation 105 (CD105) on ADSCs were identified by flow cytometry. Oil Red O staining and Alizarin Red S staining were performed to identify the multipotential differentiation of ADSCs into adipocytes and bone. The levels of Methyltransferase-like 3 (METTL3), vascular endothelial growth factor C (VEGF-C) and insulin-like growth factor 2 binding protein 2 (IGF2BP2) were assessed by RT-qPCR. CCK-8, Transwell and tubule formation assays were conducted to assess lymphatic endothelial cell (LEC) viability, migration and tubule formation ability, respectively. RIP and RNA pulldown assays were conducted to assess the interaction between IGF2BP2 and VEGF-C. The levels of VEGF-C, VEGFR3, LYVE-1 and IGF2BP2 proteins were assessed by Western blotting. The levels of VEGF-C in LECs were measured by ELISA.ResultsOur findings illustrated that ADSCs accelerate LEC proliferation, migration and lymphangiogenesis via the METTL3 pathway and regulate VEGF-C expression via the METTL3/IGF2BP2-m6A pathway VEGF-C-mediated lymphangiogenesis via the METTL3/IGF2BP2-m6A pathway in DFU mice.ConclusionADSCs enhance VEGFR3-mediated lymphangiogenesis via METTL3-mediated VEGF-C m6A modification to improve wound healing in DFUs, indicating that ADSCs may be regarded as a promising therapeutic strategy to promote wound healing in DFUs.

Coexpression of TLR9 and VEGF-C is associated with lymphatic metastasis in prostate cancer.

Prostate cancer (PCa) is one of the most frequent cancers in men, and its biomolecular targets have been extensively studied. This study aimed to analyze the expression of toll-like receptor 9 (TLR9) and vascular endothelial growth factor C (VEGF-C) and the clinical value of the coexpression of TLR9 and VEGF-C in PCa. We retrospectively evaluated 55 patients with clinically localized, intermediate-risk, or high-risk PCa who underwent laparoscopic radical prostatectomy (LRP) and extended pelvic lymph node dissection (ePLND) without neoadjuvant hormonal therapy at a single institution from June 2013 to December 2016. In all 55 patients, the median number of lymph nodes (LNs) resected was 23 (range: 18–31), and a total of 1269 LNs were removed, of which 78 LNs were positive. Seventeen patients had positive LNs, with a positive rate of 30.9%. In addition, the immunohistochemical results in the above patients revealed that high TLR9 expression was correlated with higher Gleason score (GS) (P = 0.049), increased LN metastasis (P = 0.004), and more perineural invasion (PNI) (P = 0.033). Moreover, VEGF-C expression was associated with GS (P = 0.040), pathological stage (pT stage) (P = 0.022), LN metastasis (P = 0.003), and PNI (P = 0.001). Furthermore, a significant positive correlation between TLR9 and VEGF-C was found (P < 0.001), and the TLR9/VEGF-C phenotype was associated with LN metastasis (P = 0.047). Collectively, we propose that TLR9 stimulation may promote LN metastasis in PCa cells through the upregulation of VEGF-C expression, thereby affecting the prognosis of PCa patients. Therefore, these markers may serve as valuable targets for the treatment of PCa.

MicroRNA-203a-3p affects the biological characteristics of nasopharyngeal carcinoma by targeting vascular endothelial growth factor-C.

