S100A6 drives lymphatic metastasis of liver cancer via activation of the RAGE/NF-kB/VEGF-D pathway.

Patients diagnosed with lymph node (LN) metastatic liver cancer face an exceedingly grim prognosis. In-depth analysis of LN metastatic patients' characteristics and tumor cells' interactions with human lymphatic endothelial cells (HLECs), can provide important biological and therapeutic insights. Here we identify at the single-cell level that S100A6 expression differs between primary tumor and their LN metastasis. Of particular significance, we uncovered the disparity in S100A6 expression between tumors and normal tissues is greater in intrahepatic cholangiocarcinoma (ICC) patients, frequently accompanied by LN metastases, than that in hepatocellular carcinoma (HCC), with rare occurrence of LN metastasis. Furthermore, in the infrequent instances of LN metastasis in HCC, heightened S100A6 expression was observed, suggesting a critical role of S100A6 in the process of LN metastasis. Subsequent experiments further uncovered that S100A6 secreted from tumor cells promotes lymphangiogenesis by upregulating the expression and secretion of vascular endothelial growth factor-D (VEGF-D) in HLECs through the RAGE/NF-kB/VEGF-D pathway while overexpression of S100A6 in tumor cells also augmented their migration and invasion. Taken together, these data reveal the dual effects of S100A6 in promoting LN metastasis in liver cancer, thus highlighting its potential as a promising therapeutic target.

Determinants of Progression and Mortality in Lymphangioleiomyomatosis.

BACKGROUND: Lymphangioleiomyomatosis is a progressive diffuse cystic lung disease with approximately 85% survival at 10 years. The determinants of disease progression and mortality after the introduction of sirolimus therapy and vascular endothelial growth factor D (VEGF-D) as a biomarker have not been well defined. RESEARCH QUESTION: Which factors, including VEGF-D and sirolimus therapy, influence disease progression and survival prognosis in patients with lymphangioleiomyomatosis? STUDY DESIGN AND METHODS: The progression dataset and the survival dataset included 282 and 574 patients, respectively, from Peking Union Medical College Hospital, Beijing, China. A mixed-effects model was used to compute the rate of decline in FEV1, and generalized linear models were used to identify variables affecting FEV1 decline. A Cox proportional hazards model was used to explore the association between clinical variables and the outcomes of death or lung transplantation in patients with lymphangioleiomyomatosis. RESULTS: VEGF-D levels and sirolimus treatment were associated with FEV1 changes and survival prognosis. Compared with patients with VEGF-D of < 800 pg/mL at baseline, patients with VEGF-D of ≥ 800 pg/mL lost FEV1 faster (SE, -38.86 mL/y; 95% CI, -73.90 to -3.82 mL/y; P = .031). The 8-year cumulative survival rates of patients with VEGF-D of ≥ 2,000 pg/mL and < 2,000 pg/mL were 82.9% and 95.1%, respectively (P = .014). The generalized linear regression model also demonstrated the benefit of delaying the decline of FEV1 by 65.56 mL/y (95% CI, 29.06-102.06 mL/y) in patients treated with sirolimus compared with those without sirolimus (P < .001). The 8-year risk of death was reduced by 85.1% (hazard ratio, 0.149; 95% CI, 0.075-0.299) after sirolimus treatment. After inverse treatment probability weighting, the risks of death in the sirolimus group were reduced by 85.6%. CT scan results of grade III severity were associated with worse progression than results of grades I or II severity. Patients with baseline FEV1 of 70% predicted or St. George's Respiratory Questionnaire Symptoms domain 50 or higher predicted a higher risk of worse survival. INTERPRETATION: Serum VEGF-D levels, a biomarker of lymphangioleiomyomatosis, are associated with disease progression and survival. Sirolimus therapy is associated with slower disease progression and better survival in patients with lymphangioleiomyomatosis. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03193892; URL: www. CLINICALTRIALS: gov.

