Vascular Endothelial Growth factor-C Research Articles

Hydroxysafflor yellow A regulates lymphangiogenesis and inflammation via the inhibition of PI3K on regulating AKT/mTOR and NF-κB pathway in macrophages to reduce atherosclerosis in ApoE-/- mice

BackgroundMacrophage-mediated inflammatory infiltration and pathological lymphangiogenesis around atherosclerotic plaques are newly highlighted treatment targets of atherosclerosis. Although the effect of Hydroxysafflor yellow A(HSYA) on atherosclerosis was clear, few studies focus on the regulation of HSYA on such mechanisms. PurposeThis study aimed to uncover the key site of HSYA on improving atherosclerosis by regulating macrophage-induced inflammation and lymphangiogenesis. Study designThis study was designed to explore the new mechanism of HSYA on alleviating atherosclerosis in vitro and in vivo. MethodsWe determined the expression of vascular endothelial growth factor C(VEGF-C) in Raw264.7 cells and high-fat diet fed ApoE knockout (ApoE−/−) mice. Raw264.7 cells were treated with HSYA under the stimulation of LPS and ox-LDL. HFD induced ApoE−/− mice were given different concentrations of HSYA-saline solution by tail vein injection and ATV-saline suspension by gavage. C57/B6j mice fed with chow diet were used for the control group. H&E, oil red O and immunofluorescence staining analysis were used for visualizing the pathological changes. The biological impact of HSYA was evaluated by body weight, lipid metabolism, inflammation levels, and corresponding function indexes of kidney and liver. RT-qPCR and western blot methods were conducted to determine the expression of the inflammation and lymphangiogenesis factors. Molecular docking and microscale thermophoresis analysis were used to verify the combination of HSYA and PI3K. ResultsIn vivo, HSYA reduced the plaque formation, hepatic steatosis and inflammation-related lymphangiogenesis (IAL). It also changed the serum levels of inflammation (VEGF-C, TNF-α, IL-6, VCAM1, MCP1), lipid indexes (LDL, CHOL, TRIG) and relevant lymphangiogenesis (VEGF-C and LYVE-1) and inflammation (VCAM-1 and IL-6) signals in the aorta. In vitro, HSYA regulated Akt/mTOR and NF-κB activation by the inhibition of PI3K in macrophages. ConclusionHSYA affects inflammation and inflammation-associated lymphangiogenesis via suppressing PI3K to affect AKT/mTOR and NF-B pathway activation in macrophages, showing a comprehensive protective effect on atherosclerosis.

Diagnostic and prognostic value of plasma cell-free DNA combined with VEGF-C in laryngeal squamous cell carcinoma

BackgroundCirculating cell-free DNA (cfDNA) and vascular endothelial growth factor-C (VEGF-C) can be utilized to detect cancer and predict its prognosis. However, their potential application in laryngeal squamous cell carcinoma (LSCC) is unclear. PurposeThis study aimed to identify the diagnostic and prognostic value of cfDNA and VEGF-C in LSCC patients. MethodsThe plasma cfDNA of 148 LSCC patients and 43 non-tumor patients were isolated. Quantitative real-time PCR (qRT-PCR) was performed to assess long and short DNA fragments in plasma by amplifying the ALU repeats. ALU-qPCR results (ALU247/ALU115) were used to calculate cfDNA integrity index. Vascular endothelial growth factor-C (VEGF-C) level was detected by ELISA assay. Correlation between cfDNA and clinical features was analyzed. For detecting the sensitivity and specificity of cfDNA and VEGF-C alone or in combination for diagnosing LSCC, receiver operator characteristic (ROC) was established. For evaluating the overall survival (OS) of LSCC, Kaplan-Meier curves were established. ResultsLSCC patients had significantly higher levels of plasma cfDNA (ALU115, ALU247, and cfDNA integrity index) and VEGF-C than those without cancer (p < 0.05), showing area under the curve (AUC) values of 0.79, 0.74, 0.62 and 0.80, when cutoff value was correspondingly defined at 2.14 ng/mL, 1.39 ng/mL, 0.73 and 412.90 pg/mL, respectively. The AUC for distinguishing LSCC patients from non-tumor patients by plasma cfDNA combined with VEGF-C was 0.89 (95% CI: 0.83–0.94). A significant correlation was found between plasma cfDNA levels and Ki-67, tumor size, pT stage, and smoking history (p < 0.05). Based on survival analysis, low VEGF-C concentration groups had longer OS than those with high VEGF-C concentration (p = 0.02). ConclusionIndicators such as plasma cfDNA and VEGF-C may be used to diagnose and monitor LSCC for its noninvasiveness and rapid accessibility.

