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Abstract Clear-cell renal cell carcinoma (ccRCC) is the most common origin of pancreatic metastases (PM). Distinct genomic aberrations, favorable prognosis, and clinical observations on high angiogenesis, and succeeding tyrosine kinase inhibitor (TKI) sensitivity have been reported in PM-ccRCC. However, no functional or single-cell studies have been conducted thus far. We recruited five PM-ccRCC patients and investigated the genomic, single-cell transcriptomic, and drug sensitivity profiles of their patient-derived cells (PDCs). The PM depicted both expected and novel genomic alterations. Further, the transcriptomics differed from both primary and metastatic ccRCC, with upregulations of the PI3K/mTOR and – supporting the clinical observations – angiogenesis pathways. Data integration at pathway level showed that transcriptomics explained drug sensitivities the best. Accordingly, PM-ccRCC PDCs shared sensitivity to many PI3K/mTOR inhibitors. Altogether, we show distinct genomic and transcriptomic signatures in PM-ccRCC, highlight the superiority of transcriptomics in interpreting drug sensitivities, and encourage the use of TKIs and PI3K/mTOR inhibitors in PM-ccRCC.

3-hydroxy-L-kynurenamine (3-HKA) is a metabolite deriving from a lateral pathway of tryptophan catabolism. Here the authors identify 3-HKA as a biogenic amine and show it has anti-inflammatory properties that can protect mice against psoriasis and nephrotoxic nephritis.

Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.

Understanding the pathological mechanisms unfolding after chronic traumatic brain injury (TBI) could reveal new therapeutic entry points. During the post-TBI sequel, the involvement of cerebrospinal fluid drainage through the meningeal lymphatic vessels was proposed. Here, we used K14-VEGFR3-Ig transgenic mice to analyze whether a developmental dysfunction of meningeal lymphatic vessels modifies post-TBI pathology. To this end, a moderate TBI was delivered by controlled cortical injury over the temporal lobe in male transgenic mice or their littermate controls. We performed MRI and a battery of behavioral tests over time to define the post-TBI trajectories. In vivo analyses were integrated by ex-vivo quantitative and morphometric examinations of the cortical lesion and glial cells.In post-TBI K14-VEGFR3-Ig mice, the recovery from motor deficits was protracted compared to littermates. This outcome is coherent with the observed slower hematoma clearance in transgenic mice during the first two weeks post-TBI. No other genotype-related behavioral differences were observed, and the volume of cortical lesions imaged by MRI in vivo, and confirmed by histology ex-vivo, were comparable in both groups. However, at the cellular level, post-TBI K14-VEGFR3-Ig mice exhibited an increased percentage of activated Iba1 microglia in the hippocampus and auditory cortex, areas that are proximal to the lesion.Although not impacting or modifying the structural brain damage and post-TBI behavior, a pre-existing dysfunction of meningeal lymphatic vessels is associated with morphological microglial activation over time, possibly representing a sub-clinical pathological imprint or a vulnerability factor. Our findings suggest that pre-existing mLV deficits could represent a possible risk factor for the overall outcome of TBI pathology.

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses.

Arterial flow regulates artery diameter but other mechanisms may also affect this. Here, the authors show that the guanine nucleotide exchange factor Trio and GTPases Rac1 and RhoG, triggers F-actin remodeling in arterial endothelial cells, independent of flow, to enhance lumen diameter in zebrafish and cell models.

Intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. Here the authors show that VEGF-C expression in specialized IntSCs is regulated by YAP/TAZ, and VEGF-C is responsible for maintaining lacteal integrity, thus influencing dietary fat drainage into lacteals.

Lymphatic malformation (LM) is a debilitating often incurable vascular disease. Using a mouse model of LM driven by a disease-causative PIK3CA mutation, the authors show that vascular growth is dependent on the upstream lymphangiogenic VEGF-C signalling, permitting effective therapeutic intervention.

The lymphatic vasculature is essential to maintain fluid homeostasis and immune surveillance, including in the brain where lymphatic vessels were only recently identified. Here, Jacob et al. provide an anatomical map of lymphatic vessels in the vertebral column, where they find these contribute to fluid drainage and immune responses.