Human gingival epithelial cells stimulate proliferation, migration, and tube formation of lymphatic endothelial cells in vitro.

OBJECTIVE: The aim of this study was to investigate the response of gingival epithelial cells to microbial and inflammatory signals. BACKGROUND: The gingival epithelial barrier provides the first line of defense and supports tissue homeostasis by maintaining the cross-talk between gingival epithelium, oral microbiota, and immune cells. Lymphatic vessels are essential to sustaining this homeostasis. The gingival epithelial cells have been shown to produce prolymphangiogenic factors during physiologic conditions, but their role in response to microbial and inflammatory signals is unknown. METHODS: Immortalized human gingival epithelial cells (HGEC) and human dermal lymphatic microvascular endothelial cells (LEC) were cultured. HGEC were exposed to Porphyromonas gingivalis derived-LPS, human IL-1 beta/IL-1F2 protein, or recombinant human IL-6/IL-6R. Levels of vascular growth factors (VEGF-A, VEGF-C, and VEGF-D) in cell supernatants were determined by ELISA. LEC were grown to confluence, and a scratch was induced in the monolayer. Uncovered area was measured up to 48 h after exposure to conditioned medium (CM) from HGEC. Tube formation assays were performed with LEC cocultured with labelled HGEC or exposed to CM. RESULTS: VEGF-A, VEGF-C, and low levels of VEGF-D were constitutively expressed by HGEC. The expression of VEGF-C and VEGF-D, but not VEGF-A, was upregulated in response to proinflammatory mediators. VEGF-C was upregulated in response to P. gingivalis LPS, but not to Escherichia coli LPS. A scratch migration assay showed that LEC migration was significantly increased by CM from HGEC. Both the CM and coculture with HGEC induced significant tube formation of LEC. CONCLUSIONS: HGEC can regulate production of lymphangiogenic/angiogenic factors during inflammatory insults and can stimulate proliferation, migration, and tube formation of LEC in vitro in a paracrine manner.

Cutaneous vascular structure and perfusion in patients with chronic plaque psoriasis.

BACKGROUND: Vascular dysfunction is a significant contributor to the pathophysiology of psoriasis. Some individuals have variation within the gene for vascular endothelial growth factor-A (VEGF-A), which confers an increased risk of developing psoriasis and having a severe disease phenotype, and may determine responsiveness to treatment. AIM: To determine whether patients with psoriasis have alterations in cutaneous microvascular anatomy and physiology due to expression of VEGF and whether laser Doppler imaging has utility in the assessment of this. METHODS: Twelve adult volunteers with Type 1 chronic plaque psoriasis underwent laser Doppler imaging of plaque and uninvolved skin. Skin biopsies were taken from the areas imaged for immunohistochemistry, including blood and lymphatic vessel markers, and VEGF-A isotype analysis (VEGF-A121, VEGF-A165 and VEGF-D). Venous blood was collected for DNA extraction, VEGF-A genotyping and peripheral blood mononuclear cell culture. RESULTS: Mean blood vessel area (P < 0·01), number of blood vessels (P < 0·001), number of lymphatic vessels (P < 0·001) and blood flow (P < 0·001) was significantly increased in psoriasis plaques, as was expression of VEGF-A121 (P < 0·01), VEGF-A165 (P < 0·04) and VEGF-D (P < 0·01). Blood flow within psoriasis plaques was independent of their increased vascularity (P < 0·01) and may be associated with baseline productivity of VEGF. The number of blood vessels within uninvolved skin in patients with psoriasis was associated with the VEGF-A (rs833061) genotype (P = 0·01), in a relationship suggesting an allele dosing effect. CONCLUSION: Noninvasive imaging of blood flow may help determine the cutaneous vascular signature for individual patients. This may be a useful prognostic indicator of psoriasis susceptibility and severity, and thus support selection of treatments.

Safety and Efficacy of Combined Resveratrol and Sirolimus in Lymphangioleiomyomatosis.

