DsbA-L ameliorates renal aging and renal fibrosis by maintaining mitochondrial homeostasis.

Renal fibrosis is the final pathological change in renal disease, and aging is closely related to renal fibrosis. Mitochondrial dysfunction has been reported to play an important role in aging, but the exact mechanism remains unclear. Disulfide-bond A oxidoreductase-like protein (DsbA-L) is mainly located in mitochondria and plays an important role in regulating mitochondrial function and endoplasmic reticulum (ER) stress. However, the role of DsbA-L in renal aging has not been reported. In this study, we showed a reduction in DsbA-L expression, the disruption of mitochondrial function and an increase in fibrosis in the kidneys of 12- and 24-month-old mice compared to young mice. Furthermore, the deterioration of mitochondrial dysfunction and fibrosis were observed in DsbA-L-/- mice with D-gal-induced accelerated aging. Transcriptome analysis revealed a decrease in Flt4 expression and inhibition of the PI3K-AKT signaling pathway in DsbA-L-/- mice compared to control mice. Accelerated renal aging could be alleviated by an AKT agonist (SC79) or a mitochondrial protector (MitoQ) in mice with D-gal-induced aging. In vitro, overexpression of DsbA-L in HK-2 cells restored the expression of Flt4, AKT pathway factors, SP1 and PGC-1α and alleviated mitochondrial damage and cell senescence. These beneficial effects were partially blocked by inhibiting Flt4. Finally, activating the AKT pathway or improving mitochondrial function with chemical reagents could alleviate cell senescence. Our results indicate that the DsbA-L/AKT/PGC-1α signaling pathway could be a therapeutic target for age-related renal fibrosis and is associated with mitochondrial dysfunction.

Therapeutic potential of FLT4-targeting peptide in acute myeloid leukemia.

Previously, we found that dysfunctional natural killer (NK) cells with low interferon gamma (IFN-γ) were restored in acute myeloid leukemia (AML) by the FLT4 antagonist MAZ51. Here, we developed 12 peptides targeting FLT4 for clinical application and examined whether they restored the frequency of lymphocytes, especially T cells and NK cells, and high IFN-γ expression, as MAZ51 treatment did in our previous study. Although clinical data from using peptides are currently available, peptides targeting FLT4 to modulate immune cells have not been fully elucidated. In this study, we focus on novel peptide 4 (P4) from the intracellular domain of FLT4 because it had dominant negative activity. Similar to MAZ51, high IFN-γ levels were expressed in AML-mononuclear cells exposed to P4. Additionally, T and NK cell levels were restored, as were high IFN-γ levels, in a leukemic environment when P4 was treated. Interestingly, the regulatory T cells were significantly decreased by P4, implying the role of peptide in tumor niche. Overall, we demonstrated the therapeutic value of functionally modulating lymphocytes using a peptide targeting FLT4 and proposed the development of advanced therapeutic approaches against AML by using immune cells.

Visualization of Organ-Specific Lymphatic Growth: An Efficient Approach to Labeling Molecular Markers in Cleared Tissues.

Organ-specific lymphatics are essential for the maintenance of healthy organ function and lymphatic dysfunction can lead to the development of various diseases. However, the precise role of those lymphatic structures remains unknown, mainly due to inefficient visualization techniques. Here, we present an efficient approach to visualizing organ-specific lymphatic growth. We used a modified CUBIC protocol to clear mouse organs and combined it with whole-mount immunostaining to visualize lymphatic structures. We acquired images using upright, stereo and confocal microscopy and quantified them with AngioTool, a tool for the quantification of vascular networks. Using our approach, we then characterized the organ-specific lymphatic vasculature of the Flt4kd/+ mouse model, showing symptoms of lymphatic dysfunction. Our approach enabled us to visualize the lymphatic vasculature of organs and to analyze and quantify structural changes. We detected morphologically altered lymphatic vessels in all investigated organs of Flt4kd/+ mice, including the lungs, small intestine, heart and uterus, but no lymphatic structures in the skin. Quantifications showed that these mice have fewer and dilated lymphatic vessels in the small intestine and the lungs. Our results demonstrate that our approach can be used to investigate the importance of organ-specific lymphatics under both physiological and pathophysiological conditions.