Expression of VEGF-C in Breast Carcinoma and its Association with HER2/neu and Oestrogen/ Progesterone Receptors: A Cross-sectional Study

Introduction: Breast carcinoma is the most common malignant tumour and is the leading cause of carcinoma death in women. Assessing Vascular Endothelial Growth Factor-C (VEGF-C) is important because the expression of VEGF-C is one of the factors determining the aggressiveness of breast carcinoma. Aim: To evaluate the expression of VEGF-C in breast carcinoma and its association with HER2/neu and Oestrogen Receptors (ER)/Progesterone Receptors (PR). Materials and Methods: A cross-sectional observational study was carried out in Department of Pathology of a tertiary care center of Burdwan Medical College and Hospital, West Bengal from 1st January 2018 to 30th June 2019. Patients attending Outpatient Department (OPD) and were diagnosed clinically as having breast cancer, operated upon, and subsequently sent for Histopathological Examination (HPE) in Pathology. The samples which were positive for breast carcinoma by histopathology were further studied with Immunohistochemical (IHC) markers and the positivity of IHC expression was reported using standard procedure and scoring pattern. The assosciation of VEGF-C expression with respect to histological type, grade, and stage of cancer as well as the HER2/neu and ER/PR status was studied. Using the Statistical Package for Social Sciences (SPSS) version 18.0, Chi-square was applied for the significance of p-value and the significance level was considered at a p-value &lt;0.05. Results: This study consisted of 44 cases of malignant breast lesions and of which 27 cases were associated with axillary lymph nodes. After IHC staining, 25 (56.82%) were VEGF-C positive and the rest 19 (43.18%) were VEGF-C negative. There was a significant association between VEGF-C and histologic grades of breast carcinoma. VEGF-C expression was significantly associated with lymph node metastasis and with HER2/neu. VEGF-C seems to be significantly associated with ER status of breast carcinoma and was not associated with the PR status of breast carcinoma. Conclusion: Assessment of VEGF-C is recommended because expression of VEGF-C is one of the factors determining the aggressiveness of breast carcinoma. If VEGF-C is positive in node- negative breast carcinoma, then it may help in targeted therapy of breast carcinoma before lymphatic metastasis develops.

Role of Vascular Endothelial Growth Factor-C during Stem Cell Therapy Using Autologous Bone Marrow Mononuclear Cells in Patients with Lower Limb Lymphedema

Introduction: Vascular endothelial growth factor-C (VEGF-C) is the primary lymphangiogenic factor that stimulates lymphangiogenesis by signaling via specific receptor, vascular endothelial growth factor receptor 3 (VEGFR3). This study was conducted to evaluate the change in the level of VEGF-C before and after autologous bone marrow mononuclear cell transplantation for treatment of Lower limb lymphedema. Patient and methods: Forty patients with lower limb lymphedema were divided into two groups. Group I included 20 patients with chronic lower limb lymphedema who underwent autologous bone marrow mononuclear cell transplantation. Group II included 20 patients with chronic lower limb lymphedema who were exposed only to compression therapy as a control group. VEGF-C level in the diseased limbs was measured in both groups at the beginning of the study then 3 and 6 months respectively. Results: Group I included 20 patients, 8 patients were male (40%) and 12 patients were females (60%) with mean age 29.5 ± 12.15 while group II included 20, 10 patients were male (50%) and 10 patients were females (50%) with mean age 39.5 ± 11.5. In group I, the specimens were taken at 3 and 6 months after transplantation showed a marked decrease in the VEGF-C level with statistically significant p value, 0.02 and 0.001 respectively. In group II the level of VEGF-C after compression therapy alone at 3 and 6 months interval showed fluctuation with statistically non-significant p value, 0.64 and 0.55 respectively. Conclusion: VEGF-C is essential for regulation of lymphangiogenesis. The level of VEGF-C was found elevated in patients with lymphedema and decrease after autologous mononuclear bone marrow cells, however these results were statically non-significant.

Local Adenoviral Delivery of Vascular Endothelial Growth Factor C Induces Lymphangiogenesis in the Conjunctiva in Rabbits