BACKGROUND: A critical need exists to develop remission-inducing therapies for lymphangioleiomyomatosis. RESEARCH QUESTION: Is the addition of resveratrol safe and more efficacious than sirolimus alone in patients with lymphangioleiomyomatosis? STUDY DESIGN AND METHODS: We conducted a phase 2, dose-escalating, open-label trial of resveratrol in patients with lymphangioleiomyomatosis receiving a stable regimen of sirolimus. Resveratrol was started at 250 mg/d and escalated every 8 weeks to maximum dose of 1,000 mg/d over 24 weeks. The primary outcome was ≥ 42% decline in serum vascular endothelial growth factor D (VEGF-D) levels on combined therapy compared with baseline VEGF-D levels on sirolimus. Secondary objectives included an assessment of the safety profile and the effect on lung function and health-related quality of life (HRQOL). Longitudinal change in outcome measures was assessed using linear mixed models. Adverse effects were tabulated using the National Cancer Institute's Common Terminology Criteria for Adverse Events version 4. RESULTS: Twenty-five patients with lymphangioleiomyomatosis with a median age of 51 years were enrolled. Pulmonary function parameters at study inclusion were: FEV1: median absolute, 1.72 L; 64% predicted; FVC: median absolute, 2.99 L; 96% predicted; and diffusing capacity of the lungs for carbon monoxide: median absolute, 14.68 mL/mm Hg/min; 37% predicted. The median serum VEGF-D value at baseline was 617 pg/mL. Patients entered the study with a median sirolimus dose of 2 mg/d with median trough level of 6.3 ng/mL. Despite some GI side effects, the addition of resveratrol was well tolerated. Although the primary outcome was not met, a statistically significant reduction in serum VEGF-D levels and improvement in HRQOL during the study was found. INTERPRETATION: The addition of resveratrol was safe and well tolerated in patients with lymphangioleiomyomatosis taking sirolimus and was associated with modest improvement in HRQOL. Larger controlled trials of this combination might be warranted to assess definitively the usefulness of resveratrol as an additive therapy in lymphangioleiomyomatosis. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT03253913; URL: www. CLINICALTRIALS: gov.

EGFL7 loss correlates with increased VEGF-D expression, upregulating hippocampal adult neurogenesis and improving spatial learning and memory.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

Comprehensive transcriptional profiling and functional study of lymphangiogenic growth factor in placentome of early-stage pregnant water buffalo.

The aim of the present study was to evaluate the expression and localization of lymphangiogenic factors (VEGF-C and VEGF-D), their receptor (VEGFR3) and lymphatic endothelial marker (LYVE1) in buffalo placenta during early pregnancy [EP], and to investigate the functional role of lymphangiogenic growth factors in placental lymphangiogenesis. The mRNA and protein expression of VEGF-C, VEGF-D, their receptor VEGFR3 and LYVE1 showed significant expression in EP1 (29-42 days) and EP2 stages (51-82 days) both in caruncle (maternal part) and cotyledon (foetal part) of the buffalo placenta. Immunoreactivity of VEGF-C, VEGF-D and LYVE1 was observed around the endometrial gland, in lymphatics and trophoblast cells, whereas VEGFR3 mainly localized in lymphatics of the caruncle and cotyledons. Cultured trophoblast cells were treated with VEGF-C/VEGF-D (50, 100 and 150 ng/ml) and combined doses of VEGF-C and VEGF-D (150 ng/ml) each for different time durations (24, 48 and 72 h). The mRNA expression of LYVE1 and PCNA was significantly (p < .001) upregulated with VEGF-C and VEGF-D and combined treatment (@150 ng/ml), as well as significantly downregulating Caspase-3 at 48 and 72 h. Thus, the present study provides evidence that lymphangiogenic factors are expressed in buffalo placental compartments and they may play a significant role in the regulation of placental function in water buffaloes.