Vegfd modulates both angiogenesis and lymphangiogenesis during zebrafish embryonic development.

Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, giving rise to veins and lymphatics. Vegfd has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that, during primary angiogenesis, Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context-specific indispensable and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis.

Analysis and Differential Expression of Primo Genes Using RNA-Seq and qRT-PCR Experiments.

Although the existence of the primo vasculature system has been shown in many species, including mice, rats, rabbits and humans, the biological role of this system, including expression of genes and proteins, has not yet been investigated. Especially the transcriptional action by mRNA, which is required for biological action, needs to be studied in primo vasculature biology. Differentially expressed genes in both isolated primo vessels and lymphatic vessels of rabbits were analyzed by RNA sequencing experiments. Primer efficiency and RNA purity of the primo vessels under lipopolysaccharides were confirmed prior to performing real-time qRT-PCR analysis following RNA extraction. We demonstrated that FLT4 was enriched in primo vessels and that several genes, including HSPH1 and EPHB2, were highly expressed in primo vessels compared with lymphatic vessels. Our data show that almost all genes, except HSPA4, were increased or sustained in isolated primo vessels compared with lymphatic vessels (FLT4 2.58 fold, HSPH1 1.83 fold, EPHB2 1.52 fold; whereas HSPA4 decreased 0.50 fold), suggesting primo vessels as a central regulator in diverse physiology. This implies that FLT4, HSPH1, and EPHB2 in high amounts may be involved in the functional activity of primo vessels. Our experimental data show that several genes are highly enriched in primo vessels in the lymphatic vessels of the rabbit.

ETS transcription factor Etsrp / Etv2 is required for lymphangiogenesis and directly regulates vegfr3 / flt4 expression.

The molecular mechanisms initiating the formation of the lymphatic system, lymphangiogenesis, are still poorly understood. Here we have identified a novel role in lymphangiogenesis for an ETS transcription factor, Etv2/Etsrp, a known regulator of embryonic vascular development. Through the use of fully validated photoactivatable morpholinos we show that inducible Etv2 inhibition in zebrafish embryos at 1 day post-fertilization (dpf) results in significant inhibition of lymphangiogenesis, while development of blood vessels is unaffected. In Etv2-inhibited embryos and larvae, the number of lymphatic progenitors is greatly reduced, the major lymphatic vessel, the thoracic duct, is absent or severely fragmented, and lymphangiogenesis-associated marker expression, including lyve1b, prox1a, and vegfr3/flt4, is strongly downregulated. We also demonstrate that lymphatic progenitors in Etv2 deficient embryos fail to respond to Vegfc signaling. Chromatin immunoprecipitation and sequencing (ChIP-Seq) studies using differentiated mouse embryonic stem (ES) cells as well as luciferase reporter studies in the ES cells and in zebrafish embryos argue that Etv2 directly binds the promoter/enhancer regions of Vegfc receptor Vegfr3/Flt4 and lymphatic marker Lyve1, and promotes their expression. Together these data support a model where Etv2 initiates lymphangiogenesis by directly promoting the expression of flt4 within the posterior cardinal vein.

Vegfc acts through ERK to induce sprouting and differentiation of trunk lymphatic progenitors.

