VEGFR3 tyrosine kinase inhibition aggravates cisplatin nephrotoxicity.

Expansion of renal lymphatic networks, or lymphangiogenesis (LA), is well recognized during development and is now being implicated in kidney diseases. Although LA is associated with multiple pathological conditions, very little is known about its role in acute kidney injury. The purpose of this study was to evaluate the role of LA in a model of cisplatin-induced nephrotoxicity. LA is predominately regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D, ligands that exert their function through their cognate receptor VEGF receptor 3 (VEGFR3). We demonstrated that use of MAZ51, a selective VEGFR3 inhibitor, caused significantly worse structural and functional kidney damage in cisplatin nephrotoxicity. Apoptotic cell death and inflammation were also increased in MAZ51-treated animals compared with vehicle-treated animals following cisplatin administration. Notably, MAZ51 caused significant upregulation of intrarenal phospho-NF-κB, phospho-JNK, and IL-6. Cisplatin nephrotoxicity is associated with vascular congestion due to endothelial dysfunction. Using three-dimensional tissue cytometry, a novel approach to explore lymphatics in the kidney, we detected significant vascular autofluorescence attributed to erythrocytes in cisplatin alone-treated animals. Interestingly, no such congestion was detected in MAZ51-treated animals. We found increased renal vascular damage in MAZ51-treated animals, whereby MAZ51 caused a modest decrease in the endothelial markers endomucin and von Willebrand factor, with a modest increase in VEGFR2. Our findings identify a protective role for de novo LA in cisplatin nephrotoxicity and provide a rationale for the development of therapeutic approaches targeting LA. Our study also suggests off-target effects of MAZ51 on the vasculature in the setting of cisplatin nephrotoxicity.NEW & NOTEWORTHY Little is known about injury-associated LA in the kidney and its role in the pathophysiology of acute kidney injury (AKI). Observed exacerbation of cisplatin-induced AKI after LA inhibition was accompanied by increased medullary damage and cell death in the kidney. LA inhibition also upregulated compensatory expression of LA regulatory proteins, including JNK and NF-κB. These data support the premise that LA is induced during AKI and lymphatic expansion is a protective mechanism in cisplatin nephrotoxicity.

Discovery, Synthesis, and Evaluation of Highly Selective Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) Inhibitor for the Potential Treatment of Metastatic Triple-Negative Breast Cancer.

We herein report the identification, structural optimization, and structure-activity relationship of thieno[2,3-d]pyrimidine derivatives as a novel kind of selective vascular endothelial growth factor receptor 3 (VEGFR3) inhibitors. N-(4-Chloro-3-(trifluoromethyl)phenyl)-4-(6-(4-(4-methylpiperazin-1-yl)phenyl)thieno[2,3-d]pyrimidin-4-yl)piperazine-1-carboxamide (38k) was the most potent VEGFR3 inhibitor (IC50 = 110.4 nM) among developed compounds. Compared with VEGFR1 and VEGFR2, VEGFR3 was approximately 100 times more selective. Here, compound 38k significantly inhibited proliferation and migration of VEGF-C-induced human dermal lymphatic endothelial cells (HDLEC), MDA-MB-231, and MDA-MB-436 cells by inactivating the VEGFR3 signaling pathway. Additionally, 38k induced cell apoptosis and a prolonged G1/S-phase in MDA-MB-231 and MDA-MB-436 cells. It also presented acceptable pharmacokinetic characteristics in Sprague-Dawley (SD) rats with an oral bioavailability of 30.9%. In the xenograft model in vivo, 38k effectively inhibited breast cancer growth by suppressing the VEGFR3 signaling pathway. 38k pronouncedly resisted the formation of pulmonary metastatic nodules in mice. Collectively, 38k may be a promising therapeutic agent of metastatic breast cancer.

Presence of Donor Lymph Nodes Within Vascularized Composite Allotransplantation Ameliorates VEGF-C-mediated Lymphangiogenesis and Delays the Onset of Acute Rejection.

