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Molecular markers of sorafenib efficacy in patients with metastatic renal cell carcinoma (mRCC) are not available. The purpose of this study was to discover genetic markers of survival in patients with mRCC treated with sorafenib. Germline variants from 56 genes were genotyped in 295 patients with mRCC. Variant-overall survival (OS) associations were tested in multivariate regression models. Mechanistic studies were conducted to validate clinical associations. VEGFA rs1885657, ITGAV rs3816375, and WWOX rs8047917 (sorafenib arm), and FLT4 rs307826 and VEGFA rs3024987 (sorafenib and placebo arms combined) were associated with shorter OS. FLT4 rs307826 increased VEGFR-3 phosphorylation, membrane trafficking, and receptor activation. VEGFA rs1885657 and rs58159269 increased transcriptional activity of the constructs containing these variants in endothelial and RCC cell lines, and VEGFA rs58159269 increased endothelial cell proliferation and tube formation. FLT4 rs307826 and VEGFA rs58159269 led to reduced sorafenib cytotoxicity. Genetic variation in VEGFA and FLT4 could affect survival in sorafenib-treated patients with mRCC. These markers should be examined in additional malignancies treated with sorafenib and in other angiogenesis inhibitors used in mRCC.Significance:Clinical and mechanistic data identify germline genetic variants in VEGFA and FLT4 as markers of survival in patients with metastatic renal cell carcinoma.

Expression of WT and T494A VEGFR-3 in HUVECs (A) and replication of phosphorylation experiments testing the effect of FLT4 rs307826 (A>G, T494A) on VEGFR-3 phosphorylation (B).

SNP selection criteria, imputation and bioinformatic analyses

Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor–positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.

Lymphedema can occur when tissue fluid cannot enter or leaks from the lymphatic system into surrounding tissues. Some genetic causes of primary lymphedema are known, but these currently explain a minority of cases. Previous studies have shown that dominant-negative mutations in angiopoietin 2 (ANGPT2), which is involved in lymphatic vessel formation and maturation, promote lymphangiogenesis in mice. Leppänen et al. now show that inactivating mutations in angiopoietin 2 associate with primary lymphedema in humans.Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain

The homeobox transcription factor PROX1 is induced by high Wnt/β-catenin activity in intestinal adenomas and colorectal cancer, where it promotes tumor progression. Here we report that in LGR5+ colorectal cancer cells, PROX1 suppresses the Notch pathway, which is essential for cell fate in intestinal stem cells. Pharmacologic inhibition of Notch in ex vivo 3D organoid cultures from transgenic mouse intestinal adenoma models increased Prox1 expression and the number of PROX1-positive cells. Notch inhibition led to increased proliferation of the PROX1-positive colorectal cancer cells, but did not affect their ability to give rise to PROX1-negative secretory cells. Conversely, PROX1 deletion increased Notch target gene expression and NOTCH1 promoter activity, indicating reciprocal regulation between PROX1 and the Notch pathway in colorectal cancer. PROX1 interacted with the nucleosome remodeling and deacetylase (NuRD) complex to suppress the Notch pathway. Thus, our data suggests that PROX1 and Notch suppress each other and that PROX1-mediated suppression of Notch mediates its stem cell function in colorectal cancer. Significance: These findings address the role of the PROX1 homeobox factor as a downstream effector of Wnt/β-catenin singling in colorectal cancer stem cells and show that PROX1 inhibits the Notch pathway and helps to enforce the stem cell phenotype and inhibit differentiation. Cancer Res; 78(20); 5820–32. ©2018 AACR.

The endothelial cell (EC)-specific receptor tyrosine kinases Tie1 and Tie2 are necessary for the remodeling and maturation of blood and lymphatic vessels. Angiopoietin-1 (Ang1) growth factor is a Tie2 agonist, whereas Ang2 functions as a context-dependent agonist/antagonist. The orphan receptor Tie1 modulates Tie2 activation, which is induced by association of angiopoietins with Tie2 in cis and across EC–EC junctions in trans. Except for the binding of the C-terminal angiopoietin domains to the Tie2 ligand-binding domain, the mechanisms for Tie2 activation are poorly understood. We report here the structural basis of Ang1-induced Tie2 dimerization in cis and provide mechanistic insights on Ang2 antagonism, Tie1/Tie2 heterodimerization, and Tie2 clustering. We find that Ang1-induced Tie2 dimerization and activation occurs via the formation of an intermolecular β-sheet between the membrane-proximal (third) Fibronectin type III domains (Fn3) of Tie2. The structures of Tie2 and Tie1 Fn3 domains are similar and compatible with Tie2/Tie1 heterodimerization by the same mechanism. Mutagenesis of the key interaction residues of Tie2 and Tie1 Fn3 domains decreased Ang1-induced Tie2 phosphorylation and increased the basal phosphorylation of Tie1, respectively. Furthermore, the Tie2 structures revealed additional interactions between the Fn 2 (Fn2) domains that coincide with a mutation of Tie2 in primary congenital glaucoma that leads to defective Tie2 clustering and junctional localization. Mutagenesis of the Fn2–Fn2 interface increased the basal phosphorylation of Tie2, suggesting that the Fn2 interactions are essential in preformed Tie2 oligomerization. The interactions of the membrane-proximal domains could provide new targets for modulation of Tie receptor activity.

Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in ApcAn adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcIntestines from ApcExpression of RSPO1 in Apc

Primary lymphedema is caused by developmental and functional defects of the lymphatic vascular system that result in accumulation of protein-rich fluid in tissues, resulting in edema. The 28 currently known genes causing primary lymphedema can explain30% of cases. Angiopoietin 1 (ANGPT1) and ANGPT2 function via the TIE1-TIE2 (tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 1 and 2) receptor complex and α5β1 integrin to form an endothelial cell signaling pathway that is critical for blood and lymphatic vessel formation and remodeling during embryonic development, as well as for homeostasis of the mature vasculature. By screening a cohort of 543 individuals affected by primary lymphedema, we identified one heterozygous de novo

Organ damage and innate immunity during heart transplantation may evoke adaptive immunity with serious consequences. Because lymphatic vessels bridge innate and adaptive immunity, they are critical in immune surveillance; however, their role in ischemia-reperfusion injury (IRI) in allotransplantation remains unknown. We investigated whether the lymphangiogenic VEGF-C/VEGFR3 pathway during cardiac allograft IRI regulates organ damage and subsequent interplay between innate and adaptive immunity. We found that cardiac allograft IRI, within hours, increased graft VEGF-C expression and lymphatic vessel activation in the form of increased lymphatic VEGFR3 and adhesion protein expression. Pharmacological VEGF-C/VEGFR3 stimulation resulted in early lymphatic activation and later increase in allograft inflammation. In contrast, pharmacological VEGF-C/VEGFR3 inhibition during cardiac allograft IRI decreased early lymphatic vessel activation with subsequent dampening of acute and chronic rejection. Genetic deletion of VEGFR3 specifically in the lymphatics of the transplanted heart recapitulated the survival effect achieved by pharmacological VEGF-C/VEGFR3 inhibition. Our results suggest that tissue damage rapidly changes lymphatic vessel phenotype, which, in turn, may shape the interplay of innate and adaptive immunity. Importantly, VEGF-C/VEGFR3 inhibition during solid organ transplant IRI could be used as lymphatic-targeted immunomodulatory therapy to prevent acute and chronic rejection.

Molecular markers of sorafenib efficacy in patients with metastatic renal cell carcinoma (mRCC) are not available. The purpose of this study was to discover genetic markers of survival in patients with mRCC treated with sorafenib. Germline variants from 56 genes were genotyped in 295 patients with mRCC. Variant-overall survival (OS) associations were tested in multivariate regression models. Mechanistic studies were conducted to validate clinical associations. VEGFA rs1885657, ITGAV rs3816375, and WWOX rs8047917 (sorafenib arm), and FLT4 rs307826 and VEGFA rs3024987 (sorafenib and placebo arms combined) were associated with shorter OS. FLT4 rs307826 increased VEGFR-3 phosphorylation, membrane trafficking, and receptor activation. VEGFA rs1885657 and rs58159269 increased transcriptional activity of the constructs containing these variants in endothelial and RCC cell lines, and VEGFA rs58159269 increased endothelial cell proliferation and tube formation. FLT4 rs307826 and VEGFA rs58159269 led to reduced sorafenib cytotoxicity. Genetic variation in VEGFA and FLT4 could affect survival in sorafenib-treated patients with mRCC. These markers should be examined in additional malignancies treated with sorafenib and in other angiogenesis inhibitors used in mRCC. Significance: Clinical and mechanistic data identify germline genetic variants in VEGFA and FLT4 as markers of survival in patients with metastatic renal cell carcinoma.

Vascular endothelial growth factors (VEGFs) are best known for their involvement in orchestrating the development and maintenance of the blood and lymphatic vascular systems. VEGFs are secreted by a variety of cells and they bind to their cognate tyrosine kinase VEGF receptors (VEGFRs) in endothelial cells to elicit various downstream effects. In recent years, there has been tremendous progress in elucidating different VEGF/VEGFR signaling functions in both the blood and lymphatic vascular systems. Here, and in the accompanying poster, we present key elements of the VEGF/VEGFR pathway and highlight the classical and newly discovered functions of VEGF signaling in blood and lymphatic vessel development and pathology.