Vascular endothelial growth factor C (Vegfc) activates its receptor, Flt4, to induce lymphatic development. However, the signals that act downstream of Flt4 in this context in vivo remain unclear. To understand Flt4 signaling better, we generated zebrafish bearing a deletion in the Flt4 cytoplasmic domain that eliminates tyrosines Y1226 and 1227. Embryos bearing this deletion failed to initiate sprouting or differentiation of trunk lymphatic vessels and did not form a thoracic duct. Deletion of Y1226/7 prevented ERK phosphorylation in lymphatic progenitors, and ERK inhibition blocked trunk lymphatic sprouting and differentiation. Conversely, endothelial autonomous ERK activation rescued lymphatic sprouting and differentiation in flt4 mutants. Interestingly, embryos bearing the Y1226/7 deletion formed a functional facial lymphatic network enabling them to develop normally to adulthood. By contrast, flt4 null larvae displayed hypoplastic facial lymphatics and severe lymphedema. Thus, facial lymphatic vessels appear to be the first functional lymphatic network in the zebrafish, whereas the thoracic duct is initially dispensable for lymphatic function. Moreover, distinct signaling pathways downstream of Flt4 govern lymphatic morphogenesis and differentiation in different anatomical locations.

Haploinsufficiency of vascular endothelial growth factor related signaling genes is associated with tetralogy of Fallot.

PURPOSE: To determine disease-associated single-gene variants in conotruncal defects, particularly tetralogy of Fallot (TOF). METHODS: We analyzed for rare loss-of-function and deleterious variants in FLT4 (VEGFR3) and other genes in the vascular endothelial growth factor (VEGF) pathway, as part of a genome sequencing study involving 175 adults with TOF from a single site. RESULTS: We identified nine (5.1%) probands with novel FLT4 variants: seven loss-of-function, including an 8-kb deletion, and two predicted damaging. In ten other probands we found likely disruptive variants in VEGF-related genes: KDR (VEGFR2; two stopgain and two nonsynonymous variants), VEGFA, FGD5, BCAR1, IQGAP1, FOXO1, and PRDM1. Detection of VEGF-related variants (19/175, 10.9%) was associated with an increased prevalence of absent pulmonary valve (26.3% vs. 3.4%, p < 0.0001) and right aortic arch (52.6% vs. 29.1%, p = 0.029). Extracardiac anomalies were rare. In an attempt to replicate findings, we identified three loss-of-function or damaging variants in FLT4, KDR, and IQGAP1 in ten independent families with TOF. CONCLUSION: Loss-of-function variants in FLT4 and KDR contribute substantially to the genetic basis of TOF. The findings support dysregulated VEGF signaling as a novel mechanism contributing to the pathogenesis of TOF.

Rare genetic variants potentially involved in ovarian hyperstimulation syndrome.

PURPOSE: We aim to investigate whether there is a genetic predisposition in women who developed ovarian hyperstimulation syndrome (OHSS) after GnRH antagonist protocol with GnRH agonist trigger and freeze-all approach. METHODS: Four patients with OHSS after GnRH agonist trigger and freeze-all approach were gathered from the worldwide patient population. These patients were analyzed through Whole Exome Sequencing. In this study known causes of OHSS were investigated and new causes present in at least two individuals were searched for. RESULTS: In the first part of the study, we evaluated the presence of mutations in genes already known to be involved in OHSS. In PGR and TP53, heterozygous alterations were detected. PGR is predicted to be involved in progesterone resistance with a recessive inheritance pattern and is, therefore, not considered as being causal. The consequences of the variant detected in TP53 currently remain unknown. In part 2 of the study, we assessed the clinical significance of variants in genes previously not linked to OHSS. We especially focused on genes with variants present in ≥ 2 patients. Two patients have variants in the FLT4 gene. Mutations in this gene are linked to hereditary lymphedema, but no link to OHSS has been described. CONCLUSIONS: Defining a genetic predisposition for OHSS is essential in view of prevention. In this study, a potential link between the FLT4 gene and OHSS has been suggested. Future functional studies are essential to define a more precise involvement of the detected variants in the development of OHSS.

Systemic Sclerosis Serum Significantly Impairs the Multi-Step Lymphangiogenic Process: in Vitro Evidence.