Abstract Background Angiopoietin growth factors (Angs) regulate angiogenesis and lymphangiogenesis by binding to the endothelial Tie2 receptor. Ang2 expression is elevated in tissue hypoxia and inflammation, which also induce cleavage of the extracellular domain of the orphan Tie1 receptor. Here we have examined if the concentrations of Ang2 and the soluble extracellular domain of Tie1 in patient plasma are associated with the prognosis of patients with metastatic breast cancer. Methods Plasma Tie1 and Ang2 levels were measured in metastatic breast cancer patients treated in a phase II trial with a taxane-bevacizumab combination chemotherapy in the first-line treatment setting. They were analyzed before treatment, after 6 weeks and 6 months of treatment, and at the final study visit. Using the median concentrations as cutoffs, Tie1 and Ang2 data were dichotomized into low and high concentration groups. Additionally, we analyzed Tie1 concentrations in plasma from 10 healthy women participating in a breast cancer primary prevention study. Results Plasma samples were available from 58 (89%) of the 65 patients treated in the trial. The baseline Tie1 levels of the healthy controls were significantly lower than those of the metastatic patients (p

Abstract Background Alzheimer’s disease is characterized by two main neuropathological hallmarks: extracellular plaques of amyloid-β (Aβ) protein and intracellular aggregates of tau protein. Although tau is normally a soluble monomer that bind microtubules, in disease it forms insoluble, hyperphosphorylated aggregates in the cell body. Aside from its role in AD, tau is also involved in several other neurodegenerative disorders collectively called tauopathies, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), some forms of frontotemporal dementia, and argyrophilic grain disease (AGD). The prion hypothesis suggests that after an initial trigger event, misfolded forms of tau are released into the extracellular space, where they spread through different brain regions, enter cells, and seeding previously normal forms. Thus understanding mechanisms regulating the clearance of extracellular tau from the CNS is important. The discovery of a true lymphatic system in the dura and its potential role in mediating Aβ pathology prompted us to investigate its role in regulating extracellular tau clearance. Methods To study clearance of extracellular tau from the brain, we conjugated monomeric human tau with a near-infrared dye cypate, and injected this labeled tau in the parenchyma of both wild-type and K14-VEGFR3-Ig transgenic mice, which lack a functional CNS lymphatic system. Following injection we performed longitudinal imaging using fluorescence molecular tomography (FMT) and quantified fluorescence to calculate clearance of tau from the brain. To complement this, we also measured tau clearance to the periphery by measuring plasma tau in both groups of mice. Results Our results show that a significantly higher amount of tau is retained in the brains of K14-VEGFR3-Ig vs. wild type mice at 48 and 72 h post-injection and its subsequent clearance to the periphery is delayed. We found that clearance of reference tracer human serum albumin (HSA) was also significantly delayed in the K14-VEGFR3-Ig mice. Conclusions The dural lymphatic system appears to play an important role in clearance of extracellular tau, since tau clearance is impaired in the absence of functional lymphatics. Based on our baseline characterization of extracellular tau clearance, future studies are warranted to look at the interaction between tau pathology and efficiency of lymphatic function.

Background: Prosthetic vascular grafts in humans characteristically lack confluent endothelialization regardless of the duration of implantation. Use of high-porosity grafts has been proposed as a way to induce endothelialization through transgraft capillarization, although early experiments failed to show increased healing in man. Objectives: We hypothesized that transduction of tissues around the prosthetic conduit with vectors encoding VEGF receptor-2 (VEGFR2) ligands would augment transinterstitial capillarization and induce luminal endothelialization of high-porosity ePTFE grafts. Methods: Fifty-two NZW rabbits received 87 ePTFE uni- or bilateral end-to-end interposition grafts in carotid arteries. Rabbits were randomized to local therapy with adenoviruses encoding AdVEGF-A165, AdVEGF-A109 or control AdLacZ and analyzed at 6 and 28 days after surgery by contrast-enhanced ultrasound and histology. Results: AdVEGF-A165 and AdVEGF-A109 dramatically increased perfusion in perigraft tissues at 6 days (14.2 ± 3.6 or 16.7 ± 2.6-fold increases, P < 0.05 and P < 0.01). At 28 days, the effect was no longer significantly higher than baseline. At 6 days, no luminal endothelialization was observed in any of the groups. At 28 days, AdVEGF-A109- and AdVEGF-A165-treated animals showed enhanced ingrowth of transinterstitial capillaries (66.0 ± 13.7% and 77.4 ± 15.7% of graft thickness vs 44.7 ± 24.4% in controls, P < 0.05) and improved luminal endothelialization (11.2 ± 26.3% and 11.4 ± 22.2%, AdVEGF-A109 and AdVEGF-A165 vs 0% in controls, P < 0.05). No increased stenosis was observed in the treatment groups as compared to LacZ controls. Conclusions: This study suggests that transient local overexpression of VEGFR2 ligands in the peri-implant tissues at the time of graft implantation is a novel strategy to increase endothelialization of high-porosity ePTFE vascular grafts and improve the patency of small-diameter vascular prostheses.

