Visfatin Facilitates VEGF-D-Induced Lymphangiogenesis through Activating HIF-1α and Suppressing miR-2277-3p in Human Chondrosarcoma.

Chondrosarcoma is a malignant bone tumor that arises from abnormalities in cartilaginous tissue and is associated with lung metastases. Lymphangiogenesis plays an essential role in cancer metastasis. Visfatin is an adipokine reported to enhance tumor metastasis, but its relationship with VEGF-D generation and lymphangiogenesis in chondrosarcoma remains undetermined. Our results from clinical samples reveal that VEGF-D levels are markedly higher in chondrosarcoma patients than in normal individuals. Visfatin stimulation promotes VEGF-D-dependent lymphatic endothelial cell lymphangiogenesis. We also found that visfatin induces VEGF-D production by activating HIF-1α and reducing miR-2277-3p generation through the Raf/MEK/ERK signaling cascade. Importantly, visfatin controls chondrosarcoma-related lymphangiogenesis in vivo. Therefore, visfatin is a promising target in the treatment of chondrosarcoma lymphangiogenesis.

Vascular endothelial growth factor-D plasma levels and VEGFD genetic variants are independently associated with outcomes in patients with cardiovascular disease.

AIMS: The vascular endothelial growth factor (VEGF) family is involved in pathophysiological mechanisms underlying cardiovascular (CV) diseases. The aim of this study was to investigate the associations between circulating VEGF ligands and/or soluble receptors and CV outcome in patients with acute coronary syndrome (ACS) and chronic coronary syndrome (CCS). METHODS AND RESULTS: Levels of VEGF biomarkers, including bFGF, Flt-1, KDR (VEGFR2), PlGF, Tie-2, VEGF-A, VEGF-C, and VEGF-D, were measured in the PLATO ACS cohort (n = 2091, discovery cohort). Subsequently, VEGF-D was also measured in the STABILITY CCS cohort (n = 4015, confirmation cohort) to verify associations with CV outcomes. Associations between plasma VEGF-D and outcomes were analysed by multiple Cox regression models with hazard ratios (HR [95% CI]) comparing the upper vs. the lower quartile of VEGF-D. Genome-wide association study (GWAS) of VEGF-D in PLATO identified SNPs that were used as genetic instruments in Mendelian randomization (MR) meta-analyses vs. clinical endpoints. GWAS and MR were performed in patients with ACS from PLATO (n = 10 013) and FRISC-II (n = 2952), and with CCS from the STABILITY trial (n = 10 786). VEGF-D, KDR, Flt-1, and PlGF showed significant association with CV outcomes. VEGF-D was most strongly associated with CV death (P = 3.73e-05, HR 1.892 [1.419, 2.522]). Genome-wide significant associations with VEGF-D levels were identified at the VEGFD locus on chromosome Xp22. MR analyses of the combined top ranked SNPs (GWAS P-values; rs192812042, P = 5.82e-20; rs234500, P = 1.97e-14) demonstrated a significant effect on CV mortality [P = 0.0257, HR 1.81 (1.07, 3.04) per increase of one unit in log VEGF-D]. CONCLUSION: This is the first large-scale cohort study to demonstrate that both VEGF-D plasma levels and VEGFD genetic variants are independently associated with CV outcomes in patients with ACS and CCS. Measurements of VEGF-D levels and/or VEGFD genetic variants may provide incremental prognostic information in patients with ACS and CCS.

Vascular inflammation and endothelial injury in SARS-CoV-2 infection: the overlooked regulatory cascades implicated by the ACE2 gene cluster.

Coronavirus disease 2019 (COVID-19) has presented physicians with an unprecedented number of challenges and mortality. The basic question is why, in contrast to other 'respiratory' viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in such multi-systemic, life-threatening complications and a severe pulmonary vasculopathy. It is widely known that SARS-CoV-2 uses membrane-bound angiotensin-converting enzyme 2 (ACE2) as a receptor, resulting in internalization of the complex by the host cell. We discuss the evidence that failure to suppress coronaviral replication within 5 days results in sustained downregulation of ACE2 protein expression and that ACE2 is under negative-feedback regulation. We then expose openly available experimental repository data that demonstrate the gene for ACE2 lies in a novel cluster of inter-regulated genes on the X chromosome including PIR encoding pirin (quercetin 2,3-dioxygenase), and VEGFD encoding the predominantly lung-expressed vascular endothelial growth factor D. The five double-elite enhancer/promoters pairs that are known to be operational, and shared read-through lncRNA transcripts, imply that ongoing SARS-CoV-2 infection will reduce host defences to reactive oxygen species, directly generate superoxide O2·- and H2O2 (a ' ROS storm'), and impair pulmonary endothelial homeostasis. Published cellular responses to oxidative stress complete the loop to pathophysiology observed in severe COVID-19. Thus, for patients who fail to rapidly suppress viral replication, the newly appreciated ACE2 co-regulated gene cluster predicts delayed responses that would account for catastrophic deteriorations. We conclude that ACE2 homeostatic drives provide a unified understanding that should help optimize therapeutic approaches during the wait until safe, effective vaccines and antiviral therapies for SARS-CoV-2 are delivered.