Nasopharyngeal carcinoma (NPC) has a high incidence in Southeast Asia and China. This study aims to investigate the effect of miR-203a-3p and vascular endothelial growth factor-C (VEGF-C) on nasopharyngeal carcinoma. In this study, we investigate the transfection of miR-203a-3p mimics and the ability of miR-203a-3p to inhibit in nasopharyngeal carcinoma cell line 5-8F and C666-1, the expression levels of protein AKT, p-AKT in VEGF-C and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signal pathway by plate clone formation experiment, flow cytometer PI staining method, transwell cell experiment, vasculogenic mimicry experiment, and Western blot. The results showed that miR-203a-3p in the nasopharyngeal carcinoma cell line significantly decreased, while VEGF-C in nasopharyngeal carcinoma tissues and cell lines significantly increased. Furthermore, miR-203a-3p inhibited the proliferation of nasopharyngeal carcinoma cells and blocked the cell cycle of nasopharyngeal carcinoma in the G0/G1 phase, reduced the vasculogenic phenomena and epithelial-mesenchymal transition of nasopharyngeal carcinoma cells, and effectively inhibited the growth of nasopharyngeal carcinoma in vivo. The low expression of VEGF-C can inhibit the proliferation, vasculogenic mimicry, and epithelial-mesenchymal transition of nasopharyngeal carcinoma cells. We found that miR-203a-3p was expressed to a low degree in nasopharyngeal carcinoma. Furthermore, miR-203a-3p regulated the PI3K/AKT signal pathway by downregulating the expression of VEGF-C, thereby inhibiting the proliferation, migration, invasion, vasculogenic mimicry, epithelial-mesenchymal transition, and other malignant biological characteristics of nasopharyngeal carcinoma cells.

VEGF-C/VEGFR-3 axis protects against pressure-overload induced cardiac dysfunction through regulation of lymphangiogenesis.

Prolonged pressure overload triggers cardiac hypertrophy and frequently leads to heart failure (HF). Vascular endothelial growth factor‐C (VEGF‐C) and its receptor VEGFR‐3 are components of the central pathway for lymphatic vessel growth (also known as lymphangiogenesis), which has crucial functions in the maintenance of tissue fluid balance and myocardial function after ischemic injury. However, the roles of this pathway in the development of cardiac hypertrophy and dysfunction during pressure overload remain largely unknown. Eight‐ to 10‐week‐old male wild‐type (WT) mice, VEGFR‐3 knockdown (VEGFR‐3f/−) mice, and their WT littermates (VEGFR‐3f/f) were subjected to pressure overload induced by transverse aortic constriction (TAC) for 1–6 weeks. We found that cardiac lymphangiogenesis and the protein expression of VEGF‐C and VEGFR‐3 were upregulated in the early stage of cardiac hypertrophy but were markedly reduced in failing hearts. Moreover, TAC for 6 weeks significantly reduced cardiac lymphangiogenesis by inhibiting activation of VEGFR‐3‐mediated signals (AKT/ERK1/2, calcineurin A/NFATc1/FOXc2, and CX43), leading to increased cardiac edema, hypertrophy, fibrosis, apoptosis, inflammation, and dysfunction. These effects were further aggravated in VEGFR‐3f/− mice and were dose‐dependently attenuated by delivery of recombinant VEGF‐C156S in WT mice. VEGF‐C156s administration also reversed pre‐established cardiac dysfunction induced by sustained pressure overload. Thus, these results demonstrate, for the first time, that activation of the VEGF‐C‐VEGFR‐3 axis exerts a protective effect during the transition from cardiac hypertrophy to HF and highlight selective stimulation of cardiac lymphangiogenesis as a potential new therapeutic approach for hypertrophic heart diseases.

Qingjie Fuzheng Granule suppresses lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway

ScopeTo investigate the effect of Qingjie Fuzheng Granule (QFG) on lymphangiogenesis and lymphatic metastasis in colorectal cancer. MethodsThe effects of QFG on the expression and secretion of vascular endothelial growth factor-C (VEGF-C) in HCT-116 cells were investigated both in vitro and in vivo. HCT-116 cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. The VEGF-C expression level was determined using RT-qPCR and western blotting, and the VEGF-C concentration in supernatant was measured by ELISA. Tumor xenograft models of HCT-116 cells were generated using BALB/c nude mice, and the mice were randomly divided into a control group (gavaged with normal saline) and QFG group (gavaged with 2 g/kg QFG). The effect of QFG on tumor growth was evaluated by comparing the volume and weight of tumors between two groups. Immunohistochemistry (IHC) and RT-qPCR were performed to detect the expression levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1). ELISA was performed to measure the concentration of serum VEGF-C. TMT proteomics technology and Reactome pathway analysis were used to explore the mechanism of QFG inhibiting lymphangiogenesis in tumor. The VEGF-C (5 ng/mL)-stimulated human lymphatic endothelial cell (HLEC) model was conducted to evaluate the effect of QFG on lymphangiogenesis in vitro. The model cells were treated with different concentrations (0.2, 0.5, and 1.0 mg/mL) of QFG. Cell viability was then determined using an MTT assay. The cell migration, invasion, and tube-formation ability were analyzed using transwell migration, matrigel invasion and tube formation assays, respectively. The underlying mechanism was uncovered, the levels of VEGFR-3, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9), p-PI3K/PI3K, p-AKT/AKT and p-mTOR/ mTOR were detected using western blotting. ResultsQFG significantly reduced VEGF-C expression and secretion in HCT-116 cells. QFG evidently suppressed in vivo tumor growth and the expression of VEGF-C, VEGFR-3, and LYVE-1. The serum VEGF-C level was also reduced by QFG. Moreover, TMT proteomics technology and Reactome pathway analysis identified 95 differentially expressed protein and multiple enriched pathway about matrix metalloproteinase and extracellular matrix, which is direct associate with lymphangiogenesis. In vitro experiment, QFG inhibited the viability, migration, invasion and tube formation of HLECs. Additionally, QFG reduced the VEGFR-3, MMP-2, MMP-9 expression levels, and the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/ mTOR ratios. ConclusionQFG can exert its effect on both tumor cells and HLECs, exhibiting ani- lymphangiogenesis in colorectal cancer via the VEGF-C/VEGFR-3 dependent PI3K/AKT pathway pathway.

Co-expression of VEGF-C and survivin predicts poor prognosis in esophageal squamous cell carcinoma.

BackgroundLymphatic metastasis is one of the main factors affecting prognosis in esophageal squamous cell carcinoma (ESCC). Vascular endothelial growth factor-C (VEGF-C) is an important factor that promotes lymphangiogenesis. Survivin also plays a significant role in lymphatic invasion. However, the role and mechanism of their co-expression are still unclear in ESCC. The purpose of this study was to investigate whether the co-expression of VEGF-C and survivin could be a potential marker to predict patient prognosis and survival in ESCC.MethodsThe levels of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR-3), survivin, and Ki-67 were determined by immunohistochemistry (IHC) in 97 ESCC patient tumors. The correlations of co-expression of VEGF-C and survivin with pathological features and survival results were also assessed.ResultsHigh VEGF-C expression was observed in 64.9% of the patients and significantly correlated with T stage (P=0.024), node status (P=0.038), and lymph node metastasis (P=0.015). High survivin expression was significantly associated with T stage (P=0.013), N stage (P=0.016), lymph node metastasis (P=0.005), and differentiation (P=0.044) in 67.0% of the patients. Co-expression of VEGF-C and survivin (V+S+) was significantly associated with T stage (P<0.001), N stage (P=0.015), lymph node metastasis (P=0.003), differentiation (P=0.0045), and Ki-67 levels (P=0.024). High expression of VEGF-C or survivin was associated significantly with worse disease-free survival (DFS) and overall survival (OS) (P<0.05). Moreover, the V+S+ group had a worse DFS (P<0.001) and OS (P=0.001) than any other group (i.e., V–S–, V+S–, V–S+). Furthermore, multivariate DFS analyses (95% CI: 1.147–2.220, P=0.006) and multivariate OS analyses (95% CI: 1.080–2.193, P=0.017) revealed that co-expression of VEGF-C and survivin was an independent prognostic factor in ESCC patients.ConclusionsCo-expression of VEGF-C and survivin was predictive of poor prognosis in ESCC. Combined detection of VEGF-C and survivin could represent a feasible and effective marker to predict the prognosis and survival of ESCC patients.

C-Myc promotes lymphatic metastasis of pancreatic neuroendocrine tumor through VEGFC upregulation.