In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.

Modulation of Endothelial Bone Morphogenetic Protein Receptor Type 2 Activity by Vascular Endothelial Growth Factor Receptor 3 in Pulmonary Arterial Hypertension.

BACKGROUND: Bone morphogenetic protein (BMP) signaling has multiple roles in the development and function of the blood vessels. In humans, mutations in BMP receptor type 2 (BMPR2), a key component of BMP signaling, have been identified in the majority of patients with familial pulmonary arterial hypertension (PAH). However, only a small subset of individuals with BMPR2 mutation develops PAH, suggesting that additional modifiers of BMPR2 function play an important role in the onset and progression of PAH. METHODS: We used a combination of studies in zebrafish embryos and genetically engineered mice lacking endothelial expression of Vegfr3 to determine the interaction between vascular endothelial growth factor receptor 3 (VEGFR3) and BMPR2. Additional in vitro studies were performed by using human endothelial cells, including primary lung endothelial cells from subjects with PAH. RESULTS: Attenuation of Vegfr3 in zebrafish embryos abrogated Bmp2b-induced ectopic angiogenesis. Endothelial cells with disrupted VEGFR3 expression failed to respond to exogenous BMP stimulation. Mechanistically, VEGFR3 is physically associated with BMPR2 and facilitates ligand-induced endocytosis of BMPR2 to promote phosphorylation of SMADs and transcription of ID genes. Conditional, endothelial-specific deletion of Vegfr3 in mice resulted in impaired BMP signaling responses, and significantly worsened hypoxia-induced pulmonary hypertension. Consistent with these data, we found significant decrease in VEGFR3 expression in pulmonary arterial endothelial cells from human PAH subjects, and reconstitution of VEGFR3 expression in PAH pulmonary arterial endothelial cells restored BMP signaling responses. CONCLUSIONS: Our findings identify VEGFR3 as a key regulator of endothelial BMPR2 signaling and a potential determinant of PAH penetrance in humans.

A Novel Splice-Site Mutation in VEGFC Is Associated with Congenital Primary Lymphoedema of Gordon.

Lymphedema is characterized by chronic swelling of any body part caused by malfunctioning or obstruction in the lymphatic system. Primary lymphedema is often considered genetic in origin. VEGFC, which is a gene encoding the ligand for the vascular endothelial growth factor receptor 3 (VEGFR3/FLT4) and important for lymph vessel development during lymphangiogenesis, has been associated with a specific subtype of primary lymphedema. Through Sanger sequencing of a proband with bilateral congenital pedal edema resembling Milroy disease, we identified a novel mutation (NM_005429.2; c.361+5G>A) in VEGFC. The mutation induced skipping of exon 2 of VEGFC resulting in a frameshift and the introduction of a premature stop codon (p.Ala50ValfsTer18). The mutation leads to a loss of the entire VEGF-homology domain and the C-terminus. Expression of this Vegfc variant in the zebrafish floorplate showed that the splice-site variant significantly reduces the biological activity of the protein. Our findings confirm that the splice-site variant, c.361+5G>A, causes the primary lymphedema phenotype in the proband. We examine the mutations and clinical phenotypes of the previously reported cases to review the current knowledge in this area.

Evaluation of epigenetic inactivation of vascular endothelial growth factor receptors in head and neck squamous cell carcinoma.