Molecular markers of sorafenib efficacy in metastatic renal cell carcinoma (mRCC) patients are not available. The purpose of this study was to discover genetic markers of survival in mRCC patients treated with sorafenib. Germline variants from 56 genes were genotyped in 295 mRCC patients. Variant-overall survival (OS) associations were tested in multivariate regression models. Mechanistic studies were conducted to validate clinical associations. VEGFA rs1885657, ITGAV rs3816375, and WWOX rs8047917 (sorafenib arm), and FLT4 rs307826 and VEGFA rs3024987 (sorafenib and placebo arms combined) were associated with shorter OS. FLT4 rs307826 increased VEGFR-3 phosphorylation, membrane trafficking and receptor activation. VEGFA rs1885657 and rs58159269 increased transcriptional activity of the constructs containing these variants in endothelial and RCC cell lines, and VEGFA rs58159269 increased endothelial cell proliferation and tube formation. FLT4 rs307826 and VEGFA rs58159269 led to reduced sorafenib cytotoxicity. Genetic variation in VEGFA and FLT4 could affect survival in sorafenib-treated mRCC patients. These markers should be examined in additional malignancies treated with sorafenib, and in other angiogenesis inhibitors used in mRCC.

Rationale : Collagen- and calcium-binding EGF domain–containing protein 1 (CCBE1) is essential for lymphangiogenesis in vertebrates and has been associated with Hennekam syndrome. Recently, CCBE1 has emerged as a crucial regulator of vascular endothelial growth factor-C (VEGFC) signaling. Objective : CCBE1 is a secreted protein characterized by 2 EGF domains and 2 collagen repeats. The functional role of the different CCBE1 protein domains is completely unknown. Here, we analyzed the functional role of the different CCBE1 domains in vivo and in vitro. Methods and Results : We analyzed the functionality of several CCBE1 deletion mutants by generating knock-in mice expressing these mutants, by analyzing their ability to enhance Vegfc signaling in vivo in zebrafish, and by testing their ability to induce VEGFC processing in vitro. We found that deleting the collagen domains of CCBE1 has a much stronger effect on CCBE1 activity than deleting the EGF domains. First, although CCBE1ΔCollagen mice fully phenocopy CCBE1 knock-out mice, CCBE1ΔEGF knock-in embryos still form rudimentary lymphatics. Second, Ccbe1ΔEGF, but not Ccbe1ΔCollagen, could partially substitute for Ccbe1 to enhance Vegfc signaling in zebrafish. Third, CCBE1ΔEGF, similarly to CCBE1, but not CCBE1ΔCollagen could activate VEGFC processing in vitro. Furthermore, a Hennekam syndrome mutation within the collagen domain has a stronger effect than a Hennekam syndrome mutation within the EGF domain. Conclusions: We propose that the collagen domains of CCBE1 are crucial for the activation of VEGFC in vitro and in vivo. The EGF domains of CCBE1 are dispensable for regulation of VEGFC processing in vitro, however, they are necessary for full lymphangiogenic activity of CCBE1 in vivo.

Angiopoietins signal through TIE receptors to control both developmental and homeostatic processes that can go awry in genetic diseases and cancer. This SnapShot illustrates key elements of angiopoietin signaling in normal and disease contexts.

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.

Colorectal cancers (CRCs) most commonly develop from adenomas harboring a mutant APC gene that leads to aberrant activation of the canonical Wnt/ß-catenin pathway. One of the mechanisms of adenoma progression involves the PROX1 transcription factor that promotes tumor dysplasia and progression in CRC. In the normal intestinal epithelial cells, ligands of the R-Spondin (RSPO) family are able to enhance the canonical Wnt pathway activity by binding to receptor proteins of the LGR family, such as LGR5. Tumorigenic RSPO gene fusions have recently been discovered in CRCs, but the role of RSPOs in cancer is incompletely known. We previously reported that TGFß induced apoptosis of APC-mutant organoids, including the LGR5+ stem cells, was mediated by the proapoptotic protein BIM, whereas wild-type intestinal crypts were markedly less sensitive to TGFß. The KRAS oncogene increased apoptosis resistance in the adenomas via activation of the Erk1/2 kinase pathway, which led to Bim down-regulation and increased resistance to TGFß. A subsequent study showed that RSPO/LGR5 can inhibit the in vitro growth of cultured human CRC cell line by activating the TGFß/SMAD signaling pathway. Because we were interested in analyzing the effect of exogenous Wnt signals on PROX1 expression in intestinal tumorigenesis, we constructed AAV-vectors for systemic expression of a soluble RSPO1 protein in ApcMin/+ mice. We found that the RSPO1-Fc fusion protein suppresses the Wnt/ß-catenin signaling activity in intestinal adenomas and in adenoma-derived intestinal organoids ex vivo, but not in normal intestinal epithelial cells. In the Apc mutant cells, the RSPO1-Fc fusion protein activated the TGFß/SMAD signaling pathway to suppress several Wnt target genes and adenoma growth, which effect was rescued suppressed by the TGFß receptor kinase inhibitor SB-431542. Simultaneously, RSPO1-Fc induced proliferation of the normal intestinal stem cells, giving them a growth advantage over the mutant cells, which enabled the intestinal epithelium to eventually outgrow the adenoma cells. Prolonged systemic expression of AAV-RSPO1-Fc decreased significantly the number of the intestinal adenomas and improved the overall survival of ApcMin/+ mice. Thus RSPO1-Fc provides the normal intestinal epithelial cells a growth advantage when compared to the adenoma cells, which eventually leads to the extrusion of the adenomatous tissue. An attractive idea now is to exploit such differential response of normal vs. cancer cells in cancer therapy. Citation Format: Marianne Lähde, Sarika Heino, Jenny Högström, Veli-Matti Leppänen, Seppo Kaijalainen, Olli Ritvos, Owen Sansom, Kari Alitalo. Systemic RSPO1 delivery leads to regression of intestinal adenomas via activation of the TGFβ/SMAD pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-094.