Pancreatic neuroendocrine tumor (pNET) is a pancreatic neoplasm with neuroendocrine differentiation. pNET in early stage can be treated with surgical resection with long‐term survival, whereas the prognosis of pNET with locoregional or distant metastasis is relatively poor. Lymphangiogenesis is essential for tumor metastasis via the lymphatic system and may overhead distant metastasis. c‐Myc overexpression is involved in tumorigenesis. The role of c‐Myc in lymphangiogenesis is unclear. In this study, we evaluated the mechanism and effect of c‐Myc on lymphangiogenesis of pNET via interaction of lymphatic endothelial cells (LECs) and pNET cells. Lymph node metastasis was evaluated in pNET xenograft mice. Potential target agents to inhibit lymph node metastasis were evaluated in an animal model. We found that vascular endothelial growth factor C (VEGFC) expression and secretion was increased in pNET cell lines with c‐Myc overexpression. c‐Myc transcriptionally upregulates VEGFC expression and the secretion of pNET cells by directly binding to the E‐box of the VEGFC promoter and enhances VEGF receptor 3 phosphorylation and the tube formation of LECs. c‐Myc overexpression is associated with lymph node metastasis in pNET xenograft mice. Combinational treatment with an mTOR inhibitor and c‐Myc inhibitor or VEGFC‐neutralizing chimera protein reduced lymph node metastasis in the mice with c‐Myc overexpression. The mTOR inhibitor acts on lymphangiogenesis by reducing VEGFC expression in pNET cells and inhibiting the tube formation of LECs. In conclusion, mTOR and c‐Myc are important for lymphangiogenesis of pNET and are potential therapeutic targets for prevention and treatment of lymph node metastasis in pNET.

Amplified LncRNA PVT1 promotes lung cancer proliferation and metastasis by facilitating VEGFC expression.

Although the abundance of long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) in lung cancer has been well researched, the underlying mechanisms behind its effects were unknown. Here we investigated the molecular events regulating PVT1 in lung cancer. The pro-proliferative property of PVT1 was examined using a xenograft tumor model. Transwell chambers were used to analyze the impact of PVT1 expression on cell invasiveness and migration. In vivo metastasis was examined by tail-vein-injection in mice. Direct binding of miR-128 to PVT1 was investigated using a probe pulldown assay. The relative expression levels of miR-128 and PVT1 were quantified by real-time polymerase chain reaction and Western blotting. We show here that when PVT1 is amplified, there is a poor survival prognosis for patients with lung cancer. Elevated levels of PVT1 promoted lung cancer cell proliferation and metastasis, both in vitro and in vivo. Mechanistically, we found that PVT1 competes endogenously with miR-128 in the regulation of vascular endothelial growth factor C (VEGFC) expression, which is significantly associated with an unfavorable prognosis in lung cancer. We identified that copy number amplification significantly contributes to the high level of PVT1 transcripts in lung cancer, which promotes cell proliferation and metastatic behavior via modulating VEGFC expression by endogenous competition with miR-128.

Prognostic value of lymphatic vessel density in oral squamous cell carcinoma

AimsLymphatic 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 methods128 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 findingsTumor 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). SignificanceThis 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.

Expression of vascular endothelial growth factor C in renal cell carcinoma and its correlation with pathological parameters and prognosis.