The aim of this study was to determine the methylation status of the genes encoding the vascular endothelial growth factor receptors and to evaluate the usefulness of VEGFR methylation as a prognostic indicator in head and neck squamous cell carcinoma. VEGFR messenger RNA expression and promoter methylation were examined in a panel of cell lines via quantitative reverse transcription and methylation-specific polymerase chain reaction, respectively. Promoter methylation was compared with clinical characteristics in 128 head and neck squamous cell carcinoma samples. The normalized methylation values for the VEGFR1, VEGFR2 and VEGFR3 promoters tended to be higher in the tumour cell lines than in normal tonsil samples, whereas amounts of VEGFR1, VEGFR2 and VEGFR3 messenger RNA were significantly higher. Methylation of the VEGFR1 promoter (p = 0.003; 66/128 head and neck squamous cell carcinoma samples, 52%) and VEGFR3 promoter (p = 0.043; 53/128 head and neck squamous cell carcinoma samples, 41%) significantly correlated with recurrence, whereas methylation of the VEGFR2 promoter significantly correlated with lymph node metastasis (p = 0.046; 47/128 head and neck squamous cell carcinoma samples, 37%). Concurrent methylation of the VEGFR1 and VEGFR3 promoters significantly correlated with reduced disease-free survival (log-rank test, p = 0.009). In a multivariate logistic regression analysis, methylation of the VEGFR1, VEGFR3 and both the VEGFR1 and VEGFR3 promoters independently predicted recurrence (odds ratios and 95% confidence intervals: 3.19, 1.51-6.75 (p = 0.002); 2.24, 1.06-4.76 (p = 0.035); and 2.56, 1.09-6.05 (p = 0.032), respectively). Methylation of the VEGFR promoters predicts poor prognosis in head and neck squamous cell carcinoma patients.

Blockade of FLT4 suppresses metastasis of melanoma cells by impaired lymphatic vessels.

The metastatic spread of tumor cells via lymphatic vessels affects the relapse of tumor patients. New lymphatic vessel formation, including lymphangiogenesis, is promoted in the tumor environment. The lymphangiogenic factor VEGF-C can mediate lymphatic vessel formation and induce tumor metastasis by binding with FLT4. In melanoma, metastasis via lymphatics such as lymph nodes is one of the main predictors of poor outcome. Thus, we investigated whether blockade of FLT4 can reduce metastasis via the suppression of lymphatic capillaries. Proliferative lymphatic capillaries in melanoma were estimated by immunohistochemistry using FLT4 antibody after the injection of the FLT4 antagonist MAZ51. The numbers of tumor modules in metastasised lungs were calculated by gross examination and lymphatic related factors were examined by qRT-PCR. MAZ51 injection resulted in the suppression of tumor size and module number and the inhibition of proliferative lymphatic vessels in the intratumoral region in the lung and proliferating melanoma cells in the lung compared to those of untreated groups. Additionally, high FLT4 and TNF-alpha were detected in melanoma-induced tissue, while lymphatic markers such as VEGF-C, FLT4 and Prox-1 were significantly decreased in MAZ51 treated groups, implying that anti-lymphangiogenesis by MAZ51 may provide a potential strategy to prevent tumor metastasis in melanoma and high number of lymphatic capillaries could be used diagnosis for severe metastasis.

Inhibition of FAK and VEGFR-3 binding decreases tumorigenicity in neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for over 15% of pediatric cancer deaths. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. Vascular endothelial growth factor receptor-3 (VEGFR-3), another tyrosine kinase, has also been found to be important in the development of many human tumors including neuroblastoma. Recent reports have found that FAK and VEGFR-3 interact, and we have previously shown that both of these kinases interact in neuroblastoma. We have hypothesized that interruption of the FAK-VEGFR-3 interaction would lead to decreased neuroblastoma cell survival. In the current study, we examined the effects of a small molecule, chloropyramine hydrochloride (C4), designed to disrupt the FAK-VEGFR-3 interaction, upon cellular attachment, migration, and survival in two human neuroblastoma cell lines. We also utilized a murine xenograft model to study the impact of C4 upon tumor growth. In these studies, we showed that disruption of the FAK-VEGFR-3 interaction led to decreased cellular attachment, migration, and survival in vitro. In addition, treatment of murine xenografts with chloropyramine hydrochloride decreased neuroblastoma xenograft growth. Further, this molecule acted synergistically with standard chemotherapy to further decrease neuroblastoma xenograft growth. The findings from this current study help to further our understanding of the regulation of neuroblastoma tumorigenesis, and may provide novel therapeutic strategies and targets for neuroblastoma and other solid tumors of childhood.