Introduction: The purpose of this study was to determine if conjunctival lymphangiogenesis can be induced using adenoviral delivery of vascular endothelial growth factor C (VEGF-C). Methods: Seventeen New Zealand white rabbits received a subconjunctival injection containing 3.5 × 107 plaque-forming units of an adenoviral vector containing the gene-encoding VEGF-C (Ad-VEGF-C). The contralateral eye was used for control experiment (the same volume of either saline or an empty vector). After 2 weeks, the animals were examined with trypan blue conjunctival lymphangiography, and the eyes were harvested for histology and immunohistochemistry (podoplanin and CD31). Results: Trypan blue conjunctival lymphangiography revealed significantly more extensive conjunctival vessel network in the Ad-VEGF-C group compared with control: 1.35 ± 0.67 versus 0.28 ± 0.17 vessel length/analysed area (p = <0.0001). This finding was confirmed with immunohistochemistry, where a significant increase in the number of lymphatic vessels was found compared to control; 34 ± 9 per mm2 versus 13 ± 8 per mm2 (p = 0.0019). Furthermore, there was a significant increase in lymphatic cross-sectional area; 32,500 ± 7,900 µm2 per mm2 versus 17,600 ± 9,700 µm2 per mm2 (p = 0.0149). Quantification of blood vessels revealed no significant difference in blood vessel density between Ad-VEGF-C and control; 19 ± 9 per mm2 versus 14 ± 8 per mm2 (p = 0.1971). There was no significant difference in total blood vessel area; 13,200 ± 7,600 µm2 per mm2 versus 7,100 ± 3,000 µm2 per mm2 (p = 0.0715). Eyes treated with an adenoviral vector (VEGF-C or empty vector) responded with a reactive cellular response, predominantly lymphocytes, towards the vector. Conclusion: The study demonstrates the feasibility of inducing conjunctival lymphangiogenesis with a single subconjunctival injection of Ad-VEGF-C. Future studies will explore how this can be used with a therapeutic purpose.

PS-B12-1: EPLERENONE INHIBITS UUO-INDUCED LYMPHANGIOGENESIS AND CARDIAC FIBROSIS BY ATTENUATING INFLAMMATORY INJURY

Objective: Cardiorenal syndrome (CRS) is the leading cause of death associated with chronic kidney disease (CKD) and end-stage renal disease (ESRD), however, the underlying mechanism of CKD-induced cardiac injury is incompletely understood. We reported that aldosterone is associated with cardiac fibrosis, and that inflammatory injury and lymphangiogenesis are involved. This pathogenic mechanism CKD-induced cardiac fibrosis still needs further investigation. Design and method: The unilateral ureteral obstruction (UUO) model was used to replicate the CRS type 4 model. Following induction of renal injury, the rats received eplerenone for 180-day. For in-vitro studies, we exposed human lymphatic endothelial cells (hLEC) to aldosterone and eplerenone. Changes in cardiac tissue related to lymphangiogenesis and inflammatory were analyzed in vivo. Changes in lymphangiogenesis, LEC phenotypic transformation and activation of mineralocorticoid receptor (MR)/ transforming growth factor (TGF)-beta/ vascular endothelial growth factor C (VEGFC) signaling axis involved in these changes were analyzed in vivo and in vitro. Results: We found that eplerenone ameliorated cardiac inflammation and fibrosis in UUO rats. Moreover, eplerenone inhibited MR activation, attenuated lyphangiogenesis and LEC phenotypic transformation, and regulated TGF-beta/CTGF/VEGFC signaling axis in vivo and in vitro. The levels of podoplanin, VEGFR-3 and VEGFC confirmed the role of lymphangiogenesis and inflammatory injury. Co-expression of LEC marker LYVE-1 and myofibroblast marker alpha-SMA validated EndMT in cardiac fibrosis. Treatment with eplerenone, clarified the role of aldosterone in UUO-induced cardiac fibrosis. Conclusions: We confirmed that eplerenone can alleviate UUO-induced cardiac fibrosis by inhibiting inflammation and lymphangiogenesis via MR/TGF-beta/VEGFC signaling axis. These findings provide an in-depth understanding of pathogenesis of UUO-induced cardiac injury and reveal novel therapeutic targets.

Exosomal miR-10527-5p Inhibits Migration, Invasion, Lymphangiogenesis and Lymphatic Metastasis by Affecting Wnt/β-Catenin Signaling via Rab10 in Esophageal Squamous Cell Carcinoma

Cancer cell-derived exosomal microRNAs (miRNAs) play critical role in orchestrating intercellular communication between tumor cells and tumor microenvironmental factors, including lymphatic endothelial cells (LECs). Nevertheless, the functions and underlying mechanisms of exosomal miRNAs in lymphatic metastasis and lymphangiogenesis in esophageal squamous cell carcinoma (ESCC) remain unclear. Small RNA sequencing, Gene Expression Omnibus (GEO) analysis and qRT‒PCR were performed to identify the candidate exosomal miRNAs involved in ESCC metastasis. Receiver operating characteristic curve analysis was conducted to evaluate the diagnostic potential of exosomal miR-10527-5p in predicting lymph node metastasis (LNM) status. An in vitro coculture system was used to investigate the effects of exosomal miR-10527-5p on ESCC cells and human LECs (HLECs), followed by a popliteal LNM assay in vivo. The relationship between miR-10527-5p and Rab10 was identified by dual-luciferase reporter, fluorescence in situ hybridization and qRT‒PCR assays. Then, a series of rescue assays were performed to further investigate whether Rab10 is involved in exosomal miR-10527-5p mediated ESCC metastasis. MiR-10527-5p was found to be notably reduced in both the plasma exosomes and tumor tissues of ESCC patients with LNM, and plasma exosomal miR-10527-5p had a high sensitivity and specificity for discrimination of LNM status. Moreover, exosome-shuttled miR-10527-5p suppressed the migration, invasion and epithelial-to-mesenchymal transition (EMT) of ESCC cells as well as the migration and tube formation of HLECs via Wnt/β-catenin signaling in vitro and in vivo. Further investigation revealed that Rab10 was a direct target of miR-10527-5p, and re-expression of Rab10 neutralized the inhibitory effects of exosomal miR-10527-5p. Our study demonstrated that exosomal miR-10527-5p had a strong capability to predict preoperative LNM status and anti-lymphangiogenic effect. Exosomal miR-10527-5p inhibited lymphangiogenesis and lymphatic metastasis of ESCC in a vascular endothelial growth factor-C (VEGF-C)-independent manner, showing potential as a therapeutic target for ESCC patients.