The effect of a 18 bp deletion/insertion variant of VEGF gene on the FMF development.

Objective: Familial Mediterranean fever (FMF) is one of the most common inherited autoinflammatory diseases. Angiogenesis is a feature of inflammatory activation and part of pathogenic processes in autoimmune diseases. Therefore, this study aimed to investigate the role of the Vascular endothelial growth factor (VEGF) gene insertion/deletion (I/D) functional variant in FMF Turkish patients. Methods: MEFV gene mutations were detected in all patients. The FMF patients (N:105) and the healthy controls (N:100) were genotyped for the VEGF I/D variant using PCR followed by agarose gel electrophoresis. The results were statistically analyzed by calculating the odds ratios (OR) and their 95% confidence intervals (95% CI) using the χ2-tests. Results: The mean age of patients was 25.46 ± 10.09. Fifty-nine patients (56.2%) had two or more MEFV gene mutations. The most common MEFV mutation was M694V/M694V. The VEGF I/D variant genotype distribution exhibited a statistically significant difference between the patients and the controls. VEGF I/D genotype was higher in controls compared to patients, while D/D genotype was higher in patients compared to the controls (p = 0.003, p = 0.013, respectively). When we examined the clinical findings, joint pain was more common in patients with VEGF D/D and I/D genotypes compared to I/I genotype (p = 0.043). Although not statistically significant, the most common genotype in patients with two or more MEFV mutations was VEGF D/D (28.6%). Conclusion: The results provided evidence supporting that the D/D genotype of the VEGF I/D variant is associated with an increased risk of FMF in a group of Turkish populations.

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.

Gene mutations in sporadic lymphangioleiomyomatosis and genotype-phenotype correlation analysis.

BACKGROUND: Sporadic lymphangioleiomyomatosis (S-LAM) is a rare neoplasm with heterogeneous clinical features that is conventionally considered to be related to TSC2. This study serves to elucidate the mutation landscape and potential correlation between S-LAM genomic profiles and clinical phenotypes. METHODS: Genomic profiles of 22 S-LAM patients were obtained by sequencing genomic DNA and cell-free DNA from various specimens using an NGS (next-generation sequencing)-based tumor-driver gene panel. Detected mutations were summarized. Symptoms, serum vascular endothelial growth factor D (VEGF-D) values, pulmonary function, and six-minute walk distance (6MWD) were compared among groups with different TSC2 status and genotypes to analyze genotype-phenotype correlations. RESULTS: 67 Variants in 43 genes were detected, with a TSC2 mutation detection rate of 68.2%. The TSC2 detection rate was similar in specimens obtained either through transbronchial lung biopsy (TBLB) or surgical lung biopsy (70.0% vs. 69.2%, p > 0.05). A novel mutation in VEZF1 (c.A659G) was detected in four participants and may represent a mild disease state. TSC2 mutation was significantly related to a shorter 6MWD (p < 0.05), and a higher percentage of VEGF-D over 800 pg/mL (p < 0.05); stop-gain mutation was significantly related to a higher prevalence of pneumothorax. CONCLUSIONS: Tumor-driver mutations in genes other than TSC2 may have a role in S-LAM, and TBLB specimens are practical alternatives for genomic analysis. TSC2 mutation detectability and types are related to the disease severity and phenotypes of S-LAM.

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.

Intramyocardial adenoviral vascular endothelial growth factor-D∆N∆C gene therapy does not induce ventricular arrhythmias.