VEGF-C drives lymphangiogenesis through binding to its receptors VEGFR-2 and VEGFR-3. Here, Durré et al. identify uPARAP/Endo180 as a critical regulator of VEGFR-2/VEGFR-3 heterodimerisation and downstream signaling in response to VEGF-C, and show that uPARAP deletion leads to the formation of hyperbranched vasculatures in pathological conditions.

Abstract The transcription factor PROX1 is essential for development and cell fate specification. Its function in cancer is context-dependent since PROX1 has been shown to play both oncogenic and tumour suppressive roles. Here, we show that PROX1 suppresses the transcription of MMP14, a metalloprotease involved in angiogenesis and cancer invasion, by binding and suppressing the activity of MMP14 promoter. Prox1 deletion in murine dermal lymphatic vessels in vivo and in human LECs increased MMP14 expression. In a hepatocellular carcinoma cell line expressing high endogenous levels of PROX1, its silencing increased both MMP14 expression and MMP14-dependent invasion in 3D. Moreover, PROX1 ectopic expression reduced the MMP14-dependent 3D invasiveness of breast cancer cells and angiogenic sprouting of blood endothelial cells in conjunction with MMP14 suppression. Our study uncovers a new transcriptional regulatory mechanism of cancer cell invasion and endothelial cell specification.

VEGF-C is a key regulator of lymphatic development. Here, Zhang et al. show that while complete loss of its receptor VEGFR3 results in vessel hypoplasia, mosaic loss of VEGFR3 leads to hyperplasia through induction of cell proliferation in neighboringnon-targeted cells, uncovering cell- and non-cell-autonomous roles for VEGFR3 during lymphatic vessel growth.

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2–3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 × 105 PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.

Though coronary arteries are crucial for heart function, the mechanisms guiding their formation are largely unknown. Here, Wang et al. identify a unique, endocardially-derived angiogenic precursor cell population for coronary artery formation in mice and show that a DLL4/NOTCH1/VEGFA/VEGFR2 signaling axis is key for coronary artery development.

Abstract The collagen- and calcium-binding EGF domains 1 (CCBE1) protein is necessary for lymphangiogenesis. Its C-terminal collagen-like domain was shown to be required for the activation of the major lymphangiogenic growth factor VEGF-C (Vascular Endothelial Growth Factor-C) along with the ADAMTS3 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-3) protease. However, it remained unclear how the N-terminal domain of CCBE1 contributed to lymphangiogenic signaling. Here, we show that efficient activation of VEGF-C requires its C-terminal domain both in vitro and in a transgenic mouse model. The N-terminal EGF-like domain of CCBE1 increased VEGFR-3 signaling by colocalizing pro-VEGF-C with its activating protease to the lymphatic endothelial cell surface. When the ADAMTS3 amounts were limited, proteolytic activation of pro-VEGF-C was supported by the N-terminal domain of CCBE1, but not by its C-terminal domain. A single amino acid substitution in ADAMTS3, identified from a lymphedema patient, was associated with abnormal CCBE1 localization. These results show that CCBE1 promotes VEGFR-3 signaling and lymphangiogenesis by different mechanisms, which are mediated independently by the two domains of CCBE1: by enhancing the cleavage activity of ADAMTS3 and by facilitating the colocalization of VEGF-C and ADAMTS3. These new insights should be valuable in developing new strategies to therapeutically target VEGF-C/VEGFR-3-induced lymphangiogenesis.