Lymphangiogenesis, a potential treatment target for myocardial injury.

The lymphatic vascular system is crucial for the regulation of tissue fluid homeostasis, lipid metabolism, and immune function. Cardiac injury quickly leads to myocardial edema, cardiac lymphatic dysfunction, which ultimately results in myocardial fluid imbalance and cardiac dysfunction. Therefore, lymphangiogenesis-targeted therapy may improve the recovery of myocardial function post cardiac ischemia as observed in myocardial infarction (MI). Indeed, a promising strategy for the clinical treatment of MI relies on vascular endothelial growth factor-C (VEGF-C)-targeted therapy, which promotes lymphangiogenesis. However, much effort is needed to identify the mechanisms of lymphatic transport in response to heart disease. This article reviews regulatory factors of lymphangiogenesis, and discusses the effects of lymphangiogenesis on cardiac function after cardiac injury and its regulatory mechanisms. The involvement of stem cells on lymphangiogenesis was also discussed as stem cells could differentiate into lymphatic endothelial cells (LECs) and stimulate phenotype of LECs.

The Effect of Lymphangiogenesis in Transplant Arteriosclerosis.

Transplant arteriosclerosis is a major complication in long-term survivors of heart transplantation. Increased lymph flow from donor heart to host lymph nodes has been reported to play a role in transplant arteriosclerosis, but how lymphangiogenesis affects this process is unknown. Vascular allografts were transplanted among various combinations of mice, including wild-type, Lyve1-CreERT2;R26-tdTomato, CAG-Cre-tdTomato, severe combined immune deficiency, Ccr2KO, Foxn1KO, and lghm/lghdKO mice. Whole-mount staining and 3-dimensional reconstruction identified lymphatic vessels within the grafted arteries. Lineage tracing strategies delineated the cellular origin of lymphatic endothelial cells. Adeno-associated viral vectors and a selective inhibitor were used to regulate lymphangiogenesis. Lymphangiogenesis within allograft vessels began at the anastomotic sites and extended from preexisting lymphatic vessels in the host. Tertiary lymphatic organs were identified in transplanted arteries at the anastomotic site and lymphatic vessels expressing CCL21 (chemokine [C-C motif] ligand 21) were associated with these immune structures. Fibroblasts in the vascular allografts released VEGF-C (vascular endothelial growth factor C), which stimulated lymphangiogenesis into the grafts. Inhibition of VEGF-C signaling inhibited lymphangiogenesis, neointima formation, and adventitial fibrosis of vascular allografts. These studies identified VEGF-C released from fibroblasts as a signal stimulating lymphangiogenesis extending from the host into the vascular allografts. Formation of lymphatic vessels plays a key role in the immune response to vascular transplantation. The inhibition of lymphangiogenesis may be a novel approach to prevent transplant arteriosclerosis.

Deciphering the Role of miR-200c-3p in Type 1 Diabetes (Subclinical Cardiovascular Disease) and Its Correlation with Inflammation and Vascular Health.

Uncomplicated type 1 diabetes (T1DM) displays all features of subclinical cardiovascular disease (CVD) as is associated with inflammation, endothelial dysfunction and low endothelial progenitor cells. MiR-200c-3p has been shown in animal tissues to be pro-atherogenic. We aimed to explore the role of miR-200c-3p in T1DM, a model of subclinical CVD. 19 samples from T1DM patients and 20 from matched controls (HC) were analyzed. MiR-200c in plasma and peripheral blood mononuclear cells (PBMCs) was measured by real-time quantitative polymerase chain reaction. The results were compared with the following indices of vascular health: circulating endothelial progenitor cells, (CD45dimCD34+VEGFR-2+ or CD45dimCD34+CD133+) and proangiogenic cells (PACs). MiR-200c-3p was significantly downregulated in PBMCs but not in plasma in T1DM. There was a significant negative correlation between the expression of miR-200c-3p and HbA1c, interleukin-7 (IL-7), vascular endothelial growth factor-C (VEGF-C), and soluble vascular cell adhesion molecule-1, and a positive correlation with CD45dimCD34+VEGFR-2+, CD45dimCD34+CD133+ and PACs. Receiver operating curve analyses showed miR-200c-3p as a biomarker for T1DM with significant downregulation of miR-200c-3p, possibly defining subclinical CVD at HbA1c > 44.8 mmol/mol (6.2%). In conclusion, downregulated miR-200c-3p in T1DM correlated with diabetic control, VEGF signaling, inflammation, vascular health and targeting VEGF signaling, and may define subclinical CVD. Further prospective studies are necessary to validate our findings in a larger group of patients.