Atorvastatin attenuates NS1 (Non-structural protein-1) of dengue type-2 serotype-induced expressions of matrix metalloproteinases in HL-60 cells, differentiated to neutrophils: Implications for the immunopathogenesis of dengue viral disease.

BACKGROUND: The dengue is a vector borne viral infection in humans. Bite of mosquito infected with a dengue virus transmits the disease. The neutrophils support more to the innate immune response by switching to infected tissues and triggering immunomodulatory mechanisms including the release of proteases and host defence peptides. METHODS: Cell viability by MTT and trypan blue dye exclusion assay, bright field microscopy for assessment of cell morphology, cytokines measurements by ELISA, estimation of protein by Bradford assay were done. Assessments of matrix metalloproteinase genes mRNA expressions were done using real-time PCR. RESULTS: In the present study, we have for the first time unveiled that, NS1 antigen of dengue type-2 serotype, induce and stimulate the neutrophils cells to express high levels of matrix metalloproteases. NS1 exposure of HL-60 cells differentiated to neutrophils affected cell morphology and in 24 h of exposure. We have demonstrated that, the NS1 antigen has induced MMP-2, MMP-14 and MMP-9 expressions in neutrophils in a 24hrs exposure time. NS1 exposure has also further upregulated MMP-1, MMP-13, and MMP-8 expressions in neutrophils in a 24hrs exposure time. Notably, treatment with atorvastatin concentrations downregulated the expression profile of the all matrix metalloprotease significantly. Importantly, NS1 antigen has significantly increased the IL-6, IL-13 release by the HL,60 cells which was reversed by atorvastatin. On the other hand, NS1 exposure enhanced the mRNA expressions of VEGF-A and VEGF-D which was reversed by atorvastatin. However, we found that, NS1 exposure reduced the mRNA expressions profile of VEGF-C, which was reversed by atorvastatin. CONCLUSION: In conclusion, we report that, neutrophils associated matrix metalloprotease are involved in the pathogenesis of dengue viral disease. VEGF growth factors may also be released by the neutrophils which may subsequently participate in the endothelial dysfunctions leading to dengue shock syndrome.

Suppression of VEGFD expression by S-nitrosylation promotes the development of lung adenocarcinoma.

BACKGROUND: Vascular endothelial growth factor D (VEGFD), a member of the VEGF family, is implicated in angiogenesis and lymphangiogenesis, and is deemed to be expressed at a low level in cancers. S-nitrosylation, a NO (nitric oxide)-mediated post-translational modification has a critical role in angiogenesis. Here, we attempt to dissect the role and underlying mechanism of S-nitrosylation-mediated VEGFD suppression in lung adenocarcinoma (LUAD). METHODS: Messenger RNA and protein expression of VEGFD in LUAD were analyzed by TCGA and CPTAC database, respectively, and Assistant for Clinical Bioinformatics was performed for complex analysis. Mouse models with urethane (Ure)-induced LUAD or LUAD xenograft were established to investigate the role of S-nitrosylation in VEGFD expression and of VEGFD mutants in the oncogenesis of LUAD. Molecular, cellular, and biochemical approaches were applied to explore the underlying mechanism of S-nitrosylation-mediated VEGFD suppression. Tube formation and wound healing assays were used to examine the role of VEGFD on the angiogenesis and migration of LUAD cells, and the molecular modeling was applied to predict the protein stability of VEGFD mutant. RESULTS: VEGFD mRNA and protein levels were decreased to a different extent in multiple primary malignancies, especially in LUAD. Low VEGFD protein expression was closely related to the oncogenesis of LUAD and resultant from excessive NO-induced VEGFD S-nitrosylation at Cys277. Moreover, inhibition of S-nitrosoglutathione reductase consistently decreased the VEGFD denitrosylation at Cys277 and consequently promoted angiogenesis of LUAD. Finally, the VEGFDC277S mutant decreased the secretion of mature VEGFD by attenuating the PC7-dependent proteolysis and VEGFDC277S mutant thus reversed the effect of VEGFD on angiogenesis of LUAD. CONCLUSION: Low-expression of VEGFD positively correlates with LUAD development. Aberrant S-nitrosylation of VEGFD negates itself to induce the tumorigenesis of LUAD, whereas normal S-nitrosylation of VEGFD is indispensable for its secretion and repression of angiogenesis of LUAD.