Inhibition of the focal adhesion kinase and vascular endothelial growth factor receptor-3 interaction leads to decreased survival in human neuroblastoma cell lines.

Neuroblastoma continues to be a devastating childhood solid tumor and is responsible for over 15% of all childhood cancer-related deaths. Focal adhesion kinase (FAK) and vascular endothelial growth factor receptor-3 (VEGFR-3) are protein tyrosine kinases that are overexpressed in a number of human cancers, including neuroblastoma. These two kinases can directly interact and provide survival signals to cancer cells. In this study, we utilized siRNA to VEGFR-3 to demonstrate the biologic importance of this kinase in neuroblastoma cell survival. We also used confocal microscopy and immunoprecipitation to show that FAK and VEGFR-3 bind in neuroblastoma. Finally, employing a 12-amino-acid peptide (AV3) specific to VEGFR-3, we showed that the colocalization between FAK and VEGFR-3 could be disrupted, and that disruption resulted in decreased neuroblastoma cell survival. These studies provide insight to the FAK-VEGFR-3 interaction in neuroblastoma and demonstrate its importance in this tumor type. Focusing upon the FAK-VEGFR-3 interaction may provide a novel therapeutic target for the development of new strategies for treatment of neuroblastoma.

Association study of FLT4 and HYDIN single nucleotide polymorphisms with atrial septal defect susceptibility in the Han Chinese population of Southwest China.

BACKGROUND: Atrial septal defect (ASD) is a common form of congenital heart disease. Although several genes related to ASD have been found, the genetic factors of ASD remain unclear. This study aimed to evaluate the correlation between 10 candidate single nucleotide polymorphisms (SNPs) and sporadic atrial septal defects. METHODS: Based on the results of 34 individual whole exome sequences, 10 candidate SNPs were selected. In total, 489 ASD samples and 420 normal samples were collected. The 10 SNPs in the case group and the control group were identified through Snapshot genotyping technology. The χ2-test and unconditional regression model were used to evaluate the relationship between ASD and each candidate SNP. Haploview software was used to perform linkage disequilibrium and haplotype analysis. RESULTS: The χ2 results showed that the FLT4 rs383985 (P = 0.003, OR = 1.115-1.773), HYDIN rs7198975 (P = 0.04621, OR = 1.003-1.461), and HYDIN rs1774266 (P = 0.04621, OR = 1.003-1.461) alleles were significantly different between the control group and the case group (P < 0.05). Only the association with the FLT4 polymorphism was statistically significant after adjustment for multiple comparisons. CONCLUSION: These findings suggest that a possible molecular pathogenesis associated with sporadic ASD is worth exploring in future studies.

Dauricine Inhibits Non-small Cell Lung Cancer Development by Regulating PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2 Pathways in a FLT4-dependent Manner.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is still a solid tumor with high malignancy and poor prognosis. Vascular endothelial growth factor receptor 3 (FLT4, VEGFR3) is overexpressed in NSCLC cells, making it a potential target for NSCLC treatment. In this study, we aimed to explore the anti-cancer effects of dauricine on NSCLC cells and its mechanism targeting FLT4. METHODS: We found that dauricine inhibited the growth of NCI-H1299 cells by blocking the cycle in the G2/M phase through flow cytometry analysis. In addition， dauricine also inhibited the migration of NCI-H1299 cells by wound healing assay and transwell migration assay. More importantly, our empirical analysis found the anti-cancer effect of dauricine on NCI-H1299 cells and the protein level of FLT4 had a distinctly positive correlation, and this effect was weakened after FLT4 knockdown. RESULTS: It is suggested that dauricine suppressed the growth and migration of NCI-H1299 cells by targeting FLT4. Furthermore, dauricine inhibited FLT4 downstream pathways, such as PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2, thereby regulating cell migration-related molecule MMP3 and cell cycle-related molecules (CDK1, pCDK1-T161, and cyclin B1). CONCLUSION: Dauricine may be a promising FLT4 inhibitor for the treatment of NSCLC.