Abstract Thyroid gland vasculature has a distinguishable characteristic of endothelial fenestrae, a critical component for proper molecular transport. However, the signaling pathway that critically governs the maintenance of thyroid vascular integrity, including endothelial fenestrae, is poorly understood. Here, we found profound and distinct expression of follicular epithelial VEGF‐A and vascular VEGFR2 that were precisely regulated by circulating thyrotropin, while there were no meaningful expression of angiopoietin–Tie2 system in the thyroid gland. Our genetic depletion experiments revealed that VEGFR2, but not VEGFR3, is indispensable for maintenance of thyroid vascular integrity. Notably, blockade of VEGF‐A or VEGFR2 not only abrogated vascular remodeling but also inhibited follicular hypertrophy, which led to the reduction of thyroid weights during goitrogenesis. Importantly, VEGFR2 blockade alone was sufficient to cause a reduction of endothelial fenestrae with decreases in thyrotropin‐responsive genes in goitrogen‐fed thyroids. Collectively, these findings establish follicular VEGF‐A–vascular VEGFR2 axis as a main regulator for thyrotropin‐dependent thyroid angiofollicular remodeling and goitrogenesis.

Vascular endothelial growth factor-C (VEGF-C) acts primarily on endothelial cells, but also on non-vascular targets, for example in the CNS and immune system. Here we describe a novel, unique VEGF-C form in the human reproductive system produced via cleavage by kallikrein-related peptidase 3 (KLK3), aka prostate-specific antigen (PSA). KLK3 activated VEGF-C specifically and efficiently through cleavage at a novel N-terminal site. We detected VEGF-C in seminal plasma, and sperm liquefaction occurred concurrently with VEGF-C activation, which was enhanced by collagen and calcium binding EGF domains 1 (CCBE1). After plasmin and ADAMTS3, KLK3 is the third protease shown to activate VEGF-C. Since differently activated VEGF-Cs are characterized by successively shorter N-terminal helices, we created an even shorter hypothetical form, which showed preferential binding to VEGFR-3. Using mass spectrometric analysis of the isolated VEGF-C-cleaving activity from human saliva, we identified cathepsin D as a protease that can activate VEGF-C as well as VEGF-D.

Skeletal muscle has remarkable adaptive and regenerative capacity. Here the authors show that the transcription factor Prox1 is necessary for maintenance of slow muscle fibre types via activation of NFAT signalling, and for myoblast differentiation via cross-talk with the Notch signalling pathway.

Vascular endothelial growth factor D (VEGF-D) promotes the lymph node metastasis of cancer by inducing the growth of lymphatic vasculature, but its specific roles in tumorigenesis have not been elucidated. We monitored the effects of VEGF-D in cutaneous squamous cell carcinoma (cSCC) by subjecting transgenic mice overexpressing VEGF-D in the skin (K14-mVEGF-D) and VEGF-D knockout mice to a chemical skin carcinogenesis protocol involving 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate treatments. In K14-mVEGF-D mice, tumor lymphangiogenesis was significantly increased and the frequency of lymph node metastasis was elevated in comparison with controls. Most notably, the papillomas regressed more often in K14-mVEGF-D mice than in littermate controls, resulting in a delay in tumor incidence and a remarkable reduction in the total tumor number. Skin tumor growth and metastasis were not obviously affected in the absence of VEGF-D; however, the knockout mice showed a trend for reduced lymphangiogenesis in skin tumors and in the untreated skin. Interestingly, K14-mVEGF-D mice showed an altered immune response in skin tumors. This consisted of the reduced accumulation of macrophages, mast cells, and CD4+ T-cells and an increase of cytotoxic CD8+ T-cells. Cytokine profiling by flow cytometry and quantitative real time PCR revealed that elevated VEGF-D expression results in an attenuated Th2 response and promotes M1/Th1 and Th17 polarization in the early stage of skin carcinogenesis, leading to an anti-tumoral immune environment and the regression of primary tumors. Our data suggest that VEGF-D may be beneficial in early-stage tumors since it suppresses the pro-tumorigenic inflammation, while at later stages VEGF-D-induced tumor lymphatics provide a route for metastasis.