BACKGROUND: The lymphatic system plays an active role in modulating inflammation in autoimmune diseases and organ rejection. In this work, we hypothesized that the transfer of donor lymph node (LN) might be used to promote lymphangiogenesis and influence rejection in vascularized composite allotransplantation (VCA). METHODS: Hindlimb transplantations were performed in which (1) recipient rats received VCA containing donor LN (D:LN+), (2) recipient rats received VCA depleted of all donor LN (D:LN-), and (3) D:LN+ transplantations were followed by lymphangiogenesis inhibition using a vascular endothelial growth factor receptor-3 (VEGFR3) blocker. RESULTS: Our data show that graft rejection started significantly later in D:LN+ transplanted rats as compared to the D:LN- group. Moreover, we observed a higher level of VEGF-C and a quicker and more efficient lymphangiogenesis in the D:LN+ group as compared to the D:LN- group. The presence of donor LN within the graft was associated with reduced immunoactivation in the draining LN and increased frequency of circulating and skin-resident donor T regulatory cells. Blocking of the VEGF-C pathway using a VEGFR3 blocker disrupts the lymphangiogenesis process, accelerates rejection onset, and interferes with donor T-cell migration. CONCLUSIONS: This study demonstrates that VCA LNs play a pivotal role in the regulation of graft rejection and underlines the potential of specifically targeting the LN component of a VCA to control graft rejection.

Tumor-derived exosomal BCYRN1 activates WNT5A/VEGF-C/VEGFR3 feedforward loop to drive lymphatic metastasis of bladder cancer.

BACKGROUND: Patients with lymph node (LN) metastatic bladder cancer (BCa) present with extremely poor prognosis. BCa-derived exosomes function as crucial bioactive cargo carriers to mediate the signal transduction in tumor microenvironment triggering tumor metastasis. However, the mechanisms underlying exosome-mediated LN metastasis in BCa are unclear. METHODS: We conducted the high-throughput sequencing to explore the expression profile of long noncoding RNA (lncRNA) in urinary exosomes (urinary-EXO) from patients with BCa and further evaluated the clinical relevance of exosomal lncRNA BCYRN1 in a larger 210-case cohort. The functional role of exosomal BCYRN1 was evaluated through the migration and tube formation assays in vitro and the footpad-popliteal LN metastasis model in vivo. RNA pull-down assays, luciferase assays, and actinomycin assays were conducted to detect the regulatory mechanism of exosomal BCYRN1. RESULTS: LncRNA BCYRN1 was substantially upregulated in urinary-EXO from patients with BCa, and associated with the LN metastasis of BCa. We demonstrated that exosomal BCYRN1 markedly promoted tube formation and migration of human lymphatic endothelial cells (HLECs) in vitro and lymphangiogenesis and LN metastasis of BCa in vivo. Mechanistically, BCYRN1 epigenetically upregulated WNT5A expression by inducing hnRNPA1-associated H3K4 trimethylation in WNT5A promoter, which activated Wnt/ß-catenin signaling to facilitate the secretion of VEGF-C in BCa. Moreover, exosomal BCYRN1 was transmitted to HLECs to stabilize the VEGFR3 mRNA and thus formed an hnRNPA1/WNT5A/VEGFR3 feedforward regulatory loop, ultimately promoting the lymphatic metastasis of BCa. Importantly, blocking VEGFR3 with specific inhibitor, SAR131675 significantly impaired exosomal BCYRN1-induced the LN metastasis in vivo. Clinically, exosomal BCYRN1 was positively associated with the shorter survival of BCa patients and identified as a poor prognostic factor of patients. CONCLUSION: Our results uncover a novel mechanism by which exosomal BCYRN1 synergistically enhances VEGF-C/VEGFR3 signaling-induced lymphatic metastasis of BCa, indicating that BCYRN1 may serve as an encouraging therapeutic target for patients with BCa.

Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro.

The contribution of many neuronal kinases to the adaptation of nerve cells to ischemic damage and their effect on functional neural network activity has not yet been studied. The aim of this work is to study the role of the four kinases belonging to different metabolic cascades (SRC, Ikkb, eEF2K, and FLT4) in the adaptive potential of the neuron-glial network for modeling the key factors of ischemic damage. We carried out a comprehensive study on the effects of kinases blockade on the viability and network functional calcium activity of nerve cells under ischemic factor modeling in vitro. Ischemic factor modelling was performed on day 14 of culturing primary hippocampal cells obtained from mouse embryos (E18). The most significant neuroprotective effect was shown in the blockade of FLT4 kinase in the simulation of hypoxia. The studies performed revealed the role of FLT4 in the development of functional dysfunction in cerebrovascular accidents and created new opportunities for the study of this enzyme and its blockers in the formation of new therapeutic strategies.

Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling.

Objective: Lymphatic metastasis is one of the leading causes of malignancy dispersion in various types of cancer. However, few anti-lymphangiogenic drugs have been approved for clinical use to date. Therefore, new therapies to block lymphangiogenesis are urgently required. Methods: Immunohistochemistry, immunofluorescence, Western blot, migration assays, and lymphangiogenesis and lymphatic metastasis assays were used. Results: Anlotinib, a receptor tyrosine kinase inhibitor, suppressed the rate of new metastatic lesions (31.82% in the placebo arm and 18.18% in the anlotinib arm) in patients with advanced lung adenocarcinoma who were enrolled in our ALTER-0303 study. D2-40+-lymphatic vessel density was strongly correlated with disease stage, metastasis, and poor prognosis in 144 Chinese patients with lung adenocarcinoma. In mice bearing A549EGFP tumors, tumor lymphatic vessel density, tumor cell migration to lymph nodes, and the number of distant metastatic lesions were lower in the anlotinib group than in the controls. Anlotinib inhibited the growth and migration of human lymphatic endothelial cells (hLECs) and lymphangiogenesis in vitro and in vivo. Treatment of hLECs with anlotinib downregulated phosphorylated vascular endothelial growth factor receptor 3 (VEGFR-3). Conclusions: Anlotinib inhibits lymphangiogenesis and lymphatic metastasis, probably through inactivating VEGFR-3 phosphorylation. The results indicate that anlotinib may be beneficial for treatment in avoiding lymphangiogenesis and distant lymphatic metastasis in lung adenocarcinoma. (Trial registration: ALTER0303; NCT02388919; March 17, 2015.).

Lymphoma Angiogenesis Is Orchestrated by Noncanonical Signaling Pathways.

Tumor-induced remodeling of the microenvironment relies on the formation of blood vessels, which go beyond the regulation of metabolism, shaping a maladapted survival niche for tumor cells. In high-grade B-cell lymphoma, angiogenesis correlates with poor prognosis, but attempts to target established proangiogenic pathways within the vascular niche have been inefficient. Here, we analyzed Myc-driven B-cell lymphoma-induced angiogenesis in mice. A few lymphoma cells were sufficient to activate the angiogenic switch in lymph nodes. A unique morphology of dense microvessels emerged without obvious tip cell guidance and reliance on blood endothelial cell (BEC) proliferation. The transcriptional response of BECs was inflammation independent. Conventional HIF1α or Notch signaling routes prevalent in solid tumors were not activated. Instead, a nonconventional hypersprouting morphology was orchestrated by lymphoma-provided VEGFC and lymphotoxin (LT). Interference with VEGF receptor-3 and LTß receptor signaling pathways abrogated lymphoma angiogenesis, thus revealing targets to block lymphomagenesis. SIGNIFICANCE: In lymphoma, transcriptomes and morphogenic patterns of the vasculature are distinct from processes in inflammation and solid tumors. Instead, LTßR and VEGFR3 signaling gain leading roles and are targets for lymphomagenesis blockade.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/80/6/1316/F1.large.jpg.

Brain-to-cervical lymph node signaling after stroke.

After stroke, peripheral immune cells are activated and these systemic responses may amplify brain damage, but how the injured brain sends out signals to trigger systemic inflammation remains unclear. Here we show that a brain-to-cervical lymph node (CLN) pathway is involved. In rats subjected to focal cerebral ischemia, lymphatic endothelial cells proliferate and macrophages are rapidly activated in CLNs within 24 h, in part via VEGF-C/VEGFR3 signalling. Microarray analyses of isolated lymphatic endothelium from CLNs of ischemic mice confirm the activation of transmembrane tyrosine kinase pathways. Blockade of VEGFR3 reduces lymphatic endothelial activation, decreases pro-inflammatory macrophages, and reduces brain infarction. In vitro, VEGF-C/VEGFR3 signalling in lymphatic endothelial cells enhances inflammatory responses in co-cultured macrophages. Lastly, surgical removal of CLNs in mice significantly reduces infarction after focal cerebral ischemia. These findings suggest that modulating the brain-to-CLN pathway may offer therapeutic opportunities to ameliorate systemic inflammation and brain injury after stroke.

Isolation of a Novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library.

Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.