BACKGROUND: The phase I KAT301 trial investigated the use of intramyocardial adenoviral vascular endothelial growth factor-DΔNΔC (AdVEGF-D) gene therapy (GT) to alleviate symptoms in refractory angina (RA) patients. In KAT301, 30 patients with RA were randomized to AdVEGF-D or the control group in 4:1 ratio. The treatment was found to be feasible, increasing myocardial perfusion and reducing angina symptoms at 1-year follow-up. However, there is some evidence suggesting that the intramyocardial delivery route and overexpression of (vascular endothelial growth) VEGFs might induce ventricular arrhythmias. Thus, we investigated whether intramyocardial AdVEGF-D GT increases the risk of ventricular arrhythmias in patients treated for RA. METHODS: We analyzed non-invasive risk predictors of ventricular arrhythmias from 12-lead electrocardiography (ECG) as well as heart rate variability (HRV) and the incidence of arrhythmias from 24 h ambulatory ECG at baseline and 3 and 12 months after the GT. In addition, we analyzed the incidence of new-onset arrhythmias and pacemaker implantations during 8.2 years (range 6.3-10.4 years) of follow-up. RESULTS: We found no significant increase in arrhythmias, including supraventricular and ventricular ectopic beats, atrial fibrillation, non-sustained ventricular tachycardias, and life-threatening tachycardias, nor changes in the non-invasive risk predictors of ventricular arrhythmias in the AdVEGF-D treated patients. Instead, we found a significant improvement in the very low and high-frequency bands of HRV suggestive of improved cardiac autonomic regulation after GT. CONCLUSIONS: In conclusion, our results suggest that AdVEGF-D GT does not predispose to arrhythmias and might improve HRV metrics.

LncRNA CRART16/miR-122-5p/FOS axis promotes angiogenesis of gastric cancer by upregulating VEGFD expression.

BACKGROUND: We previously identified a novel lncRNA, CRART16, that could induce cetuximab resistance in colorectal cancer cells. This study explored the relationship of CRART16 expression to gastric cancer progression and the molecular mechanisms involved. METHODS: We evaluated CRART16 expression in gastric cancer tissues and adjacent normal tissues from the TCGA database and our hospital. Besides, we assessed its relationship with the overall survival (OS) of patients with gastric cancer. The effects of CRART16 on gastric cancer angiogenesis were determined by endothelial tube formation assay, spheroid sprouting assay, HUVEC invasion assay, and chick embryo chorioallantoic membrane (CAM) assay. The involvement of the lncRNA CRART16/miR-122-5p/FOS axis was analyzed by western blotting and dual-luciferase reporter assay. The functions of CRART16 were confirmed in xenograft mouse models. RESULTS: We found that CRART16 was substantially overexpressed in gastric cancer tissues compared with normal tissues, based on the TCGA database and our clinical samples. High expression of CRART16 correlated with more advanced tumor stages and poor prognosis. Overexpression of CRART16 in gastric cancer cells promoted proliferation, colony formation, angiogenesis, and bevacizumab resistance in vitro, and it promoted tumor growth and angiogenesis in vivo, and vice versa. CRART16 was found to downregulate miR-122-5p by acting as a sponge, upregulating the target oncogene FOS. Afterward, the increased FOS expression led to the upregulation of VEGFD. CONCLUSION: Our findings demonstrate that CRART16 promotes angiogenesis in vitro and in vivo, and CRART16 is a prognostic marker and therapeutic target in gastric cancer.

Plasma Protein Biomarkers in Advanced or Metastatic Colorectal Cancer Patients Receiving Chemotherapy With Bevacizumab or Cetuximab: Results from CALGB 80405 (Alliance).

PURPOSE: CALGB 80405 compared the combination of first-line chemotherapy with cetuximab or bevacizumab in the treatment of advanced or metastatic colorectal cancer (mCRC). Although similar clinical outcomes were observed in the cetuximab-chemotherapy group and the bevacizumab-chemotherapy group, biomarkers could identify patients deriving more benefit from either biologic agent. PATIENTS AND METHODS: In this exploratory analysis, the Angiome, a panel of 24 soluble protein biomarkers were measured in baseline plasma samples in CALGB 80405. Prognostic biomarkers were determined using univariate Cox proportional hazards models. Predictive biomarkers were identified using multivariable Cox regression models including interaction between biomarker level and treatment. RESULTS: In the total population, high plasma levels of Ang-2, CD73, HGF, ICAM-1, IL6, OPN, TIMP-1, TSP-2, VCAM-1, and VEGF-R3 were identified as prognostic of worse progression-free survival (PFS) and overall survival (OS). PlGF was identified as predictive of lack of PFS benefit from bevacizumab [bevacizumab HR, 1.51; 95% confidence interval (CI), 1.10-2.06; cetuximab HR, 0.94; 95% CI, 0.71-1.25; Pinteraction = 0.0298] in the combined FOLFIRI/FOLFOX regimens. High levels of VEGF-D were predictive of lack of PFS benefit from bevacizumab in patients receiving FOLFOX regimen only (FOLFOX/bevacizumab HR, 1.70; 95% CI, 1.19-2.42; FOLFOX/cetuximab HR, 0.92; 95% CI, 0.68-1.24; Pinteraction = 0.0097). CONCLUSIONS: In this exploratory, hypothesis-generating analysis, the Angiome identified multiple prognostic biomarkers and two potential predictive biomarkers for patients with mCRC enrolled in CALGB 80405. PlGF and VEGF-D predicted lack of benefit from bevacizumab in a chemo-dependent manner. See related commentaries by Mishkin and Kohn, p. 2722 and George and Bertagnolli, p. 2725.