Abstract High‐grade gliomas are aggressive and abundantly vascular tumors, and as in most cancer types, blood vessels in advanced lesions are highly abnormal. Poor perfusion and vascular leakage in tumor tissue resulting in hypoxia, necrosis, and high interstitial fluid pressure can hamper the efficient delivery of chemotherapy. Tumor angiogenesis is known to be supported by host leukocytes recruited to the tumor microenvironment, but the mechanisms leading to dysfunctional vascular network formation are incompletely understood. In this issue of EMBO Molecular Medicine, Mathivet et al (2017) present an elegant study, where longitudinal intravital imaging gives new insight on how recruitment and polarization of tumor‐associated macrophages regulate aberrant angiogenesis in experimental gliomas. They show that macrophage targeting results in vessel normalization and improved chemotherapy response, suggesting that the combination of these therapeutic modalities could improve the outcome of glioma treatment in the clinic.

Patients with Gorham-Stout disease (GSD) have lymphatic vessels in their bones and their bones gradually disappear. Here, we report that mice that overexpress VEGF-C in bone exhibit a phenotype that resembles GSD. To drive VEGF-C expression in bone, we generated Osx-tTA;TetO-Vegfc double-transgenic mice. In contrast to Osx-tTA mice, Osx-tTA;TetO-Vegfc mice developed lymphatics in their bones. We found that inhibition of VEGFR3, but not VEGFR2, prevented the formation of bone lymphatics in Osx-tTA;TetO-Vegfc mice. Radiological and histological analysis revealed that bones from Osx-tTA;TetO-Vegfc mice were more porous and had more osteoclasts than bones from Osx-tTA mice. Importantly, we found that bone loss in Osx-tTA;TetO-Vegfc mice could be attenuated by an osteoclast inhibitor. We also discovered that the mutant phenotype of Osx-tTA;TetO-Vegfc mice could be reversed by inhibiting the expression of VEGF-C. Taken together, our results indicate that expression of VEGF-C in bone is sufficient to induce the pathologic hallmarks of GSD in mice.

Abstract Vascular endothelial growth factor C (VEGF‐C) binding to its tyrosine kinase receptor VEGFR‐3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF‐C/VEGFR‐3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF‐C in adult vasculature are unknown. We show here that VEGF‐C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF‐C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. The Vegfc‐deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high‐fat diet, the Vegfc‐deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism.

Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular endothelial growth factor receptor 3 (VEGFR-3)/p110α PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma.

Neural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60–70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito) and total K+ current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.

Colorectal cancer (CRC) initiation and growth is often attributed to stem cells, yet little is known about the regulation of these cells. We show here that a subpopulation of Prox1-transcription-factor-expressing cells have stem cell activity in intestinal adenomas, but not in the normal intestine. Using in vivo models and 3D ex vivo organoid cultures of mouse adenomas and human CRC, we found that Prox1 deletion reduced the number of stem cells and cell proliferation and decreased intestinal tumor growth via induction of annexin A1 and reduction of the actin-binding protein filamin A, which has been implicated as a prognostic marker in CRC. Loss of Prox1 also decreased autophagy and the survival of hypoxic tumor cells in tumor transplants. Thus, Prox1 is essential for the expansion of the stem cell pool in intestinal adenomas and CRC without being critical for the normal functions of the gut.

Abstract Angiogenic growth factors have recently been linked to tissue metabolism. We have used genetic gain‐ and loss‐of function models to elucidate the effects and mechanisms of action of vascular endothelial growth factor‐B (VEGF‐B) in the heart. A cardiomyocyte‐specific VEGF‐B transgene induced an expanded coronary arterial tree and reprogramming of cardiomyocyte metabolism. This was associated with protection against myocardial infarction and preservation of mitochondrial complex I function upon ischemia‐reperfusion. VEGF‐B increased VEGF signals via VEGF receptor‐2 to activate Erk1/2, which resulted in vascular growth. Akt and mTORC1 pathways were upregulated and AMPK downregulated, readjusting cardiomyocyte metabolic pathways to favor glucose oxidation and macromolecular biosynthesis. However, contrasting with a previous theory, there was no difference in fatty acid uptake by the heart between the VEGF‐B transgenic, gene‐targeted or wildtype rats. Importantly, we also show that VEGF‐B expression is reduced in human heart disease. Our data indicate that VEGF‐B could be used to increase the coronary vasculature and to reprogram myocardial metabolism to improve cardiac function in ischemic heart disease.

Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII.

Abstract The features and regulation of uterine angiogenesis and vascular remodelling during pregnancy are poorly defined. Here we show that dynamic and variable decidual angiogenesis (sprouting, intussusception and networking), and active vigorous vascular remodelling such as enlargement and elongation of ‘vascular sinus folding’ (VSF) and mural cell drop‐out occur distinctly in a spatiotemporal manner in the rapidly growing mouse uterus during early pregnancy — just after implantation but before placentation. Decidual angiogenesis is mainly regulated through VEGF‐A secreted from the progesterone receptor (PR)‐expressing decidual stromal cells which are largely distributed in the anti‐mesometrial region (AMR). In comparison, P4‐PR‐regulated VEGF‐A‐VEGFR2 signalling, ligand‐independent VEGFR3 signalling and uterine natural killer (uNK) cells positively and coordinately regulate enlargement and elongation of VSF. During the postpartum period, Tie2 signalling could be involved in vascular maturation at the endometrium in a ligand‐independent manner, with marked reduction of VEGF‐A, VEGFR2 and PR expressions. Overall, we show that two key vascular growth factor receptors — VEGFR2 and Tie2 — strikingly but differentially regulate decidual angiogenesis and vascular remodelling in rapidly growing and regressing uteri in an organotypic manner.

Abstract Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin‐1 (ANG1)‐mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1‐dependent Phd2 repression initiated a feed‐forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2‐like phenotype, suggesting possible new venues for the treatment of ischaemic disorders. →See accompanying articles http://dx.doi.org/10.1002/emmm.201302752 and http://dx.doi.org/10.1002/emmm.201302794

SUMMARY Tie-2 is a member of the receptor tyrosine kinase family and is required for vascular remodeling and maintenance of mammalian vessel integrity. A number of mutations in the human TIE2 gene have been identified in patients suffering from cutaneomucosal venous malformations and ventricular septal defects. How exactly Tie-2 signaling pathways play different roles in both vascular development and vascular stability is unknown. We have generated a zebrafish line carrying a stop mutation in the kinase domain of the Tie-2 receptor. Mutant embryos lack Tie-2 protein, but do not display any defect in heart and vessel development. Simultaneous loss of Tie-1 and Tie-2, however, leads to a cardiac phenotype. Our study shows that Tie-1 and Tie-2 are not required for early heart development, yet they have redundant roles for the maintenance of endocardial-myocardial connection in later stages. Tie-2 and its ligand Angiopoietin-1 have also been reported to play an important role in vessel stability. We used atorvastatin and simvastatin, drugs that cause bleeding in wild-type zebrafish larvae, to challenge vessel stability in tie-2 mutants. Interestingly, recent clinical studies have reported hemorrhagic stroke as a side effect of atorvastatin treatment. Exposure of embryos to statins revealed that tie-2 mutants are significantly protected from statin-induced bleeding. Furthermore, tie-2 mutants became less resistant to bleeding after VE-cadherin knockdown. Taken together, these data show that atorvastatin affects vessel stability through Tie-2, and that VE-cadherin and Tie-2 act in concert to allow vessel remodeling while playing a role in vessel stability. Our study introduces an additional vertebrate model to study in vivo the function of Tie-2 in development and disease.

Kaposi sarcoma is a tumor consisting of Kaposi sarcoma herpesvirus (KSHV)-infected tumor cells that express endothelial cell (EC) markers and viral genes like v-cyclin, vFLIP, and LANA. Despite a strong link between KSHV infection and certain neoplasms, de novo virus infection of human primary cells does not readily lead to cellular transformation. We have studied the consequences of expression of v-cyclin in primary and immortalized human dermal microvascular ECs. We show that v-cyclin, which is a homolog of cellular D-type cyclins, induces replicative stress in ECs, which leads to senescence and activation of the DNA damage response. We find that antiproliferative checkpoints are activated upon KSHV infection of ECs, and in early-stage but not late-stage lesions of clinical Kaposi sarcoma specimens. These are some of the first results suggesting that DNA damage checkpoint response also functions as an anticancer barrier in virally induced cancers.