Vascular bypass procedures in the central nervous system (CNS) remain technically challenging, hindered by complications and often failing to prevent adverse outcome such as stroke. Thus, there is an unmet clinical need for a safe and effective CNS revascularization. Vascular endothelial growth factors (VEGFs) are promising candidates for revascularization; however, their effects appear to be tissue-specific and their potential in the CNS has not been fully explored. To test growth factors for angiogenesis in the CNS, we characterized the effects of endothelium-specific growth factors on the brain vasculature and parenchyma. Recombinant adeno-associated virus (AAV) vectors encoding the growth factors were injected transcranially to the frontoparietal cerebrum of mice. Angiogenesis, mural cell investment, leukocyte recruitment, vascular permeability, reactive gliosis and neuronal patterning were evaluated by 3-dimensional immunofluorescence, electron microscopy, optical projection tomography, and magnetic resonance imaging. Placenta growth factor (PlGF) stimulated robust angiogenesis and arteriogenesis without significant side effects, whereas VEGF and VEGF-C incited growth of aberrant vessels, severe edema, and inflammation. VEGF-B, angiopoietin-1, angiopoietin-2, and a VEGF/angiopoietin-1 chimera had minimal effects on the brain vessels or parenchyma. Of the growth factors tested, PlGF emerged as the most efficient and safe angiogenic factor, hence making it a candidate for therapeutic CNS revascularization.

VEGFs activate 3 receptor tyrosine kinases, VEGFR-1, VEGFR-2, and VEGFR-3, promoting angiogenic and lymphangiogenic signaling. The extracellular receptor domain (ECD) consists of 7 Ig-homology domains; domains 2 and 3 (D23) represent the ligand-binding domain, whereas the function of D4-7 is unclear. Ligand binding promotes receptor dimerization and instigates transmembrane signaling and receptor kinase activation. In the present study, isothermal titration calorimetry showed that the Gibbs free energy of VEGF-A, VEGF-C, or VEGF-E binding to D23 or the full-length ECD of VEGFR-2 is dominated by favorable entropic contribution with enthalpic penalty. The free energy of VEGF binding to the ECD is 1.0-1.7 kcal/mol less favorable than for binding to D23. A model of the VEGF-E/VEGFR-2 ECD complex derived from small-angle scattering data provided evidence for homotypic interactions in D4-7. We also solved the crystal structures of complexes between VEGF-A or VEGF-E with D23, which revealed comparable binding surfaces and similar interactions between the ligands and the receptor, but showed variation in D23 twist angles. The energetically unfavorable homotypic interactions in D4-7 may be required for re-orientation of receptor monomers, and this mechanism might prevent ligand-independent activation of VEGFR-2 to evade the deleterious consequences for blood and lymph vessel homeostasis arising from inappropriate receptor activation.

Receptor tyrosine kinases play essential roles in tissue development and homeostasis, and aberrant signaling by these molecules is the basis of many diseases. Understanding the activation mechanism of these receptors is thus of high clinical relevance. We investigated vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which regulate blood and lymph vessel formation. We analyzed the structural changes in the extracellular receptor domain that were induced by ligand binding and that represent the initial step in transmembrane signaling, culminating in the activation of the intracellular receptor kinase domain. High-resolution structural information for the ligand binding domain became available recently, but the flexibility of the extracellular domain and inhomogeneous glycosylation of VEGFRs have prevented the production of highly diffracting crystals of the entire extracellular domain so far. Therefore, we chose to further investigate VEGFR structure by small-angle X-ray scattering in solution (SAXS). SAXS data were combined with independent distance restraint determination obtained by mass spectrometric analysis of chemically cross-linked ligand/receptor complexes. With these data, we constructed a structural model of the entire extracellular receptor domain in the unbound form and in complex with VEGF.

Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy. Refereed/Peer-reviewed

Purpose: To discover genetic determinants of overall survival (OS) in metastatic renal cell carcinoma (mRCC) patients treated with sorafenib. Patients and Methods: Information for 11,117 germline DNA variants from 56 genes was obtained, using both Illumina GoldenGate genotyping and imputation, from 295 patients originally enrolled in the phase III TARGET trial (155 sorafenib and 140 placebo patients). OS was this genotyping study's primary endpoint, and associations between variants and OS were tested using multivariate Cox regression. Associations were considered significant if P Results: Five intragenic variants significantly associated with OS. Among patients in the sorafenib arm, VEGFA rs1885657 (HR=17.3, 95% CI 5.7-52.7; P=1.4x10-4), ITGAV rs3816375 (HR=5.9, 95% CI 2.1-16.4; P=4.9x10-4), and WWOX rs8047917 (HR=4.1, 95% CI 1.9-8.8; P=3.3x10-4) associated with shorter OS. Among patients from both arms, FLT4 rs307826 (HR=13.8, 95% CI 3.0-62.6; P=1.2x10-4) and VEGFA rs3024987 (HR=3.0, 95% CI 1.7-5.4; P=8.3x10-5) associated with shorter OS. No significant associations were discovered in the placebo arm. Bioinformatic evidence was used to prioritize rs1885657, rs58159269 (in complete linkage disequilibrium with rs1885657), and rs307826. rs1885657 and rs58159269 increased luciferase activity in human endothelial (TIME, LPEC) and mRCC (Caki-1) cells, and rs58159269 increased cell proliferation/reduced sorafenib cytotoxicity, and reduced endothelial tube formation in TIME cells. rs307826 increased VEGFR-3 phosphorylation, and led to post-translational VEGFR-3 processing differences in HUVECs. Conclusions: Regulatory VEGFA and FLT4 variants associated with OS in mRCC patients induce cellular alterations that affect angiogenesis and sorafenib activity. These are novel and potentially impactful clinical biomarkers for mRCC and sorafenib, and their relevance to other multikinase inhibitors used in mRCC should be tested. Citation Format: Daniel James Crona, Andrew D. Skol, Veli-Matti Leppänen, Dylan M. Glubb, Amy S. Etheridge, Eleanor Hilliard, Carol Peña, Yuri K. Peterson, Nancy Klauber-DeMore, Kari K. Alitalo, Federico Innocenti. Clinical biomarkers of survival in renal cell carcinoma patients treated with sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1625.

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality. Signaling pathways, such as the Wnt and Notch pathways, are essential for the maintenance and differentiation of wild-type intestinal stem cells. We have shown that the Prox1 transcription promotes expansion of the colorectal cancer stem cell pool and that a subpopulation of the Prox1+ CRC cells display stem cell activity. Because of these results we have further analyzed the role of Prox1 in the regulation of CRC stem cell. Here, we report that Prox1 regulates CRC stem-like cells via bidirectional interaction with Notch1 during CRC initiation and progression. Using genetic in vivo models and ex vivo 3D organoid cultures, we show that Notch inhibition decreases the number of Lgr5+ stem cells whereas it increases expression of the Prox1. Although Notch inhibition led to increased proliferation of the Prox1 positive cells, it did not affect their ability to give rise to differentiated Prox1 negative progeny. Ectopic overexpression of the active fragment of Notch1 suppressed Prox1 expression and inhibited stem cell activity in the CRC cells. On the other hand, the PROX1-NuRD complex suppressed the Notch signaling pathway and Prox1 deletion increased Notch target gene expression and promoter activity, indicating reciprocal regulation between Prox1 and Notch1. Thus, although Prox1 and Notch suppress each other in colorectal cancer cells, Prox1+ cells can function as a stem cell population without the need for Notch pathway activity. Citation Format: Jenny Högström, Sarika Heino, Pauliina Kallio, Marianne Lähde, Veli-Matti Leppänen, Seppo Kaijalainen, Diego Balboa, Timo Otonkoski, Sylvie Robine, Zoltan Wiener, Kari Alitalo. Prox1 and Notch mark distinct colorectal cancer stem cell populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1143.

The therapeutic potential of vascular endothelial growth factor (VEGF)-C and VEGF-D in skeletal muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic activities. Previous studies have mainly used adenoviral gene delivery for short-term expression of VEGF-C and VEGF-D in pig, rabbit, and mouse skeletal muscles. Here we have used the activated mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV), which provides stable, long-lasting transgene expression in various tissues including skeletal muscle. Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood and lymph vessels, and Doppler ultrasound analysis indicated increased blood vessel perfusion. The lymphatic vessels further increased at the 4-week time point were functional, as shown by FITC-lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when compared to the other growth factors studied. We conclude that long-term expression of VEGF-C and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and lymphedema can now be evaluated using this model system.

We have solved the structures of mammalian mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). CDNF protects and repairs midbrain dopaminergic neurons in vivo; MANF supports their survival in culture and is also cytoprotective against endoplasmic reticulum (ER) stress. Neither protein structure resembles any known growth factor but the N-terminal domain is a saposin-like lipid-binding domain. MANF and CDNF may thus bind lipids or membranes. Consistent with this, there are two patches of conserved lysines and arginines. The natively unfolded MANF C-terminus contains a CKGC disulphide bridge, such as reductases and disulphide isomerases, consistent with a role in ER stress response. The structure thus explains why MANF and CDNF are bifunctional; neurotrophic activity may reside in the N-terminal domain and ER stress response in the C-terminal domain. Finally, we identified three changes, (MANF)I10-->K(CDNF), (MANF)E79-->M(CDNF) and (MANF)K88-->L(CDNF), that may account for the biological differences between the proteins.