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.

Long-term safety and efficacy of intramyocardial adenovirus-mediated VEGF-DΔNΔC gene therapy eight-year follow-up of phase I KAT301 study.

In phase I KAT301 trial, intramyocardial adenovirus-mediated vascular endothelial growth factor -DΔNΔC (AdVEGF-D) gene therapy (GT) resulted in a significant improvement in myocardial perfusion reserve and relieved symptoms in refractory angina patients at 1-year follow-up without major safety concerns. We investigated the long-term safety and efficacy of AdVEGF-D GT. 30 patients (24 in VEGF-D group and 6 blinded, randomized controls) were followed for 8.2 years (range 6.3-10.4 years). Patients were interviewed for the current severity of symptoms (Canadian Cardiovascular Society class, CCS) and perceived benefit from GT. Medical records were reviewed to assess the incidence of major cardiovascular adverse event (MACE) and other predefined safety endpoints. MACE occurred in 15 patients in VEGF-D group and in five patients in control group (21.5 vs. 24.9 per 100 patient-years; hazard ratio 0.97; 95% confidence interval 0.36-2.63; P = 0.95). Mortality and new-onset comorbidity were similar between the groups. Angina symptoms (CCS) were less severe compared to baseline in VEGF-D group (1.9 vs. 2.9; P = 0.006) but not in control group (2.2 vs. 2.6; P = 0.414). Our study indicates that intramyocardial AdVEGF-D GT is safe in the long-term. In addition, the relief of symptoms remained significant during the follow-up.

VEGF Family Gene Expression as Prognostic Biomarkers for Alzheimer's Disease and Primary Liver Cancer.

BACKGROUND: To analyze the expression of vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and cognitive impairment, explore the relationship between the expression of VEGF family genes and prognosis of patients with HCC, and evaluate the predictive ability of VEGF in cognitive impairment using computerized methods. METHODS: VEGF expression in liver cancer tissues and normal tissues was analyzed using bioinformatics methods. The Kaplan-Meier survival analysis method was also used to analyze the relationship between VEGF expression and the prognosis of patients with HCC. Furthermore, immune infiltration assessment and gene set enrichment analysis were performed. Meanwhile, the differential expression of VEGF family genes between patients with Alzheimer's disease (AD) and healthy controls was also checked. RESULTS: Based on The Cancer Genome Atlas (TCGA) database, the VEGF family genes (VEFGA, VEGFB, VEGFC, and VEGFD) were highly expressed in cancer tissues and were significantly associated with poor prognosis in HCC. In HCC, the VEGF family genes showed significant heterogeneity in their functional and immune infiltration characteristics. Finally, VEGF family genes were identified as prognostic biomarkers in AD and risk prediction markers in HCC. CONCLUSIONS: VEGF is highly expressed in patients with HCC and lowly expressed in patients with AD. VEGF has opposite opposing roles in the treatment of tumors and cognitive impairment.

Expanded renal lymphatics improve recovery following kidney injury.

Acute kidney injury (AKI) is a major cause of patient mortality and a major risk multiplier for the progression to chronic kidney disease (CKD). The mechanism of the AKI to CKD transition is complex but is likely mediated by the extent and length of the inflammatory response following the initial injury. Lymphatic vessels help to maintain tissue homeostasis through fluid, macromolecule, and immune modulation. Increased lymphatic growth, or lymphangiogenesis, often occurs during inflammation and plays a role in acute and chronic disease processes. What roles renal lymphatics and lymphangiogenesis play in AKI recovery and CKD progression remains largely unknown. To determine if the increased lymphatic density is protective in the response to kidney injury, we utilized a transgenic mouse model with inducible, kidney-specific overexpression of the lymphangiogenic protein vascular endothelial growth factor-D to expand renal lymphatics. "KidVD" mouse kidneys were injured using inducible podocyte apoptosis and proteinuria (POD-ATTAC) or bilateral ischemia reperfusion. In the acute injury phase of both models, KidVD mice demonstrated a similar loss of function measured by serum creatinine and glomerular filtration rate compared to their littermates. While the initial inflammatory response was similar, KidVD mice demonstrated a shift toward more CD4+ and fewer CD8+ T cells in the kidney. Reduced collagen deposition and improved functional recovery over time was also identified in KidVD mice. In KidVD-POD-ATTAC mice, an increased number of podocytes were counted at 28 days post-injury. These data demonstrate that increased lymphatic density prior to injury alters the injury recovery response and affords protection from CKD progression.