Identification of a Unique Resorcylic Acid Lactone Derivative That Targets Both Lymphangiogenesis and Angiogenesis.

We synthesized 11 novel L-783277 derivatives, in which a structure rigidifying phenyl ring is incorporated into the 14-membered chiral resorcylic acid lactone system. The SAR study with these substances demonstrated that 17 possesses excellent kinase selectivity against a panel of 335 kinases in contrast to L-783277 and inhibits VEGFR3, VEGFR2, and FLT3 with single-digit nanomolar IC50 values. Also, we found that 21, a stereoisomer of 17, has excellent potency (IC50 = 9 nM) against VEGFR3 and selectivity over VEGFR2 and FLT3. 17, a potent dual VEGFR3 and VEGFR2 inhibitor, effectively suppresses both lymphangiogenesis and angiogenesis in a 3D-microfluidic tumor lymphangiogenesis assay and in vivo corneal assay while SAR131675 blocks only lymphangiogenesis. In addition, 17 blocks the endothelial tube formation and suppresses proliferation of PHE tumor vascular model. 17 will be a valuable templatefor developing therapeutically active and selective substances that target both lymphangiogenesis and angiogenesis.

Blockade of Lymphangiogenesis Shapes Tumor-Promoting Adipose Tissue Inflammation.

Tumor-associated lymphangiogenesis correlates with lymph node metastasis and poor outcome in several human malignancies. In addition, the presence of functional lymphatic vessels regulates the formation of tumor inflammatory and immune microenvironments. Although lymphatic structures are often found deeply integrated into the fabric of adipose tissue, the impact of lymphangiogenesis on tumor-associated adipose tissue (AT) has not yet been investigated. Using K14-VEGFR3-Ig mice that constitutively express soluble vascular endothelial growth factor receptor (VEGFR) 3-Ig in the skin, scavenging VEGF-C and VEGF-D, the role of lymphangiogenesis in the generation of an inflammatory response within tumor-associated AT was studied. Macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 were found within peritumoral adipose tissue from melanoma-bearing K14-VEGFR3-Ig mice, which were further enriched with alternatively activated macrophages based on surface marker CD301/C-type lectin domain family 10 member A expression. The blockade of lymphangiogenesis also resulted in accumulation of the cytokine IL-6, which correlated with enhanced macrophage proliferation of the alternatively activated phenotype. Furthermore, melanomas co-implanted with freshly isolated adipose tissue macrophages grew more robustly than melanomas growing alone. In human cutaneous melanomas, adipocyte-selective FABP4 transcripts closely correlated with gene signatures of CLEC10A and were associated with poor overall survival. These data suggest that the blockade of pathways regulating lymphatic vessel formation shapes an inflammatory response within tumor-associated AT by facilitating accumulation of tumor-promoting alternatively activated macrophages.

Two Birds, One Stone: Double Hits on Tumor Growth and Lymphangiogenesis by Targeting Vascular Endothelial Growth Factor Receptor 3.

Vascular endothelial growth factor receptor 3 (VEGFR3) has been known for its involvement in tumor-associated lymphangiogenesis and lymphatic metastasis. The VEGFR3 signaling is stimulated by its main cognate ligand, vascular endothelial growth factor C (VEGF-C), which in turn promotes tumor progression. Activation of VEGF-C/VEGFR3 signaling in lymphatic endothelial cells (LECs) was shown to enhance the proliferation of LECs and the formation of lymphatic vessels, leading to increased lymphatic metastasis of tumor cells. In the past decade, the expression and pathological roles of VEGFR3 in tumor cells have been described. Moreover, the VEGF-C/VEGFR3 axis has been implicated in regulating immune tolerance and suppression. Therefore, the inhibition of the VEGF-C/VEGFR3 axis has emerged as an important therapeutic strategy for the treatment of cancer. In this review, we discuss the current findings related to VEGF-C/VEGFR3 signaling in cancer progression and recent advances in the development of therapeutic drugs targeting VEGF-C/VEGFR3.

Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice.