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor α1 (GFRα1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF2·GFRα12 complex structure at 2.35 A resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF2·GFRα12 complex and the previously published artemin2·GFRα32 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFRα interface between the two structures, in particular ones that buttress the key conserved ArgGFRα-Gluligand-ArgGFRα interaction. Second, the flexible GDNF ligand “finger” loops fit differently into the GFRαs, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand2-co-receptor2-RET2 heterohexamer complexes. Consistent with this, we showed that GDNF2·GFRα12 and artemin2·GFRα32 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFRα1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg190-Lys202 region in GFRα1 domain 2. This may explain how GDNF·GFRα1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.

Collagen IX, located on the surface of collagen fibrils, is crucial for cartilage integrity and stability. The N-terminal NC4 domain of the alpha1(IX) chain is probably important in this because it interacts with various macromolecules such as proteoglycans and cartilage oligomeric matrix protein. At least 17 distinct collagen polypeptides carry an NC4-like unit near their N terminus, but this report, describing the crystal structure of NC4 at 1.8-A resolution, represents the first atomic level structure for these domains. The structure is similar to previously characterized laminin-neurexin-sex hormone binding globulin (LNS) structures, dominated by an antiparallel beta-sheet sandwich. In addition, a zinc ion was found in a position similar to that of the metal binding site of other LNS domains. A partial backbone NMR assignment of NC4 was obtained and utilized in NMR titration studies to investigate the zinc binding in solution state and to quantitate the affinity of metal binding. The K(d) of 11.5 mM suggests a regulatory rather than a structural role for zinc in solution. NMR titration with a heparin tetrasaccharide revealed the presence of a secondary binding site for heparin on NC4, showing structural and functional conservation with thrombospondin-1, but a markedly reduced affinity for the ligand. Also the overall arrangement of the N and C termini of NC4 resembles most closely the N-terminal domain of thrombospondin-1, distinguishing the two from the majority of the published LNS structures.

In Parkinson's disease, brain dopamine neurons degenerate most prominently in the substantia nigra. Neurotrophic factors promote survival, differentiation and maintenance of neurons in developing and adult vertebrate nervous system. The most potent neurotrophic factor for dopamine neurons described so far is the glial-cell-line-derived neurotrophic factor (GDNF). Here we have identified a conserved dopamine neurotrophic factor (CDNF) as a trophic factor for dopamine neurons. CDNF, together with its previously described vertebrate and invertebrate homologue the mesencephalic-astrocyte-derived neurotrophic factor, is a secreted protein with eight conserved cysteine residues, predicting a unique protein fold and defining a new, evolutionarily conserved protein family. CDNF (Armetl1) is expressed in several tissues of mouse and human, including the mouse embryonic and postnatal brain. In vivo, CDNF prevented the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in a rat experimental model of Parkinson's disease. A single injection of CDNF before 6-OHDA delivery into the striatum significantly reduced amphetamine-induced ipsilateral turning behaviour and almost completely rescued dopaminergic tyrosine-hydroxylase-positive cells in the substantia nigra. When administered four weeks after 6-OHDA, intrastriatal injection of CDNF was able to restore the dopaminergic function and prevent the degeneration of dopaminergic neurons in substantia nigra. Thus, CDNF was at least as efficient as GDNF in both experimental settings. Our results suggest that CDNF might be beneficial for the treatment of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) all signal through the transmembrane receptor tyrosine kinase RET. The signalling complex consists of GFLs, GPI-anchored ligand binding GDNF family receptor alphas (GFRalphas) and RET. Signalling via RET is required for the development of the nervous system and the kidney, as well as for spermatogenesis. However, constitutive activation of RET is implicated as a cause in several diseases. Mutations of the RET proto-oncogene cause the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). Recently, it has been suggested that mutations in the persephin binding GFRalpha4 receptor may have a potentially modifying role in MEN 2. Several naturally occurring, different splice variants of the mammalian GFRalpha4 have been reported. A 7 bp insertion-mutation in the human GFRalpha4 gene causes a shift of reading frame and thereby changes the balance between the transcripts encoding GPI-anchored and soluble GFRalpha4 receptors. We report here that the mammalian soluble GFRalpha4 can activate RET independently of its preferential ligand, persephin. Our data show that soluble GFRalpha4 can associate with, and induce, phosphorylation of RET. In addition, our data show that this isoform of GFRalpha4 can induce downstream signalling, as well as neuronal survival and differentiation, in the absence of persephin. These results suggest that, in line with the previous report, GFRalpha4 may be a candidate gene for, or modifier of, the MEN 2 diseases.

Impaired angiogenesis has been implicated in adipose tissue dysfunction and the development of obesity and associated metabolic disorders. Here, we report the unexpected finding that vascular endothelial growth factor B (VEGFB) gene transduction into mice inhibits obesity-associated inflammation and improves metabolic health without changes in body weight or ectopic lipid deposition. Mechanistically, the binding of VEGFB to VEGF receptor 1 (VEGFR1, also known as Flt1) activated the VEGF/VEGFR2 pathway and increased capillary density, tissue perfusion, and insulin supply, signaling, and function in adipose tissue. Furthermore, endothelial Flt1 gene deletion enhanced the effect of VEGFB, activating the thermogenic program in subcutaneous adipose tissue, which increased the basal metabolic rate, thus preventing diet-induced obesity and related metabolic complications. In obese and insulin-resistant mice, Vegfb gene transfer, together with endothelial Flt1 gene deletion, induced weight loss and mitigated the metabolic complications, demonstrating the therapeutic potential of the VEGFB/VEGFR1 pathway.