BackgroundRenal cell carcinoma (RCC) is a common malignant tumor of the urinary system. This study aimed to analyze the expression of vascular endothelial growth factor C (VEGF-C) in RCC and its relationship with pathological parameters and prognosis.MethodsThe clinical data of 68 patients who underwent surgical treatment and confirmed to be RCC by pathology from February 2012 to January 2014 were collected. The specimens of carcinoma tissues of the 68 patients were collected, among which 20 patients were collected from normal tissue specimens adjacent to the cancer more than 5 cm away from the tumor as controls. The VEGF-C expression level was detected by immunohistochemistry, and the relationship between VEGF-C expression and clinical pathological parameters and prognosis of RCC was analyzed.ResultsThe positive expression rate of VEGF-C in cancer tissues of RCC patients was significantly higher than the adjacent tissues (85.29% vs. 15.00%) (P<0.05). The positive expression rate of VEGF-C in cancer tissues of RCC patients with low-to- moderate differentiation, stage III–IV, and lymph node metastasis was higher than that of RCC patients with high differentiation, stage I–II, and no lymph node metastasis (P<0.05). The survival rates of RCC patients at 1-, 3- and 5-year follow-up were 82.35% (56/68), 54.41% (37/68), and 32.35% (22/68), and the survival time of patients with positive VEGF-C expression was shorter than patients with negative expression (P<0.05). There was no significant difference in the 5-year survival rate among RCC patients according to sex or presence of muscular infiltration (P>0.05). Meanwhile, the 5-year survival rate was higher in patients with tumor diameter <5 cm, high differentiation, stage I–II, no lymph node metastasis, VEGF-C–negative expression, and aged <55 years old (P<0.05). The Cox regression model analysis showed that differentiation degree, clinical stage, lymph node metastasis, and VEGF-C expression were all independent risk factors affecting the prognosis of RCC patients (P<0.05).ConclusionsVEGF-C is highly expressed in cancer tissues of RCC patients, and is related to clinical stage, pathological differentiation, and lymph node metastasis, which maybe an effective factor of prognosis prediction.

Continuous theta burst stimulation dilates meningeal lymphatic vessels by up-regulating VEGF-C in meninges

BackgroundLymphatic vessels (LVs) of meninges and lymphatic drainage in the brain have been investigated previously. Here, we examined the role of continuous theta burst stimulation (CTBS) in the modulation of meningeal LVs. MethodsTo explore the effects of CTBS on meningeal LVs, the diameters of LVs were measured between a real CTBS group and sham CTBS group of wild-type male mice. Vascular endothelial growth factor-C (VEGF-C) expression was subsequently calculated in both groups to account for lymphatic changes after CTBS. Sunitinib was administered by 3-day oral gavage to inhibit the VEGF receptor (VEGFR), and the effects of CTBS were further examined in the following groups: vehicle with real CTBS, vehicle with sham CTBS, sunitinib treatment with real CTBS, and sunitinib treatment with sham CTBS. ResultsThe lymphatic vessels were augmented, and the level of VEGF-C in meninges increased after CTBS. CTBS dilated meningeal lymphatic vessels were impaired after the VEGF-C/VEGFR3 pathway was blocked. ConclusionsCTBS can dilate meningeal lymphatic vessels by up-regulating VEGF-C in meninges.

MiR-27b promotes angiogenesis and skin repair in scalded rats through regulating VEGF-C expression.

In this study, the effects of miR-27b on angiogenesis in skin repair procedure in rats with deep II degree scald were explored. The rat model of deep II scald was established. miR-27b mimics and inhibitor were injected daily at the wound site for 3weeks. The healing of scald was observed at 0, 3, 7, 14, and 21days after the model was established, and the pathological changes of skin were observed by HE and Masson's trichrome stains. Skin tissues were taken 14days after the operation; CD31 and Ki-67 immunohistochemistry was exerted to evaluate neovascularization and proliferation. Human microvascular endothelial cells (HMEC-1) cells were cultured in vitro. miR-27b mimics or inhibitor was transfected to construct over-expression or inhibition cell lines. MTT assay, scratch test, and angiogenesis test were used to evaluate cell proliferation, migration, and vascular regeneration. Finally, RT-PCR and Western blot were exerted to determine the expression of vascular endothelial growth factor C (VEGF-C), epidermal growth factor (EGF) mRNAs, and protein, respectively. Control, inhibitor, mi-NC, VEGF-C, inhibitor + si-NC, and inhibitor + VEGF-C siRNA groups were used to further analyze the mechanism of miR-27b on VEGF-C; the above experiments were repeated. In contrast to model group, miR-27b inhibitor could significantly promote the healing of scalded skin, alleviate the pathological status of scalded, and promote the angiogenesis and proliferation (p < 0.05). In vitro, miR-27b inhibitor evidently promoted cell proliferation, migration, and angiogenesis and increased the expression of VEGF-C, EGF genes, and protein, while miR-27b mimics significantly reversed the above trends. Further studies shown that downregulation of miR-27b expression can promote the proliferation, migration, and angiogenesis of HMEC-1 cells by promoting the expression of VEGF-C. miR-27b promotes angiogenesis and skin repair in scalded rats through regulating VEGF-C expression.