Recent studies have demonstrated that chronic inflammation-induced lymphangiogenesis plays a crucial role in the progression of various renal diseases, including diabetic nephropathy. SAR131675 is a selective vascular endothelial cell growth factor receptor-3 (VEGFR-3)-tyrosine kinase inhibitor that acts as a ligand for VEGF-C and VEGF-D to inhibit lymphangiogenesis. In this study, we evaluated the effect of SAR131675 on renal lymphangiogenesis in a mouse model of type 2 diabetes. Male C57BLKS/J db/m and db/db mice were fed either a regular chow diet or a diet containing SAR131675 for 12 weeks from 8 weeks of age. In addition, we studied palmitate-induced lymphangiogenesis in human kidney-2 (HK2) cells and RAW264.7 monocytes/macrophages, which play a major role in lymphangiogenesis in the kidneys. SAR131475 ameliorated dyslipidemia, albuminuria, and lipid accumulation in the kidneys of db/db mice, with no significant changes in glucose and creatinine levels and body weight. Diabetes-induced systemic inflammation as evidenced by increased systemic monocyte chemoattractant protein-1 and tumor necrosis factor-α level was decreased by SAR131475. SAR131475 ameliorated the accumulation of triglycerides and free fatty acids and reduced inflammation in relation to decreased chemokine expression and pro-inflammatory M1 macrophage infiltration in the kidneys. Downregulation of VEGF-C and VEGFR-3 by SAR131475 inhibited lymphatic growth as demonstrated by decreased expression of LYVE-1 and podoplanin that was further accompanied by reduced tubulointerstitial fibrosis, and inflammation in relation to improvement in oxidative stress and apoptosis. Treatment with SAR131475 improved palmitate-induced increase in the expression of VEGF-C, VEGFR-3, and LYVE-1, along with improvement in cytosolic and mitochondrial oxidative stress in RAW264.7 and HK2 cells. Moreover, the enhanced expression of M1 phenotypes in RAW264.7 cells under palmitate stress was reduced by SAR131475 treatment. The results suggest that modulation of lymphatic proliferation in the kidneys is a new treatment approach for type 2 diabetic nephropathy and that SAR131675 is a promising therapy to ameliorate renal damage by reducing lipotoxicity-induced lymphangiogenesis.

Attenuated Joint Tissue Damage Associated With Improved Synovial Lymphatic Function Following Treatment With Bortezomib in a Mouse Model of Experimental Posttraumatic Osteoarthritis.

OBJECTIVE: To investigate the roles of the synovial lymphatic system in the severity and progression of joint tissue damage and functional responses of synovial lymphatic endothelial cells (LECs) to macrophage subsets, and to evaluate the therapeutic potential of the proteasome inhibitor bortezomib (BTZ) in a mouse model of experimental posttraumatic osteoarthritis (OA). METHODS: C57BL/6J wild-type mice received a meniscal ligamentous injury to induce posttraumatic knee OA. Lymphangiogenesis was blocked by a vascular endothelial growth factor receptor 3 (VEGFR-3) neutralizing antibody. Synovial lymphatic drainage was examined by near-infrared imaging. Joint damage was assessed by histology. RNA-sequencing and pathway analyses were applied to synovial LECs. Macrophage subsets in the mouse synovium were identified by flow cytometry and immunofluorescence staining. M1 and M2 macrophages were induced from mouse bone marrow cells, and their effects on LECs were examined in cocultures in the presence or absence of BTZ. The effects of BTZ on joint damage, LEC inflammation, and synovial lymphatic drainage were examined. RESULTS: Injection of a VEGFR-3 neutralizing antibody into the joints of mice with posttraumatic knee OA reduced synovial lymphatic drainage and accelerated joint tissue damage. Synovial LECs from the mouse OA joints had dysregulated inflammatory pathways and expressed high levels of inflammatory genes. The number of M1 macrophages was increased in the knee joints of mice with posttraumatic OA, thereby promoting the expression of inflammatory genes by LECs; this effect was blocked by BTZ. Treatment with BTZ decreased cartilage loss, reduced the expression of inflammatory genes by LECs, and improved lymphatic drainage in the knee joints of mice with posttraumatic OA. CONCLUSION: Experimental posttraumatic knee OA is associated with decreased synovial lymphatic drainage, increased numbers of M1 macrophages, and enhanced inflammatory gene expression by LECs, all of which was improved by treatment with BTZ. Intraarticular administration of BTZ may represent a new therapy for the restoration of synovial lymphatic function in subjects with posttraumatic knee OA.

Docetaxel facilitates lymphatic-tumor crosstalk to promote lymphangiogenesis and cancer progression.