Nerve growth factor (NGF) is the ligand for two unrelated cellular receptors, TrkA and p75(NTR), and acts as a mediator in the development and maintenance of the mammalian nervous system. Signaling through TrkA kinase domains promotes neuronal survival, whereas activation of the p75(NTR) "death domains" induces apoptosis under correct physiological conditions. However, co-expression of these receptors leads to enhanced neuronal survival upon NGF stimulation, possibly through a ternary p75(NTR) x NGF x TrkA complex. We have expressed human p75(NTR) ligand binding domain as a secreted glycosylated protein in Trichoplusia ni cells. Following assembly and purification of soluble p75(NTR) x NGF complexes, mass spectrometry, analytical ultracentrifugation, and solution x-ray scattering measurements are indicative of 2:2 stoichiometry, which implies a symmetric complex. Molecular models of the 2:2 p75(NTR) x NGF complex based on these data are not consistent with the further assembly of either symmetric (2:2:2) or asymmetric (2:2:1) ternary p75(NTR) x NGF x TrkA complexes.

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell–cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2., Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) have opposing effects on vascular stability through their receptor Tie2, but there is evidence for Tie2-independent functions of Ang2. Here, Hakanpaa et al. show that Ang2 directly activates β1-integrin, leading to rearrangement of the actin cytoskeleton and decreased VE-cadherin in cell–cell junctions.

The endothelial TIE1 and TIE2 receptor tyrosine kinases form a distinct subfamily characterized by their unique extracellular domains. Together with the angiopoietin growth factors (ANGPT1, ANGPT2, ANGPT4, also abbreviated as ANG), the TIE receptors form an endothelial specific signaling pathway with important functions in the regulation of lymphatic and cardiovascular development and vascular homeostasis. Angiopoietins exist in multimeric forms that activate the TIE receptors via unique mechanism. In endothelial cell–cell contacts, angiopoietins induce the formation of homomeric in trans TIE receptor complexes extending across the cell junctions, whereas matrix-bound angiopoietin-1 (ANG1) activates the TIE receptors in a cis configuration. In comparison to the vascular endothelial growth factor receptors, the TIE receptors undergo little ubiquitin-mediated degradation after activation, whereas TIE2 signaling is negatively regulated by the vascular endothelial protein tyrosine phosphatase, VE-PTP. ANG1 activation of TIE2 supports vascular stabilization, whereas angiopoietin-2 (ANG2), a context-dependent weak TIE2 agonist/antagonist, promotes pathological tumor angiogenesis, vascular permeability, and inflammation. Recently, ANG2 has been found to mediate some of its vascular destabilizing and angiogenic functions via integrin signalling. The circulating levels of ANG2 are increased in cancer, and in several human diseases associated with inflammation and vascular leak, for example, in sepsis. Blocking of ANG2 has emerged as a potential novel therapeutic strategy for these diseases. In addition, preclinical results demonstrate that genetic TIE1 deletion in mice inhibits the vascularization and growth of tumor isografts and protects from atherosclerosis, with little effect on normal vascular homeostasis in adult mice. The ability of the ANG-TIE pathway to control vessel stability and angiogenesis makes it an interesting vascular target for the treatment of the various diseases.

The structure of inactive pyruvate formate-lyase in complex with a natural substrate, pyruvate, was solved at 2.7 A resolution. Both active sites of the homodimeric enzyme are occupied by pyruvate; additional binding sites were not found. Pyruvate was found in a cleft close to the active-site cysteines 418 and 419, with the carboxyl group in contact with arginines 176 and 435 and the methyl group within van der Waals distance of Phe327. It is believed that the binding site of pyruvate is not the position of pyruvate as the reaction initiates, as conformational changes occur during activation of the enzyme.

In glaucoma, aqueous outflow into the Schlemm’s canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

Vascular endothelial growth factor-B (VEGF-B), discovered over 15 years ago, has long been seen as one of the more ambiguous members of the VEGF family. VEGF-B is produced as two isoforms: one that binds strongly to heparan sulfate in the pericellular matrix and a soluble form that can acquire binding via proteolytic processing. Both forms of VEGF-B bind to VEGF-receptor 1 (VEGFR-1) and the neuropilin-1 (NRP-1) coreceptor, which are expressed mainly in blood vascular endothelial cells. VEGF-B-deficient mice and rats are viable without any overt phenotype, and the ability of VEGF-B to induce angiogenesis in most tissues is weak. This has been a puzzle, as the related placenta growth factor (PlGF) binds to the same receptors and induces angiogenesis and arteriogenesis in a variety of tissues. However, it seems that VEGF-B is a vascular growth factor that is more tissue specific and can have trophic and metabolic effects, and its binding to VEGFR-1 shows subtle but important differences compared with that of PlGF. VEGF-B has the potential to induce coronary vessel growth and cardiac hypertrophy, which can protect the heart from ischemic damage as well as heart failure. In addition, VEGF-B is abundantly expressed in tissues with highly active energy metabolism, where it could support significant metabolic functions. VEGF-B also has a role in neuroprotection, but unlike other members of the VEGF family, it does not have a clear role in tumor progression. Here we review what is hitherto known about the functions of this growth factor in physiology and disease.