Wnt/beta-catenin signaling plays a central role in guiding the differentiation of the posterior parts of the primitive gut tube into intestinal structures in vivo and some studies suggest that FGF4 is another crucial factor for intestinal development. The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids. The most prominent induction of the well-established intestinal marker gene CDX2 was achieved when hPSC-derived definitive endoderm cells were treated with Wnt agonist molecule CHIR99021 during differentiation to hindgut. FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage. When hindgut stage cells were further cultured in 3D, they formed self-renewing organoid structures containing all major intestinal cell types even without exogenous R-spondin1 (RSPO1), a crucial factor for the culture of epithelial organoids derived from adult intestine. This may be explained by the presence of a mesenchymal compartment in the hPSC-derived organoids. Addition of WNT3A increased the expression of the Paneth cell marker Lysozyme in hPSC-derived organoid cultures, whereas FGF4 inhibited both the formation and maturation of intestinal-like organoids. Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.

The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.

Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.

Fibroblast Growth Factor receptor (FGFR) activity plays crucial roles in tumor growth and patient survival. However, FGF (Fibroblast Growth Factor) signaling as a target for cancer therapy has been under-investigated compared to other receptor tyrosine kinases. Here, we studied the effect of FGFR signaling inhibition on tumor growth, metastasis and lymphangiogenesis by expressing a dominant negative FGFR (FGFR-2DN) in an orthotopic mouse mammary 66c14 carcinoma model. We show that FGFR-2DN-expressing 66c14 cells proliferate in vitro slower than controls. 66c14 tumor outgrowth and lung metastatic foci are reduced in mice implanted with FGFR-2DN-expressing cells, which also exhibited better overall survival. We found 66c14 cells in the lumen of tumor lymphatic vessels and in lymph nodes. FGFR-2DN-expressing tumors exhibited a decrease in VEGFR-3 (Vascular Endothelial Growth Factor Receptor-3) or podoplanin-positive lymphatic vessels, an increase in isolated intratumoral lymphatic endothelial cells and a reduction in VEGF-C (Vascular Endothelial Growth Factor-C) mRNA expression. FGFs may act in an autocrine manner as the inhibition of FGFR signaling in tumor cells suppresses VEGF-C expression in a COX-2 (cyclooxygenase-2) or HIF1-α (hypoxia-inducible factor-1 α) independent manner. FGFs may also act in a paracrine manner on tumor lymphatics by inducing expression of pro-lymphangiogenic molecules such as VEGFR-3, integrin α9, prox1 and netrin-1. Finally, in vitro lymphangiogenesis is impeded in the presence of FGFR-2DN 66c14 cells. These data confirm that both FGF and VEGF signaling are necessary for the maintenance of vascular morphogenesis and provide evidence that targeting FGFR signaling may be an interesting approach to inhibit tumor lymphangiogenesis and metastatic spread.

BackgroundThe family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb.Methodology/principal findingsEctopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors.Conclusions/significanceTaken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.

We have generated a transgenic mouse where hVEGF-A(165) expression has been silenced with loxP-STOP fragment, and we used this model to study the effects of hVEGF-A(165) over-expression in mice after systemic adenovirus mediated Cre-gene transfer. Unlike previous conventional transgenic models, this model leads to the expression of hVEGF-A(165) in only a low number of cells in the target tissues in adult mice. Levels of hVEGF-A(165) expression were moderate and morphological changes were found mainly in the liver, showing typical signs of active angiogenesis. Most mice were healthy without any major consequences up to 18 months after the activation of hVEGF-A(165) expression. However, one mouse with a high plasma hVEGF-A(165) level died spontaneously because of bleeding into abdominal cavity and having liver hemangioma, haemorrhagic paratubarian cystic lesions and spleen peliosis. Also, two mice developed malignant tumors (hepatocellular carcinoma and lung adenocarcinoma), which were not seen in control mice. We conclude that long-term uncontrolled hVEGF-A(165) expression in only a limited number of target cells in adult mice can be associated with pathological changes, including possible formation of malignant tumors and uncontrolled bleeding in target tissues. These findings have implications for the design of long-term clinical trials using hVEGF-A(165) gene and protein.