BACH family members regulate angiogenesis and lymphangiogenesis by modulating VEGFC expression.

Angiogenesis and lymphangiogenesis are key processes during embryogenesis as well as under physiological and pathological conditions. Vascular endothelial growth factor C (VEGFC), the ligand for both VEGFR2 and VEGFR3, is a central lymphangiogenic regulator that also drives angiogenesis. Here, we report that members of the highly conserved BACH (BTB and CNC homology) family of transcription factors regulate VEGFC expression, through direct binding to its promoter. Accordingly, down-regulation of bach2a hinders blood vessel formation and impairs lymphatic sprouting in a Vegfc-dependent manner during zebrafish embryonic development. In contrast, BACH1 overexpression enhances intratumoral blood vessel density and peritumoral lymphatic vessel diameter in ovarian and lung mouse tumor models. The effects on the vascular compartment correlate spatially and temporally with BACH1 transcriptional regulation of VEGFC expression. Altogether, our results uncover a novel role for the BACH/VEGFC signaling axis in lymphatic formation during embryogenesis and cancer, providing a novel potential target for therapeutic interventions.

MicroRNA-135a Regulates VEGFC Expression and Promotes Luteinized Granulosa Cell Apoptosis in Polycystic Ovary Syndrome.

Androgen is known to regulate microRNA-135a (miR-135a) and can be regulated by androgen, suggesting that it may contribute to polycystic ovary syndrome (PCOS) with hyperandrogenism. However, its roles and mechanisms of action in PCOS are unknown. In this study, the role and molecular mechanisms underlying miR-135a in granulosa cells (GCs) in PCOS were evaluated. miR-135a expression was upregulated in patients with PCOS and in GCs isolated from patients compared with that in the respective controls (P < 0.01), as determined by RT-qPCR. The overexpression of miR-135a inhibited GC proliferation and induced GC apoptosis, as observed by CCK-8 assay and apoptosis assay. Furthermore, miR-135a overexpression increased the expression of double-strand break maker, γH2AX, as confirmed by western blotting. Our results further suggest that these effects were mediated via downregulation of vascular endothelial growth factor C (VEGFC), which was identified as a direct target of miR-135a. Moreover, levels of VEGFC and miR-135a expression showed a negative correlation. These findings indicate that miR-135a promotes apoptosis and the DNA damage response in GCs in PCOS, likely via VEGFC signaling. This study provides novel insights into GC dysregulation in PCOS and suggests that miR-135a is a promising therapeutic target for PCOS treatment.

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.

The Association of Parametrial Invasion with the Expression of Vascular Endothelial Growth Factor-C and Other Factors in Squamous Cell Cervical Carcinoma Stage IB and IIA

BACKGROUND: The presence of parametrial invasion in early stage squamous cell cervical cancer (SCCC) indeed indicates worse prognosis and need more adjuvant treatment. This study aimed to investigate the association between parametrial invasion and clinicohystopathology variables.METHODS: This retrospective study used specimens of squamous cell carcinoma stage IB-IIA. The inclusion criteria were cervical cancer stage IB-IIA and post radical hysterectomy with lymph node dissection and exclusion criteria was damaged and or insufficient histological preparations. The clinicohystopathology variables included age, tumour size and stage, differentiation and inflammation grade, lymphatic vascular and parametrial invasion, and lymph node metastasis. Histopathology staining, immunohistochemistry examination, and vascular endothelial growth factor-C (VEGF-C) expression were evaluated according to the standard procedure. The independent-T, Chi square, and Fisher’s exact test were applied to evaluate the association. The significance was set at p&lt;0.05.RESULTS: Seventy-five cases were eligible. Parametrial invasion was found in 15 cases (20%). Three variables demonstrated a significant association with parametrial invasion, tumor size &gt;4 cm (OR 4.32, 95% CI 1.29-14.38, p=0.01), lymphatic node metastasis (OR 3.90, 95% CI 1.17-13.03, p=0.02), and VEGF-C (OR 0.75, 95% CI 0.65- 0.87, p=0.03).CONCLUSION: Tumor size of &gt;4 cm and lymph node metastasis (LNM) had a higher risk of parametrial invasion in SCCC stage IB-IIA.KEYWORDS: cervical carcinoma, parametrial invasion, vascular invasion, peritumoral stroma, VEGF-C expression