Background— Hennekam lymphangiectasia–lymphedema syndrome (Online Mendelian Inheritance in Man 235510) is a rare autosomal recessive disease, which is associated with mutations in the CCBE1 gene. Because of the striking phenotypic similarity of embryos lacking either the Ccbe1 gene or the lymphangiogenic growth factor Vegfc gene, we searched for collagen- and calcium-binding epidermal growth factor domains 1 (CCBE1) interactions with the vascular endothelial growth factor-C (VEGF-C) growth factor signaling pathway, which is critical in embryonic and adult lymphangiogenesis. Methods and Results— By analyzing VEGF-C produced by CCBE1-transfected cells, we found that, whereas CCBE1 itself does not process VEGF-C, it promotes proteolytic cleavage of the otherwise poorly active 29/31-kDa form of VEGF-C by the A disintegrin and metalloprotease with thrombospondin motifs-3 protease, resulting in the mature 21/23-kDa form of VEGF-C, which induces increased VEGF-C receptor signaling. Adeno-associated viral vector–mediated transduction of CCBE1 into mouse skeletal muscle enhanced lymphangiogenesis and angiogenesis induced by adeno-associated viral vector–VEGF-C. Conclusions— These results identify A disintegrin and metalloprotease with thrombospondin motifs-3 as a VEGF-C–activating protease and reveal a novel type of regulation of a vascular growth factor by a protein that enhances its proteolytic cleavage and activation. The results suggest that CCBE1 is a potential therapeutic tool for the modulation of lymphangiogenesis and angiogenesis in a variety of diseases that involve the lymphatic system, such as lymphedema or lymphatic metastasis.

Background: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C α radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. Results: The first structure of a fragment of PFL (residues 1–624) at 2.8A resolution shows an unusual barrel-like structure, with a catalytic β finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the β finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. Conclusions: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR α / β barrel as well as the β finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1–3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4–7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1P) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B-derived L1 loop (P-L1B) did not. Unlike P-L1B and VEGF-B, B-L1P and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1P also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1. Refereed/Peer-reviewed

Background— There is an unmet need for proangiogenic therapeutic molecules for the treatment of tissue ischemia in cardiovascular diseases. However, major inducers of angiogenesis such as vascular endothelial growth factor (VEGF/VEGF-A) have side effects that limit their therapeutic utility in vivo, especially at high concentrations. Angiopoietin-1 has been considered to be a blood vessel stabilization factor that can inhibit the intrinsic property of VEGF to promote vessel leakiness. In this study, we have designed and tested the angiogenic properties of chimeric molecules consisting of receptor-binding parts of VEGF and angiopoietin-1. We aimed at combining the activities of both factors into 1 molecule for easy delivery and expression in target tissues. Methods and Results— The VEGF–angiopoietin-1 (VA1) chimeric protein bound to both VEGF receptor-2 and Tie2 and induced the activation of both receptors. Detailed analysis of VA1 versus VEGF revealed differences in the kinetics of VEGF receptor-2 activation and endocytosis, downstream kinase activation, and VE-cadherin internalization. The delivery of a VA1 transgene into mouse skeletal muscle led to increased blood flow and enhanced angiogenesis. VA1 was also very efficient in rescuing ischemic limb perfusion. However, VA1 induced less plasma protein leakage and myeloid inflammatory cell recruitment than VEGF. Furthermore, angioma-like structures associated with VEGF expression were not observed with VA1. Conclusions— The VEGF–angiopoietin-1 chimera is a potent angiogenic factor that triggers a novel mode of VEGF receptor-2 activation, promoting less vessel leakiness, less tissue inflammation, and better perfusion in ischemic muscle than VEGF. These properties of VA1 make it an attractive therapeutic tool.

The basidiomycete Phanerochaete chrysosporium produces several isoforms of lignin peroxidase, which catalyzes the oxidative depolymerization of lignin To date, ion-exchange chromatography and preparative isoelectric focusing (IEF) have been commonly used for isolation of lignin peroxidase isoenzymes. In this work we have purified major lignin peroxidases to high purity by a one-step chromatographic method, chromatofocusing. The purified isoenzymes were identified by analytical IEF using isoenzymes purified by preparative IEF as standards. The specific activities and spectral properties of the isoenzymes were comparable with the previously published data. The predominant isoenzyme under the growth conditions used was LiP 4.65. Almost 50% of the lignin peroxidase activity applied into the column was recovered in the LiP 4.65 fraction. The total recovery of the lignin peroxidase activity was over 80%.