Novel COL4A1-VEGFD gene fusion in myofibroma.

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

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.

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.

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.

Lymphangioleiomyomatosis Association with Underlying Genotype in Patients with Tuberous Sclerosis Complex.

Rationale: Lymphangioleiomyomatosis (LAM) is a female-predominant lung disease caused by mutations in the tuberous sclerosis complex (TSC) genes TSC1 and TSC2.Objectives: To examine the association between TSC mutation subtypes and the prevalence of LAM in women with TSC.Methods: Adult women seen at the Cincinnati Children's Hospital Medical Center's TSC clinic were stratified into the following three groups: those with TSC1 mutation, those with TSC2 mutation, and those with no mutation identified (NMI). Individual TSC manifestations were ascertained by blinded review of chest computed tomographic scans (LAM, multifocal micronodular pneumocyte hyperplasia, and sclerotic bone lesions) and chart review (all other manifestations). The association between mutation status and TSC manifestations was assessed by the Wilcoxon rank-sum test.Results: Our cohort consisted of 55 TSC women, including 30/55 (55%) with TSC2, 12/55 (22%) with TSC1, and 13/55 (23%) with NMI. Twenty-three women (42%) had characteristic cysts consistent with LAM, of whom 16 had TSC2 mutations and seven had NMI. The prevalence of LAM was higher in women with TSC2 mutations compared with women with TSC1 mutations (16/29 [55%] vs. 0/12; P = 0.003). Similarly, renal angiomyolipomas were more common in women with TSC2 mutations compared with women with TSC1 mutations (29/30 [97%] vs. 6/12 [50%]; P = 0.01). There was no association between TSC mutation subtype and the presence of multifocal micronodular pneumocyte hyperplasia, sclerotic bone lesions, and skin or brain involvement. Serum VEGF-D (vascular endothelial growth factor-D) concentrations (median [95% confidence interval]) tended to be higher in patients harboring TSC2 mutations compared with patients with TSC1 mutations (725 pg/ml [612-1,317] vs. 331 pg/ml [284-406]; P = 0.03) and in patients with LAM compared with patients without LAM (725 pg/ml [563-1,609] vs. 429 pg/ml [357-773]; P = 0.02).Conclusions: LAM and angiomyolipomas are more common in women with TSC harboring TSC2 mutations compared with women with TSC1 mutations. Serum VEGF-D is a useful biomarker to suggest the presence of LAM in women with TSC.

SIRT2 modulates VEGFD-associated lymphangiogenesis by deacetylating EPAS1 in human head and neck cancer.

Sirtuin 2 (SIRT2) is one of seven mammalian homologs of silent information regulator 2 (Sir2) and an NAD+ -dependent deacetylase; however, its critical role in lymphangiogenesis remains to be explored. We investigate SIRT2 mediated regulation of vascular endothelial growth factor D (VEGFD) expression and lymphangiogenesis by deacetylating endothelial PAS domain protein 1 (EPAS1) in head and neck cancer (HNC) in vitro and in vivo. In this study, we report that SIRT2, rather than other members of the Sir2 family, reduces the expression of VEGFD and lymphangiogenesis in hypoxia-induced HNC cells and transplanted HNC mice models by reducing EPAS1 acetylation at Lys674 and decreasing the transcriptional activity of EPAS1 target genes. The expression of SIRT2 was closely related to the expression of VEGFD, lymphangiogenesis in subcutaneously transplanted mice models, and lymphangiogenesis in patients with HNC. Our results suggest that SIRT2 plays a central role in tumor lymphangiogenesis via deacetylating EPAS1 protein. Reagents targeting the NAD+ -dependent deacetylase activity of SIRT2 would be beneficial for inhibiting tumor lymphangiogenesis and treating other hypoxia-related diseases.