BACKGROUND: Infiltration into lymphatic vessels is a critical step in breast cancer metastasis. Lymphatics undergo changes that facilitate metastasis as a result of activation of the cells lining lymphatic vessels, lymphatic endothelial cells (LECs). Inhibition of activation by targeting VEGFR3 can reduce invasion toward lymphatics. To best benefit patients, this approach should be coupled with standard of care that slows tumor growth, such as chemotherapy. Little is known about how chemotherapies, like docetaxel, may influence lymphatics and conversely, how lymphatics can alter responses to therapy. METHODS: A novel 3D in vitro co-culture model of the human breast tumor microenvironment was employed to examine the contribution of LECs to tumor invasion and viability with docetaxel and anti-VEGFR3, using three cell lines, MDA-MB-231, HCC38, and HCC1806. In vivo, the 4T1 mouse model of breast carcinoma was used to examine the efficacy of combinatorial therapy with docetaxel and anti-VEGFR3 on lymph node metastasis and tumor growth. Lymphangiogenesis in these mice was analyzed by immunohistochemistry and flow cytometry. Luminex analysis was used to measure expression of lymphangiogenic cytokines. RESULTS: In vitro, tumor cell invasion significantly increased with docetaxel when LECs were present; this effect was attenuated by inhibition of VEGFR3. LECs reduced docetaxel-induced cell death independent of VEGFR3. In vivo, docetaxel significantly increased breast cancer metastasis to the lymph node. Docetaxel and anti-VEGFR3 combination therapy reduced lymph node and lung metastasis in 4T1 and synergized to reduce tumor growth. Docetaxel induced VEGFR3-dependent vessel enlargement, lymphangiogenesis, and expansion of the LEC population in the peritumoral microenvironment, but not tumor-free stroma. Docetaxel caused an upregulation in pro-lymphangiogenic factors including VEGFC and TNF-α in the tumor microenvironment in vivo. CONCLUSIONS: Here we present a counter-therapeutic effect of docetaxel chemotherapy that triggers cancer cells to elicit lymphangiogenesis. In turn, lymphatics reduce cancer response to docetaxel by altering the cytokine milieu in breast cancer. These changes lead to an increase in tumor cell invasion and survival under docetaxel treatment, ultimately reducing docetaxel efficacy. These docetaxel-induced effects can be mitigated by anti-VEGFR3 therapy, resulting in a synergism between these treatments that reduces tumor growth and metastasis.

Endothelial cells in the innate response to allergens and initiation of atopic asthma.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

VEGFC/VEGFR3 Signaling Regulates Mouse Spermatogonial Cell Proliferation via the Activation of AKT/MAPK and Cyclin D1 Pathway and Mediates the Apoptosis by affecting Caspase 3/9 and Bcl-2.

We have previously shown that the transcript levels of Vegfc and its receptor Vegfr3 were high in spermatogonia and extremely low in spermatocytes and spermatids. However, it remains unknown about the functions and the mechanisms of VEGFC/VEGFR3 signaling in regulating the fate determinations of spermatogonia. To this end, here we explored the role and signaling pathways of VEGFC/VEGFR3 by using a cell line derived from immortalized mouse spermatogonia retaining markers of mitotic germ cells, namely GC-1 cells. VEGFR3 was expressed in mouse primary spermatogonia and GC-1 cells. VEGFC stimulated the proliferation and DNA synthesis of GC-1 cells and enhanced the phosphorylation of PI3K-AKT and MAPK, whereas LY294002 (an inhibitor for AKT) and CI-1040 (an inhibitor for MAPK) blocked the effect of VEGFC on GC-1 cell proliferation. Furthermore, VEGFC increased the transcripts of c-fos and Egr1 and protein levels of cyclin D1, PCNA and Bcl-2. Conversely, the blocking of VEGFC/VEGFR3 signaling by VEGFR3 knockdown reduced the phosphorylation of AKT/MAPK and decreased the levels of cyclin D1 and PCNA. Additionally, VEGFR3 knockdown not only resulted in more apoptosis of GC-1 cells but also led to a decrease of Bcl-2 and promoted the cleavage of Caspase-3/9 and PARP. Collectively, these data suggested that VEGFC/VEGFR3 signaling promotes the proliferation of GC-1 cells via the AKT /MAPK and cyclin D1 pathway and it inhibits the cell apoptosis through Caspase-3/9, PARP and Bcl-2. Thus, this study sheds a novel insight to the molecular mechanisms underlying the fate decisions of mammalian spermatogonia.