Hemostasis stimulates lymphangiogenesis through release and activation of VEGFC

AbstractHemostasis associated with tissue injury is followed by wound healing, a complex process by which damaged cellular material is removed and tissue repaired. Angiogenic responses are a central aspect of wound healing, including the growth of new lymphatic vessels by which immune cells, protein, and fluid are transported out of the wound area. The concept that hemostatic responses might be linked to wound healing responses is an old one, but demonstrating such a link in vivo and defining specific molecular mechanisms by which the 2 processes are connected has been difficult. In the present study, we demonstrate that the lymphangiogenic factors vascular endothelial growth factor C (VEGFC) and VEGFD are cleaved by thrombin and plasmin, serine proteases generated during hemostasis and wound healing. Using a new tail-wounding assay to test the relationship between clot formation and lymphangiogenesis in mice, we find that platelets accelerate lymphatic growth after injury in vivo. Genetic studies reveal that platelet enhancement of lymphatic growth after wounding is dependent on the release of VEGFC, but not VEGFD, a finding consistent with high expression of VEGFC in both platelets and avian thrombocytes. Analysis of lymphangiogenesis after full-thickness skin excision, a wound model that is not associated with significant clot formation, also revealed an essential role for VEGFC, but not VEGFD. These studies define a concrete molecular and cellular link between hemostasis and lymphangiogenesis during wound healing and reveal that VEGFC, the dominant lymphangiogenic factor during embryonic development, continues to play a dominant role in lymphatic growth in mature animals.

Receptor-Interacting Protein Kinase 1 Promotes Cholangiocarcinoma Proliferation And Lymphangiogenesis Through The Activation Protein 1 Pathway.

PurposeReceptor-interacting protein kinase 1 (RIPK1) is an important upstream regulator of multiple cell signaling pathways including inflammatory signals. RIPK1 is reported to be closely associated with the prognostic implications of cancer, especially epithelial tumors. But its role in proliferation and lymphangiogenesis in cholangiocarcinoma (CCA) remains unclear and requires further investigation.Patients and methodsExpression of RIPK1 in human CCA tissues and CCA cell lines (QBC939, HUH28 and CCPL-1) was measured using qPCR, immunoblotting and immunohistochemistry. Silencing of RIPK1 was achieved by transduction of CCA cells via lentiviral plasmids (LV3-H1/GFP&Puro) encapsulating RIPK1 shRNA (LV-shRIPK1) or negative control shRNA (LV-shNC), and puromycin was used to select stable colonies. Proliferation and lymphangiogenesis were assessed in vitro by CCK-8 and matrigel-based tube formation assays, respectively. Activity of the activation protein-1 (AP-1) was evaluated by double-luciferase reporter gene assay. Protein expression of JNK, P38MAPK, ERK1/2, AP-1, P-AP-1, E-cadherin, N-cadherin and vascular endothelial growth factor-C (VEGF-C) was measured by immunoblotting or ELISA. An orthotopic CCA model in null mice was generated by transplanting QBC939 LV-shRIPK1, LV-shNC and control cells to further evaluate the role of RIPK1 on lymphangiogenesis in vivo. Immunohistochemistry was utilized to evaluate the expression of RIPK1 and VEGF-C, and tumor lymphatic vessels in the CCA model mice.ResultsUpregulated expression of RIPK1 in CCA tissues was closely related to tumor size, lymph node metastasis and poor prognosis. RIPK1 promoted proliferation and lymphangiogenesis in CCA cells, and regulated the activation of JNK and P38MAPK-mediated AP-1/VEGF-C pathway. Finally, in vivo animal experiments in the orthotopic CCA mouse model further confirmed the function of RIPK1 in lymphangiogenesis.ConclusionThis is the first report demonstrating the role of RIPK1 in proliferation and lymphangiogenesis through the MAPK (JNK and P38MAPK)- AP-1 pathway in CCA.

