Intranasal administration of adenoviral vaccines expressing SARS-CoV-2 spike protein improves vaccine immunity in mouse models.

The ongoing SARS-CoV-2 pandemic is controlled but not halted by public health measures and mass vaccination strategies which have exclusively relied on intramuscular vaccines. Intranasal vaccines can prime or recruit to the respiratory epithelium mucosal immune cells capable of preventing infection. Here we report a comprehensive series of studies on this concept using various mouse models, including HLA class II-humanized transgenic strains. We found that a single intranasal (i.n.) dose of serotype-5 adenoviral vectors expressing either the receptor binding domain (Ad5-RBD) or the complete ectodomain (Ad5-S) of the SARS-CoV-2 spike protein was effective in inducing i) serum and bronchoalveolar lavage (BAL) anti-spike IgA and IgG, ii) robust SARS-CoV-2-neutralizing activity in the serum and BAL, iii) rigorous spike-directed T helper 1 cell/cytotoxic T cell immunity, and iv) protection of mice from a challenge with the SARS-CoV-2 beta variant. Intramuscular (i.m.) Ad5-RBD or Ad5-S administration did not induce serum or BAL IgA, and resulted in lower neutralizing titers in the serum. Moreover, prior immunity induced by an intramuscular mRNA vaccine could be potently enhanced and modulated towards a mucosal IgA response by an i.n. Ad5-S booster. Notably, Ad5 DNA was found in the liver or spleen after i.m. but not i.n. administration, indicating a lack of systemic spread of the vaccine vector, which has been associated with a risk of thrombotic thrombocytopenia. Unlike in otherwise genetically identical HLA-DQ6 mice, in HLA-DQ8 mice Ad5-RBD vaccine was inferior to Ad5-S, suggesting that the RBD fragment does not contain a sufficient collection of helper-T cell epitopes to constitute an optimal vaccine antigen. Our data add to previous promising preclinical results on intranasal SARS-CoV-2 vaccination and support the potential of this approach to elicit mucosal immunity for preventing transmission of SARS-CoV-2.

Expression of R-Spondin 1 in ApcMin/+ Mice Suppresses Growth of Intestinal Adenomas by Altering Wnt and Transforming Growth Factor Beta Signaling.

BACKGROUND & AIMS: 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 ApcMin/+ mutant mice. METHODS: An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into ApcMin/+mice. Their intestinal crypts were isolated and cultured as organoids. which were incubated with or without RSPO1-Fc and an inhibitor of transforming growth factor beta receptor (TGFBR). Livers were collected from mice and analyzed by immunohistochemistry. Organoids and adenomas were analyzed by quantitative reverse-transcription PCR, single cell RNA sequencing, and immunohistochemistry. RESULTS: Intestines from Apc+/+ mice injected with the vector encoding RSPO1-Fc had significantly deeper crypts, longer villi, with increased EdU labeling, indicating increased proliferation of epithelial cells, in comparison to mice given control vector. AAV-RSPO1-Fc-transduced ApcMin/+ mice also developed fewer and smaller intestinal tumors and had significantly longer survival times. Adenomas of ApcMin/+ mice injected with the RSPO1-Fc vector showed a rapid increase in apoptosis and in the expression of Wnt target genes, followed by reduced expression of messenger RNAs and proteins regulated by the Wnt pathway, reduced cell proliferation, and less crypt branching than adenomas of mice given the control vector. Addition of RSPO1 reduced the number of adenoma organoids derived from ApcMin/+ mice and suppressed expression of Wnt target genes but increased phosphorylation of SMAD2 and transcription of genes regulated by SMAD. Inhibition of TGFBR signaling in organoids stimulated with RSPO1-Fc restored organoid formation and expression of genes regulated by Wnt. The TGFBR inhibitor restored apoptosis in adenomas from ApcMin/+ mice expressing RSPO1-Fc back to the same level as in the adenomas from mice given the control vector. CONCLUSIONS: Expression of RSPO1 in ApcMin/+ mice increases apoptosis and reduces proliferation and Wnt signaling in adenoma cells, resulting in development of fewer and smaller intestinal tumors and longer mouse survival. Addition of RSPO1 to organoids derived from adenomas inhibits their growth and promotes proliferation of intestinal stem cells that retain the APC protein; these effects are reversed by TGFB inhibitor. Strategies to increase the expression of RSPO1 might be developed for the treatment of intestinal adenomas.