Corneal edema resorption after injury is regulated by the transcription factor NFAT5

AbstractIn cornea, injury or inflammation leads to an ingrowth of blood‐ and lymphatic vessels associated with corneal edema. That can lead to a loss of transparency which is the second most common cause for blindness worldwide. So far, the only treatment is corneal transplantation, resulting in more than a million people suffering from corneal blindness due to shortage of donor corneas. Recently we showed a positive role of lymphatic vessels in the resolution of corneal edema. The nuclear factor of activated T‐cells 5 (NFAT5/TonEBP) is an important transcription factor regulating lymphangiogenesis (LA) by modulating the expression of pro‐lymphangiogenic vascular endothelial growth factor C (VEGF‐C) in skin macrophages. It has not been investigated whether NFAT5 is expressed in the cornea and which role it plays in the inflammatory response after corneal injury.In this study, two NFAT5‐knockout mouse models were used: tamoxifen‐inducible systemic (UbcCre/NFAT5fl/fl) and a myeloid cell specific (LysMCre/NFAT5fl/fl). Both underwent perforating corneal injury (PCI) in the central cornea. Seven days after surgery, corneal edema and its resolution were measured using in vivo optical coherence tomography (OCT). The mean corneal thickness (MCT) was evaluated using MatLab®. Corneal inflammation was assayed via immunofluorescence. Bone marrow derived macrophages (BMDMs) from LysMCre/NFAT5fl/fl mice were stimulated with LPS/IFN‐γ for 24 h and used for pinocytosis assays using FITC‐conjugated dextran. Pinocytosis capacity was measured by calculating the ratio of FITC‐saturated cells to the total number of cells using MatLab®.In naive corneas, NFAT5 was mainly expressed in fibroblasts. After PCI, NFAT5 expression in fibroblasts was downregulated and mainly expressed by macrophages (Correlation coefficient of TonEBP+/Vimentin+: 0.446 ± 0.111 in naive corneas vs 0.153 ± 0.024 after PCI [p &lt; 0.0001]; TonEBP+/F480+: 0.113 ± 0.032 in naive cornea vs. 0.446 ± 0.018 after PCI [p &lt; 0.0001]). In uninjured corneas, NFAT5 knockout did not lead to changes in corneal thickness, however, the number of macrophages was significantly increased (24.76 ± 3.54% in knockout vs. 16.14 ± 0.36%; p = 0.0094 of corneal area covered by F480+ cells). After PCI, the resolution of corneal edema was enhanced in both, systemic NFAT5 knockout mice (Mean corneal thickness: 97.1 ± 10.0 μm in knockout vs. 137.1 ± 28.8 μm [p = 0.002]) and myeloid cell NFAT5 knockout mice (Mean corneal thickness: 97.1 ± 10.0 μm in knockout vs. 126.0 ± 16.6 μm; [p &lt; 0.001]). After incubation of BMDMs from LysMCre/NFAT5fl/fl mice with fluorescent dextran, the percentage of cells saturated which incorporated the fluorescent dextran in inflammatory stimulated macrophages was significantly higher in LysMCre+/NFAT5fl/fl macrophages compared to LysMCre−/NFAT5fl/fl macrophages (1.45 ± 0.28% vs. 0.61 ± 0.18%; [p &lt; 0.001]).Our results demonstrate that NFAT5 is highly expressed in the cornea and its expression shows considerable changes after PCI. Mechanistically, we found that myeloid cell‐derived NFAT5 is crucial for controlling corneal edema, as edema resorption after PCI was significantly enhanced in mice with conditional loss of NFAT5 in the myeloid cell lineage, presumably due to increased pinocytosis of corneal macrophages. Collectively, we uncover a suppressive role for NFAT5 in corneal edema resorption, thereby identifying a novel therapeutic target to combat edema‐induced corneal blindness.

Lentiviral overexpression of VEGFC in transplanted MSCs leads to resolution of swelling in a mouse tail lymphedema model.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema. Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage. Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model. First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls. Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.

The Effect of Vascular Endothelial Growth Factor C and Adipose-Derived Stem Cells on Lymphatic Regeneration in a Rat Vascularized Lymph Node Transfer Model.