Long Noncoding RNA ASLNC07322 Functions in VEGF-C Expression Regulated by Smad4 during Colon Cancer Metastasis

Deletion and mutation of the Smad4 gene are favorable events for the progression of colon cancer, which is related to the negative regulation of vascular endothelial growth factor C (VEGF-C). However, the regulatory mechanism between Smad4 and VEGF-C remains unclear. We reported first that Smad4 can increase the transcription of miR-128-3p, a microRNA targeting VEGF-C mRNA, resulting in a negative correlation between Smad4 and VEGF-C. Moreover, we found that Smad4 combined with Smad3 can positively regulate VEGF-C during colon cancer metastasis through binding to VEGF-C gene promoter. Further, results revealed a mechanism that long noncoding RNA (lncRNA) ASLNC07322 increased specifically in metastatic colon cancer and decreased miR-128-3p as a sponge, leading to a subsequent elevation of VEGF-C. In a word, there are two pathways in the progression of colon cancer, including Smad4/miR-128-3p/VEGF-C and Smad4/VEGF-C pathways in non-metastatic and metastatic colon cancer, respectively. ASLNC07322 crucially controlled this negative and positive regulatory transformation between them. Additionally, ASLNC07322 knockdown combined with Smad4 overexpression could efficiently inhibit lymphatic endothelial cells (LECs) proliferation and tube formation in vitro, as well as tumor growth and lymphangiogenesis in vivo. These data explained the underlying mechanism of Smad4 contribution on VEGF-C expression during metastasis where ASLNC07322 functions vitally as a switch in colon cancer.

Receptor‑interacting serine/threonine‑protein kinase 1 promotes the progress and lymph metastasis of gallbladder cancer.

Receptor-interacting serine/threonine-protein kinase 1 (RIP-1) is highly expressed in gallbladder cancer, and is very important in promoting tumor proliferation and invasion. The underlying mechanism in this promotion is the RIP-1-nuclear factor κ-B (NF-κB) and activator protein 1 (AP-1)-vascular endothelial growth factor-C (VEGF-C) signaling pathways. However, the precise mechanisms by which RIP-1 regulates VEGF-C expression are still unknown. The current study aims to clarify the detailed mechanisms by which RIP-1 upregulates VEGF-C expression. In the current study, the authors constructed various VEGF-C promoter deletions, VEGF-C promoter mutations and RIP-1 overexpression plasmids, and silenced RIP-1 with a small interfering RNA. Promoter analysis, an electrophoretic mobility shift assay, a chromatin immunoprecipitation assay was then performed, and an orthotopic transplantation model in nude mice was established by modified methods previously used. The authors also found that the core region for luciferase activity in the VEGF-C promoter was −332 to −190 nt, in which there are two overlapping AP-1 sites and an NF-κB site. RIP-1 was demonstrated to activate transcription factors NF-κB and AP-1 to combine with the core region and enhance VEGF-C promoter activity. In conclusion, the current study illustrated the mechanisms by which RIP-1 regulates VEGF-C expression, by activating NF-κB and AP-1 to combine with the −332 to −190 nt area of the VEGF-C promoter. By establishing an orthotopic mouse model of gallbladder cancer tumors, it was further elucidated that RIP-1 promotes gallbladder cancer metastasis. The findings provide evidence that targeting RIP-1 may prove to be useful in the treatment of gallbladder cancer.