Ocular surface mast cells promote inflammatory lymphangiogenesis.

Mast cells, sentinel immune cells, are most abundantly expressed in vascularized tissues that interface the external environment, such as the skin and ocular surface. Our previous reports have shown mast cells reside closely with vascular endothelial cells and mediate the pathogenic angiogenic response. However, the contribution of mast cells and their underlying mechanisms on lymphangiogenesis have not been fully deciphered. Using a murine model of inflammatory corneal angiogenesis, we observed adjacent migration of activated mast cells with new lymph vessel growth. Our in vitro co-culture assays demonstrate that mast cells express high levels of of VEGF-D and directly promote lymphatic endothelial cell tube formation and proliferation. Moreover, our loss-of-function approaches, using mast cell knockout mice and cromolyn-mediated mast cell inhibition, showed mast cell deficiency suppresses the induction of inflammatory lymphangiogenesis and VEGF-D expression at the ocular surface following corneal tissue insult. Our findings suggest blockade of mast cells as a potential therapeutic strategy to inhibit pathological lymphangiogenesis.

VEGF-D-mediated signaling in tendon cells is involved in degenerative processes.

Vascular endothelial growth factor (VEGF) signaling is crucial for a large variety of cellular processes, not only related to angiogenesis but also in nonvascular cell types. We have previously shown that controlling angiogenesis by reducing VEGF-A signaling positively affects tendon healing. We now hypothesize that VEGF signaling in non-endothelial cells may contribute to tendon pathologies. By immunohistochemistry we show that VEGFR1, VEGFR2, and VEGFR3 are expressed in murine and human tendon cells in vivo. In a rat Achilles tendon defect model we show that VEGFR1, VEGFR3, and VEGF-D expression are increased after injury. On cultured rat tendon cells we show that VEGF-D stimulates cell proliferation in a dose-dependent manner; the specific VEGFR3 inhibitor SAR131675 reduces cell proliferation and cell migration. Furthermore, activation of VEGFR2 and -3 in tendon-derived cells affects the expression of mRNAs encoding extracellular matrix and matrix remodeling proteins. Using explant model systems, we provide evidence, that VEGFR3 inhibition prevents biomechanical deterioration in rat tail tendon fascicles cultured without load and attenuates matrix damage if exposed to dynamic overload in a bioreactor system. Together, these results suggest a strong role of tendon cell VEGF signaling in mediation of degenerative processes. These findings give novel insight into tendon cell biology and may pave the way for novel treatment options for degenerative tendon diseases.

Use of Vascular Endothelial Growth Factor-D As a Targeted Therapy in Lymphedema Treatment: A Comprehensive Literature Review.

Background: Lymphangiogenic growth factors, such as vascular endothelial growth factor (VEGF)-D, are subjects of interest in studies of targeted therapies in lymphedema treatment. Methods and Results: We conducted a systematic review assessing the use of VEGF-D as a targeted therapy in lymphedema treatment. We hypothesized that VEGF-D was a promising therapy to induce lymphangiogenesis. Our search yielded 90 studies in the literature, but only 4 articles fulfilled our study eligibility criteria, and they were all experimental studies using viral gene transfer. The majority of the studies were conducted on small animals (mice) and investigated the effects of VEGF-D on lymph node transfer. All authors agreed about VEGF-D's lymphangiogenic potential, but they noticed that VEGF-C induced a superior lymphangiogenesis, and one study noticed that VEGF-D induced seroma. Conclusions: The publications assessing the use of VEGF-D as a targeted therapy in lymphedema treatment were able to demonstrate its lymphangiogenic potential. Nonetheless, further studies are still necessary to investigate VEGF-D's efficacy and safety in lymphedema treatment on patients.