Lymphedema is a chronic condition characterized by progressive edema with complicated treatment. Recently, new treatment strategies inducing lymphangiogenesis were proposed. The aim of our study was to examine the effect of vascular endothelial growth factor C (VEGF-C) and adipose-derived stem cells (ADSCs) on lymphatic regeneration and drainage re-establishment in vascularized lymph node transfer (VLNT) model using a pedicled vascularized lymph node (VLN) groin flap. Female Lewis rats with groin VLN flaps were utilized as a lymphedema model. Group A served as the control. Group B received VEGF-C. Group C received both VEGF-C and ADSCs. Group D received ADSCs only. Lymphatic drainage re-establishment was evaluated by ultrasound-photoacoustic imaging (US-PAI) after indocyanine green (ICG) injection. The fastest regeneration of elevated flaps was observed in Groups B and C in all monitored periods. After the first month, ICG positivity was detected in 14.3% of animals in Group A, 71.43% of animals in Group B (odds ratio [OR] = 15; p = 0.048), and 83.33% in Group C (OR = 30; p = 0.027). On the contrary, the difference between control group and Group D (16.67%; p = 0.905) was statistically insignificant. Administration of VEGF-C, ADSC + VEGF-C, and ADSC led to full flap regeneration after 6 months. The control group had the lowest percentage of ICG positivity at all monitored time points. We found that the fastest regeneration occurred with the combination of the VLN flap and VEGF-C. The addition of ADSC had an insignificant effect in our study. Furthermore, we proved the feasibility of PAI as an assessment tool of the lymphatic drainage recovery in a VLNT model.

Vascular endothelial growth factor-C in activating vascular endothelial growth factor receptor-3 and chemokine receptor-4 in melanoma adhesion.

Vascular endothelial growth factor-C (VEGF-C) binds to receptor vascular endothelial growth factor receptor-3 (VEGFR-3) expressed on lymphatic endothelial and melanoma cells. Binding of VEGF-C to VEGFR-3 enhances receptor phosphorylation that activates mitogen-activated protein kinase (MAP-K) and phosphatidylinositol-3-kinase (PI3K). These signalling pathways regulate cell migration and adhesion in response to internal or external changes. In addition, the overexpression of VEGF-C upregulates chemokine receptor CXCR-4 in tumours (melanoma). CXCR-4 is expressed on cells of the immune system (natural killer cells) and facilitates the migration of leukocytes in response to the CXCL12 ligand. The latter is expressed by lymphatic endothelial cells and by stromal cells in the tumour microenvironment (TME). The gradient established between CXCR-4 expressed on tumour cells and CXCL12 produced by stromal and lymphatic endothelial cells enhances tumour cell metastasis. 3-(4-Dimethylamino-naphthalen-1-ylmethylene)-1, 3-dihydroindol-2-one, MAZ-51, is an indolinone-based synthetic molecule that inhibits the phosphorylation of the tyrosine kinase receptor VEGFR-3. CTCE-9908, a CXCR-4 antagonist derived from human CXCL12, hinders receptor phosphorylation and the subsequent signalling pathways that would be activated. VEGF-C is stimulated by transforming growth factor-beta 1 (TGF-β1), which facilitates cell-cell and cell-matrix adhesion by regulating cadherins through the activation of focal adhesion kinase (FAK) and mediates paxillin upregulation. Increased VEGF-C protein levels stimulated by TGF-β bound to VEGFR-3 impact on intracellular pathways that promote tumour cell adhesion. In addition, increased VEGF-C protein levels lead to enhanced CXCR-4 protein expression. Therefore, effective blocking of VEGR-3 and CXCR-4 may inhibit tumour cell metastasis by hampering intracellular proteins promoting adhesion.

Effect of electroacupuncture on myocardial inflammatory injury and apoptosis in mice with acute myocardial ischemia based on VEGF-C/VEGFR-3 pathway