FLT4 causes developmental disorders of the cardiovascular and lymphovascular systems via pleiotropic molecular mechanisms.

AIMS: Rare, deleterious genetic variants in FLT4 are associated with Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD). Distinct genetic variants in FLT4 are also an established cause of Milroy disease, the most prevalent form of primary hereditary lymphoedema. Phenotypic features of these two conditions are non-overlapping, implying pleiotropic cellular mechanisms during development. METHODS AND RESULTS: Here, we show that FLT4 variants identified in TOF patients, when expressed in primary human endothelial cells, cause aggregation of FLT4 protein in the perinuclear endoplasmic reticulum, activating proteostatic and metabolic signalling, whereas lymphoedema-associated FLT4 variants and wildtype FLT4 do not. FLT4 TOF variants display characteristic gene expression profiles in key developmental signalling pathways, revealing a role for FLT4 in cardiogenesis distinct from its role in lymphatic development. Inhibition of proteostatic signalling abrogates these effects, identifying potential avenues for therapeutic intervention. Depletion of flt4 in zebrafish caused cardiac phenotypes of reduced heart size and altered heart looping. These phenotypes were rescued with coinjection of wildtype human FLT4 mRNA, but incompletely or not at all by mRNA harbouring FLT4 TOF variants. CONCLUSIONS: Taken together, we identify a pathogenic mechanism for FLT4 variants predisposing to TOF that is distinct from the known dominant negative mechanism of Milroy-causative variants. FLT4 variants give rise to conditions of the two circulatory subdivisions of the vascular system via distinct developmental pleiotropic molecular mechanisms. TRANSLATIONAL PERSPECTIVE: Proteostatic dysfunction, if confirmed as a mechanism of CHD pathogenesis for other predisposing genes, may identify pathways to therapeutic interventions. Distinguishing mechanistically how variants in FLT4 give rise to CHD may have potential to individualise genetic counselling in affected families.

VEGFR-3 signaling in macrophages: friend or foe in disease?

Lymphatic vessels have been increasingly appreciated in the context of immunology not only as passive conduits for immune and cancer cell transport but also as key in local tissue immunomodulation. Targeting lymphatic vessel growth and potential immune regulation often takes advantage of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling to manipulate lymphatic biology. A receptor tyrosine kinase, VEGFR-3, is highly expressed on lymphatic endothelial cells, and its signaling is key in lymphatic growth, development, and survival and, as a result, often considered to be "lymphatic-specific" in adults. A subset of immune cells, notably of the monocyte-derived lineage, have been identified to express VEGFR-3 in tissues from the lung to the gut and in conditions as varied as cancer and chronic kidney disease. These VEGFR-3+ macrophages are highly chemotactic toward the VEGFR-3 ligands VEGF-C and VEGF-D. VEGFR-3 signaling has also been implicated in dictating the plasticity of these cells from pro-inflammatory to anti-inflammatory phenotypes. Conversely, expression may potentially be transient during monocyte differentiation with unknown effects. Macrophages play critically important and varied roles in the onset and resolution of inflammation, tissue remodeling, and vasculogenesis: targeting lymphatic vessel growth and immunomodulation by manipulating VEGFR-3 signaling may thus impact macrophage biology and their impact on disease pathogenesis. This mini review highlights the studies and pathologies in which VEGFR-3+ macrophages have been specifically identified, as well as the activity and polarization changes that macrophage VEGFR-3 signaling may elicit, and affords some conclusions as to the importance of macrophage VEGFR-3 signaling in disease.