High-Dose Vitamin C Exerts Its Anti-cancer Effects in a Xenograft Model of Colon Cancer by Suppressing Angiogenesis.

Several studies have been conducted to investigate the anti-cancer effects of vitamin C (VC). However, the effect of high-dose VC administration on tumor angiogenesis remains unclear. Focusing on our high-dose VC, our study investigated the effect of high-dose VC (4 g/kg) on vascular endothelial growth in mice with xenografts of a rectal cancer cell line referred to as Colon 26. Male mice harboring Colon 26 tumors were established, and high-dose VC solution was orally administered once daily for 14 d. On the final day of the study, the lower limb tumor tissues and serum samples were collected and analyzed for the expression of tumor angiogenesis related proteins as well as the levels of reactive oxygen species (ROS). Oral VC administration decreased tumor volumes and increased p53 and endostatin levels. In addition, plasma and in tumor part ROS levels and tissue hypoxia inducible factor-1α (HIF-1α) were reduced by VC administration. In addition, the levels of vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor D (VEGFD) were decreased by VC administration. These results suggest that VC exerts its anti-cancer effects by suppressing angiogenesis.

Asymptomatic COVID-19: disease tolerance with efficient anti-viral immunity against SARS-CoV-2.

The immune responses and mechanisms limiting symptom progression in asymptomatic cases of SARS-CoV-2 infection remain unclear. We comprehensively characterized transcriptomic profiles, cytokine responses, neutralization capacity of antibodies, and cellular immune phenotypes of asymptomatic patients with acute SARS-CoV-2 infection to identify potential protective mechanisms. Compared to symptomatic patients, asymptomatic patients had higher counts of mature neutrophils and lower proportion of CD169+ expressing monocytes in the peripheral blood. Systemic levels of pro-inflammatory cytokines were also lower in asymptomatic patients, accompanied by milder pro-inflammatory gene signatures. Mechanistically, a more robust systemic Th2 cell signature with a higher level of virus-specific Th17 cells and a weaker yet sufficient neutralizing antibody profile against SARS-CoV-2 was observed in asymptomatic patients. In addition, asymptomatic COVID-19 patients had higher systemic levels of growth factors that are associated with cellular repair. Together, the data suggest that asymptomatic patients mount less pro-inflammatory and more protective immune responses against SARS-CoV-2 indicative of disease tolerance. Insights from this study highlight key immune pathways that could serve as therapeutic targets to prevent disease progression in COVID-19.

Isolation and characterisation of lymphatic endothelial cells from lung tissues affected by lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare pulmonary disease characterised by the proliferation of smooth muscle-like cells (LAM cells), and an abundance of lymphatic vessels in LAM lesions. Studies reported that vascular endothelial growth factor-D (VEGF-D) secreted by LAM cells contributes to LAM-associated lymphangiogenesis, however, the precise mechanisms of lymphangiogenesis and characteristics of lymphatic endothelial cells (LECs) in LAM lesions have not yet been elucidated. In this study, human primary-cultured LECs were obtained both from LAM-affected lung tissues (LAM-LECs) and normal lung tissues (control LECs) using fluorescence-activated cell sorting (FACS). We found that LAM-LECs had significantly higher ability of proliferation and migration compared to control LECs. VEGF-D significantly promoted migration of LECs but not proliferation of LECs in vitro. cDNA microarray and FACS analysis revealed the expression of vascular endothelial growth factor receptor (VEGFR)-3 and integrin α9 were elevated in LAM-LECs. Inhibition of VEGFR-3 suppressed proliferation and migration of LECs, and blockade of integrin α9 reduced VEGF-D-induced migration of LECs. Our data uncovered the distinct features of LAM-associated LECs, increased proliferation and migration, which may be due to higher expression of VEGFR-3 and integrin α9. Furthermore, we also found VEGF-D/VEGFR-3 and VEGF-D/ integrin α9 signaling play an important role in LAM-associated lymphangiogenesis.

circEHBP1 promotes lymphangiogenesis and lymphatic metastasis of bladder cancer via miR-130a-3p/TGFßR1/VEGF-D signaling.