To observe the effect of electroacupuncture (EA) on vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), proinflammatory factors and apoptosis in myocardial tissue in mice with acute myocardial ischemia (AMI), and to explore the mechanism of EA for AMI. Fifty male C57BL/6 mice were randomly divided into a sham operation group, a model group, an EA group, an inhibitor group and an inhibitor+EA group, 10 mice in each group. Except for the sham operation group, the mice in the remaining groups were intervented with ligation at the left anterior descending (LAD) coronary artery to establish AMI model. The mice in the sham operation group were intervented without ligation after thoracotomy. The mice in the EA group were intervented with EA at "Shenmen" (HT 7) and "Tongli" (HT 5), disperse-dense wave, 2 Hz/15 Hz in frequency, 1 mA in current intensity, 30 min each time, once a day, for 3 d. The mice in the inhibitor group were treated with intraperitoneal injection of SAR 131675 (12.5 mg•kg-1•d-1, once a day for 3 d). The mice in the inhibitor+EA group were injected intraperitoneally with SAR 131675 30 min before EA. The ECG before modeling, 30 min after modeling and 3 d after intervention was detected, and the ST segment displacement was recorded; after the intervention, the ELISA method was applied to measure the contents of serum creatine kinase isoenzyme (CK-MB), aspartate aminotransferase (AST) as well as tumor necrosis factor-α (TNF-α) and interleukin-23 (IL-23) in myocardial tissue; the HE staining method was used to observe the morphological changes of myocardial tissue; the immunofluorescence double labeling method was applied to measure the number of co-expression positive cells of VEGF-C/VEGFR-3 in myocardial tissue; the TUNEL method was used to detect the level of cardiomyocyte apoptosis; the Western blot method was applied to measure the protein expressions of VEGF-C, VEGFR-3, b-lymphoma-2 (Bcl-2), activated caspase-3 (Cleaved Caspase-3) and activated poly adenosine diphosphate ribose polymerase-1 (Cleaved PARP-1). Compared with the sham operation group, in the model group the ST segment displacement was increased (P<0.01); the contents of CK-MB, AST, TNF-α and IL-23 were increased (P<0.01); the arrangement of myocardial fibers was disordered, and interstitial inflammatory cell infiltration was obvious; the number of co-expression positive cells of VEGF-C/VEGFR-3 was decreased (P<0.01); the number of cardiomyocyte apoptosis was increased (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were decreased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were increased (P<0.01). Compared with the model group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.01); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). There was no significant difference in all the indexes between the model group and the inhibitor group (P>0.05). Compared with the model group, the protein expression of VEGF-C was increased in the inhibitor+EA group (P<0.01). Compared with the inhibitor group, in the EA group the ST segment displacement was decreased (P<0.01); the contents of CK-MB, AST, TNF-α, IL-23 were decreased (P<0.01); the severity of myocardial pathological injury was reduced; the number of co-expression positive cells of VEGF-C/VEGFR-3 was increased (P<0.05); the number of cardiomyocyte apoptosis was reduced (P<0.01); the expressions of VEGF-C, VEGFR-3 and Bcl-2 were increased (P<0.01); the expressions of Cleaved Caspase-3 and Cleaved PARP-1 were reduced (P<0.01). Compared with the inhibitor+EA group, all the indexes in the EA group were improved except the protein expression of VEGF-C (P<0.01). EA could relieve the inflammatory reaction and apoptosis in AMI mice, and its mechanism may be related to activating VEGF-C/VEGFR-3 pathway and promoting lymphangion genesis.

PTEN regulates invasiveness in pancreatic neuroendocrine tumors through DUSP19-mediated VEGFR3 dephosphorylation

BackgroundPhosphatase and tensin homolog (PTEN) is a tumor suppressor. Low PTEN expression has been observed in pancreatic neuroendocrine tumors (pNETs) and is associated with increased liver metastasis and poor survival. Vascular endothelial growth factor receptor 3 (VEGFR3) is a receptor tyrosine kinase and is usually activated by binding with vascular endothelial growth factor C (VEGFC). VEGFR3 has been demonstrated with lymphangiogenesis and cancer invasiveness. PTEN is also a phosphatase to dephosphorylate both lipid and protein substrates and VEGFR3 is hypothesized to be a substrate of PTEN. Dual-specificity phosphatase 19 (DUSP19) is an atypical DUSP and can interact with VEGFR3. In this study, we investigated the function of PTEN on regulation of pNET invasiveness and its association with VEGFR3 and DUSP19.MethodsPTEN was knocked down or overexpressed in pNET cells to evaluate its effect on invasiveness and its association with VEGFR3 phosphorylation. In vitro phosphatase assay was performed to identify the regulatory molecule on the regulation of VEGFR3 phosphorylation. In addition, immunoprecipitation, and immunofluorescence staining were performed to evaluate the molecule with direct interaction on VEGFR3 phosphorylation. The animal study was performed to validate the results of the in vitro study.ResultsThe invasion and migration capabilities of pNETs were enhanced by PTEN knockdown accompanied with increased VEGFR3 phosphorylation, ERK phosphorylation, and increased expression of epithelial–mesenchymal transition molecules in the cells. The enhanced invasion and migration abilities of pNET cells with PTEN knockdown were suppressed by addition of the VEGFR3 inhibitor MAZ51, but not by the VEGFR3-Fc chimeric protein to neutralize VEGFC. VEGFR3 phosphorylation is responsible for pNET cell invasiveness and is VEGFC-independent. However, an in vitro phosphatase assay failed to show VEGFR3 as a substrate of PTEN. In contrast, DUSP19 was transcriptionally upregulated by PTEN and was shown to dephosphorylate VEGFR3 via direct interaction with VEGFR3 by an in vitro phosphatase assay, immunoprecipitation, and immunofluorescence staining. Increased tumor invasion into peripheral tissues was validated in xenograft mouse model. Tumor invasion was suppressed by treatment with VEGFR3 or MEK inhibitors.ConclusionsPTEN regulates pNET invasiveness via DUSP19-mediated VEGFR3 dephosphorylation. VEGFR3 and DUSP19 are potential therapeutic targets for pNET treatment.