VEGFR3 inhibition chemosensitizes lung adenocarcinoma A549 cells in the tumor-associated macrophage microenvironment through upregulation of p53 and PTEN.

In lung adenocarcinoma, loss of p53 and PTEN in tumors are associated with decreased response to chemotherapy and decreased survival. A means to pharmacologically upregulate p53 and PTEN protein expression could improve the prognosis of patients with p53- and PTEN-deficient tumors. In the present study we revealed that vascular endothelial growth factor receptor 3 (VEGFR3) inhibition in lung adenocarcinoma cells was associated with improved expression levels of both p53 and PTEN in the tumor-associated macrophage (TAM) microenvironment. Inhibition of VEGFR3 in lung adenocarcinoma cells was associated with growth arrest and decreased migration and invasion. The upregulation of p53 and PTEN protein expression after VEGFR3 inhibition decreased chemotherapy resistance and improved chemosensitivity in co-cultured A549 cells in which p53 and PTEN expression were decreased. Finally, we demonstrated that TAMs promoted the expression of VEGF-C and its receptor VEGFR3. Western blot analysis revealed the co-cultured A549 cells with TAMs are a primary source of VEGF-C and VEGFR3 in the tumor microenvironment. Our studies revealed that VEGFR3 inhibition may be a pharmacological means to upregulate p53 and PTEN protein expression and improve the outcome of patients with p53- and PTEN-deficient tumors.

Novel methyl indolinone-6-carboxylates containing an indole moiety as angiokinase inhibitors.

A novel series of methyl indolinone-6-carboxylates bearing an indole moiety were identified as potent angiokinase inhibitors. The most active compound, A8, potently targeted the kinase activities of vascular endothelial growth factor receptors 2 and 3, and platelet-derived growth factor receptors α and ß, with IC50 values in the nanomolar range. In addition, A8 effectively suppressed the proliferation of human umbilical vein endothelial cells, and HT-29 and MCF-7 cancer cells, by inducing apoptosis. Compound A8 is thus a promising candidate for further investigation.

Targeting VEGFR-3/-2 signaling pathways with AD0157: a potential strategy against tumor-associated lymphangiogenesis and lymphatic metastases.

BACKGROUND: Lymphatic metastasis is one of the leading causes of death in patients with different types of cancer and is the main prognostic factor for the disease survival. The formation of new lymphatic vessels (lymphangiogenesis) in primary tumors facilitates tumor cell dissemination to regional lymph nodes and correlates with distant metastases. Lymphangiogenesis has thus emerged as a suitable therapeutic target to block metastases, but no anti-lymphangiogenic compounds have been approved for clinical use to date. Therefore, new or improved therapies blocking lymphatic metastases are urgently required. METHODS: We established murine breast tumors to assess the effect of AD0157 on tumor growth, lymphangiogenesis, and lymphatic dissemination. Then, a battery of in vivo (mouse corneal neovascularization and ear sponges), ex vivo (mouse lymphatic rings and rat mesentery explants), and in vitro (proliferation, tubulogenesis, wound-healing, Boyden chambers, and spheroids) assays was used to give insight into the lymphangiogenic steps affected by AD0157. Finally, we investigated the molecular pathways controlled by this drug. RESULTS: AD0157 was found to inhibit the growth of human breast cancer xenografts in mice, to strongly reduce tumor-associated lymphangiogenesis and to block metastatic dissemination to both lymph nodes and distant organs. The high anti-lymphangiogenic potency of AD0157 was further supported by its inhibitory activity at low micromolar range in two in vivo pathological models and in two ex vivo assays. In addition, AD0157 inhibited lymphatic endothelial cell proliferation, migration and invasion, cellular sprouting, and tube formation. Mechanistically, this compound induced apoptosis in lymphatic endothelial cells and decreased VEGFR-3/-2, ERK1/2, and Akt phosphorylations. CONCLUSIONS: These findings demonstrate the suitability of AD0157 to suppress tumor-associated lymphangiogenesis. Beyond discovering a new potent anti-lymphangiogenic drug that is worth considering in future clinical settings, our study supports the interest of designing anti-lymphangiogenic therapies to avoid distant metastatic processes.