Lymphatic metastasis constitutes a leading cause of recurrence and mortality in bladder cancer. Accumulating evidence indicates that lymphangiogenesis is indispensable to trigger lymphatic metastasis. However, the specific mechanism is poorly understood. In the present study, we revealed a pathway involved in lymphatic metastasis of bladder cancer, in which a circular RNA (circRNA) facilitated lymphangiogenesis in a vascular endothelial growth factor C (VEGF-C)-independent manner. Novel circRNA circEHBP1 was markedly upregulated in bladder cancer and correlated positively with lymphatic metastasis and poor prognosis of patients with bladder cancer. circEHBP1 upregulated transforming growth factor beta receptor 1 (TGFBR1) expression through physically binding to miR-130a-3p and antagonizing the suppression effect of miR-130a-3p on the 3' UTR region of TGFBR1. Subsequently, circEHBP1-mediated TGFßR1 overexpression activated the TGF-ß/SMAD3 signaling pathway, thereby promoting the secretion of VEGF-D and driving lymphangiogenesis and lymphatic metastasis in bladder cancer. Importantly, administration of VEGF-D neutralizing antibodies remarkably blocked circEHBP1-induced lymphangiogenesis and lymphatic metastasis in vivo. Our findings highlighted that the circEHBP1/miR-130a-3p/TGFßR1/VEGF-D axis contributes to lymphangiogenesis and lymphatic metastasis of bladder cancer independent of VEGF-C, which might lead to the development of circEHBP1 as a potential biomarker and promising therapeutic target for lymphatic metastasis in bladder cancer.

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

[Figure: see text].

Endothelial cell-type-specific molecular requirements for angiogenesis drive fenestrated vessel development in the brain.

Vascular endothelial cells (vECs) in the brain exhibit structural and functional heterogeneity. Fenestrated, permeable brain vasculature mediates neuroendocrine function, body-fluid regulation, and neural immune responses; however, its vascular formation remains poorly understood. Here, we show that specific combinations of vascular endothelial growth factors (Vegfs) are required to selectively drive fenestrated vessel formation in the zebrafish myelencephalic choroid plexus (mCP). We found that the combined, but not individual, loss of Vegfab, Vegfc, and Vegfd causes severely impaired mCP vascularization with little effect on neighboring non-fenestrated brain vessel formation, demonstrating fenestrated-vEC-specific angiogenic requirements. This Vegfs-mediated vessel-selective patterning also involves Ccbe1. Expression analyses, cell-type-specific ablation, and paracrine activity-deficient vegfc mutant characterization suggest that vEC-autonomous Vegfc and meningeal fibroblast-derived Vegfab and Vegfd are critical for mCP vascularization. These results define molecular cues and cell types critical for directing fenestrated CP vascularization and indicate that vECs' distinct molecular requirements for angiogenesis underlie brain vessel heterogeneity.

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

AIM: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Efficacy and Safety of Clinical-Grade Human Vascular Endothelial Growth Factor-DΔNΔC Gene Therapy Containing Residual Replication-Competent Adenoviruses.

Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 1010 viral particles (vp): <10 RCA (VEGF-D L-RCA1), 10-100 RCA (VEGF-D H-RCA2), and 100-200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (PCR) RCA assay in a preclinical rabbit model (n = 21). ß-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 1011 vp. Efficacy of the products was tested by the combination of contrast pulse sequencing ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution, and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood cell count and body temperature as well as using quantitative real-time PCR with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral GT products may not be as important major safety issue as previously anticipated.