The Lymphangiogenic Factor CCBE1 Promotes Angiogenesis and Tumor Growth in Colorectal Cancer.

Collagen and calcium-binding EGF domain-1 (CCBE1) is essential for the development of the lymphatic vasculature and colorectal cancer (CRC) lymphangiogenesis as it enhances the proteolytic process of vascular endothelial growth factor C (VEGFC) activating VEGFR3. The fully processed mature VEGFC could also activate VEGFR2, the important endothelial-specific receptor tyrosine kinase, involved in blood vascular development and tumor angiogenesis. However, the role of CCBE1 in cancer angiogenesis remains undefined. In this paper, we find that the protein expression of CCBE1 is higher in the primary CRC tissue with distant metastasis and positively correlated with blood vessel density. The mRNA expression of CCBE1 is closely positively correlated with the vascular endothelial marker CD31 and VEGFR2 in CRC from TCGA datasets. The supernatant of the colorectal cancer cell line HCT116 with CCBE1 overexpression significantly promotes the tube formation ability of the human umbilical vein endothelial cells (HUVECs) in vitro and enhances angiogenesis and tumor growth in vivo. Knockdown of CCBE1 decreases the angiogenic ability of CRC. Our results demonstrate the angiogenic role of CCBE1 in CRC.

Bioactive VEGF-C from E. coli

Vascular endothelial growth factor-C (VEGF-C) stimulates lymphatic vessel growth in transgenic models, via viral gene delivery, and as a recombinant protein. Expressing eukaryotic proteins like VEGF-C in bacterial cells has limitations, as these cells lack specific posttranslational modifications and provisions for disulfide bond formation. However, given the cost and time savings associated with bacterial expression systems, there is considerable value in expressing VEGF-C using bacterial cells. We identified two approaches that result in biologically active Escherichia coli-derived VEGF-C. Expectedly, VEGF-C expressed from a truncated cDNA became bioactive after in vitro folding from inclusion bodies. Given that VEGF-C is one of the cysteine-richest growth factors in humans, it was unclear whether known methods to facilitate correct cysteine bond formation allow for the direct expression of bioactive VEGF-C in the cytoplasm. By fusing VEGF-C to maltose-binding protein and expressing these fusions in the redox-modified cytoplasm of the Origami (DE3) strain, we could recover biological activity for deletion mutants lacking the propeptides of VEGF-C. This is the first report of a bioactive VEGF growth factor obtained from E. coli cells circumventing in-vitro folding.

Improving the Function of Meningeal Lymphatic Vessels to Promote Brain Edema Absorption after Traumatic Brain Injury.

Brain edema is the most common and fatal complication after traumatic brain injury (TBI). Meningeal lymphatic vessels (MLVs) are the conduits that transport cerebrospinal fluid (CSF) and macromolecules to deep extracranial cervical lymph nodes (dCLNs). After TBI, the drainage function of MLVs can become impaired. However, the scenario in which the improvement of the function of MLVs can promote brain edema absorption after TBI has not been reported. Therefore, the purpose of this study was to investigate the effects of ketoprofen, 9-cis retinoic acid (RA) and vascular endothelial cell growth factor-C (VEGF-C), which promote the proliferation of peripheral lymphatic vessels, on the cerebellar medullary cistern injection of TBI rats, as well as their mechanism of action on brain edema after TBI. In the experiment, we found that ketoprofen, 9-cisRA, and VEGF-C can improve the function of MLVs, promote the extracranial drainage of CSF and the absorption of brain edema, weaken the neuroinflammatory response, reduce reactive oxygen species (ROS) production, maintain the structural integrity of MLVs, and improve neurological function. In addition, ketoprofen, 9-cisRA, and VEGF-C upregulated the lymphatic-specific proteins VEGF receptor (VEGFR)3, PROX1, forkhead box protein C2 (FOXC2), and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1). These results indicate that ketoprofen, 9-cisRA, and VEGF-C may maintain the integrity of the meningeal lymphatic wall and promote lymphatic proliferation by upregulating the expression of lymphatic vessel-specific proteins, improve meningeal lymphatic function after TBI, promote CSF drainage and brain edema absorption, reduce the immune response of the nervous system, and reduce ROS formation, thereby improving prognoses. These findings may provide new ideas for the treatment of brain edema after TBI.

Electroacupuncture ameliorates inflammatory response of mice with acute myocardial ischemia by regulating vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 pathway

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. 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. 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. 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.