VEGF-C protects the integrity of the bone marrow perivascular niche in mice.

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.

High baseline Tie1 level predicts poor survival in metastatic breast cancer.

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 < 0.001). The overall survival of the patients with a high baseline Tie1 level was significantly shorter (multivariate HR 3.07, 95% CI 1.39-6.79, p = 0.005). Additionally, the progression-free survival was shorter for patients with a high baseline Tie1 level (multivariate HR 3.78, 95% CI 1.57-9.09, p = 0.003). In contrast, the baseline Ang2 levels had no prognostic impact in a multivariate Cox proportional hazard regression analysis. The combined analysis of baseline Tie1 and Ang2 levels revealed that patients with both high Tie1 and high Ang2 baseline levels had a significantly shorter overall survival than the patients with low baseline levels of both markers (multivariate HR for overall survival 4.32, 95% CI 1.44-12.94, p = 0.009). CONCLUSIONS: This is the first study to demonstrate the prognostic value of baseline Tie1 plasma concentration in patients with metastatic breast cancer. Combined with the results of the Ang2 analyses, the patients with both high Tie1 and Ang2 levels before treatment had the poorest survival. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00979641, registration date 19-DEC-2008. The regional Ethics Committee: R08142M, registration date 18-NOV-2008.

Genetic Variants of VEGFA and FLT4 Are Determinants of Survival in Renal Cell Carcinoma Patients Treated with Sorafenib.

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.

Transcription Factor PROX1 Suppresses Notch Pathway Activation via the Nucleosome Remodeling and Deacetylase Complex in Colorectal Cancer Stem-like Cells.

The homeobox transcription factor PROX1 is induced by high Wnt/ß-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/ß-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.

Vascular endothelial growth factor signaling in development and disease.

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.

SnapShot: Angiopoietins and Their Functions.

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.

Efficient activation of the lymphangiogenic growth factor VEGF-C requires the C-terminal domain of VEGF-C and the N-terminal domain of CCBE1.

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.

VEGFB/VEGFR1-Induced Expansion of Adipose Vasculature Counteracts Obesity and Related Metabolic Complications.

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.

Functional Dissection of the CCBE1 Protein: A Crucial Requirement for the Collagen Repeat Domain.

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.

Endothelial destabilization by angiopoietin-2 via integrin ß1 activation.

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 ß1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes ß1-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 ß1-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 ß1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.

Vascular endothelial growth factor-B in physiology and disease.

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.

CCBE1 enhances lymphangiogenesis via A disintegrin and metalloprotease with thrombospondin motifs-3-mediated vascular endothelial growth factor-C activation.

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.

Receptor tyrosine kinase-mediated angiogenesis.

The endothelial cell is the essential cell type forming the inner layer of the vasculature. Two families of receptor tyrosine kinases (RTKs) are almost completely endothelial cell specific: the vascular endothelial growth factor (VEGF) receptors (VEGFR1-3) and the Tie receptors (Tie1 and Tie2). Both are key players governing the generation of blood and lymphatic vessels during embryonic development. Because the growth of new blood and lymphatic vessels (or the lack thereof) is a central element in many diseases, the VEGF and the Tie receptors provide attractive therapeutic targets in various diseases. Indeed, several drugs directed to these RTK signaling pathways are already on the market, whereas many are in clinical trials. Here we review the VEGFR and Tie families, their involvement in developmental and pathological angiogenesis, and the different possibilities for targeting them to either block or enhance angiogenesis and lymphangiogenesis.

The basis for the distinct biological activities of vascular endothelial growth factor receptor-1 ligands.

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.

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation.

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.

Comparison of vascular growth factors in the murine brain reveals placenta growth factor as prime candidate for CNS revascularization.

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.

Vascular endothelial growth factor-angiopoietin chimera with improved properties for therapeutic angiogenesis.

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.

Thermodynamic and structural description of allosterically regulated VEGFR-2 dimerization.

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.