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.

Lymphangiogenesis requires Ang2/Tie/PI3K signaling for VEGFR3 cell-surface expression.

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.

Common Laboratory Mice Are Susceptible to Infection with the SARS-CoV-2 Beta Variant.

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.

Characterization of ANGPT2 mutations associated with primary lymphedema.

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 <30% 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 α5ß1 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 ANGPT2 whole-gene deletion and four heterozygous ANGPT2 missense mutations. Functional analyses revealed three missense mutations that resulted in decreased ANGPT2 secretion and inhibited the secretion of wild-type (WT)-ANGPT2, suggesting that they have a dominant-negative effect on ANGPT2 signaling. WT-ANGPT2 and soluble mutants T299M and N304K activated TIE1 and TIE2 in an autocrine assay in human lymphatic endothelial cells. Molecular modeling and biophysical studies showed that amino-terminally truncated ANGPT subunits formed asymmetrical homodimers that bound TIE2 in a 2:1 ratio. The T299M mutant, located in the dimerization interphase, showed reduced integrin α5 binding, and its expression in mouse skin promoted hyperplasia and dilation of cutaneous lymphatic vessels. These results demonstrate that primary lymphedema can be associated with ANGPT2 mutations and provide insights into TIE1 and TIE2 activation mechanisms.

KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D.

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.

Key molecules in lymphatic development, function, and identification.

While both blood and lymphatic vessels transport fluids and thus share many similarities, they also show functional and structural differences, which can be used to differentiate them. Specific visualization of lymphatic vessels has historically been and still is a pivot point in lymphatic research. Many of the proteins that are investigated by molecular biologists in lymphatic research have been defined as marker molecules, i.e. to visualize and distinguish lymphatic endothelial cells (LECs) from other cell types, most notably from blood vascular endothelial cells (BECs) and cells of the hematopoietic lineage. Among the factors that drive the developmental differentiation of lymphatic structures from venous endothelium, Prospero homeobox protein 1 (PROX1) is the master transcriptional regulator. PROX1 maintains lymphatic identity also in the adult organism and thus is a universal LEC marker. Vascular endothelial growth factor receptor-3 (VEGFR-3) is the major tyrosine kinase receptor that drives LEC proliferation and migration. The major activator for VEGFR-3 is vascular endothelial growth factor-C (VEGF-C). However, before VEGF-C can signal, it needs to be proteolytically activated by an extracellular protein complex comprised of Collagen and calcium binding EGF domains 1 (CCBE1) protein and the protease A disintegrin and metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3). This minireview attempts to give an overview of these and a few other central proteins that scientific inquiry has linked specifically to the lymphatic vasculature. It is limited in scope to a brief description of their main functions, properties and developmental roles.

Biology of Vascular Endothelial Growth Factor C in the Morphogenesis of Lymphatic Vessels.

Because virtually all tissues contain blood vessels, the importance of hemevascularization has been long recognized in regenerative medicine and tissue engineering. However, the lymphatic vasculature has only recently become a subject of interest. Central to the task of growing a lymphatic network are lymphatic endothelial cells (LECs), which constitute the innermost layer of all lymphatic vessels. The central molecule that directs proliferation and migration of LECs during embryogenesis is vascular endothelial growth factor C (VEGF-C). VEGF-C is therefore an important ingredient for LEC culture and attempts to (re)generate lymphatic vessels and networks. During its biosynthesis VEGF-C undergoes a stepwise proteolytic processing, during which its properties and affinities for its interaction partners change. Many of these fundamental aspects of VEGF-C biosynthesis have only recently been uncovered. So far, most-if not all-applications of VEGF-C do not discriminate between different forms of VEGF-C. However, for lymphatic regeneration and engineering purposes, it appears mandatory to understand these differences, since they relate, e.g., to important aspects such as biodistribution and receptor activation potential. In this review, we discuss the molecular biology of VEGF-C as it relates to the growth of LECs and lymphatic vessels. However, the properties of VEGF-C are similarly relevant for the cardiovascular system, since both old and recent data show that VEGF-C can have a profound effect on the blood vasculature.

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.

Factors regulating the substrate specificity of cytosolic phospholipase A2-alpha in vitro.

Cytosolic phospholipase A2 alpha (cPLA2α) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA2α for AA-containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA2α towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the sn1 or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA2α. Such promiscuous binding would explain the previous finding that cPLA2α has both PLA1 and PLA2 activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA2α is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA2α.

Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive type of cancer that lacks effective targeted therapy. Despite detailed molecular profiling, no targeted therapy has been established. Hence, with the aim of gaining deeper understanding of the functional differences of TNBC subtypes and how that may relate to potential novel therapeutic strategies, we studied comprehensive anticancer-agent responses among a panel of TNBC cell lines. METHOD: The responses of 301 approved and investigational oncology compounds were measured in 16 TNBC cell lines applying a functional profiling approach. To go beyond the standard drug viability effect profiling, which has been used in most chemosensitivity studies, we utilized a multiplexed readout for both cell viability and cytotoxicity, allowing us to differentiate between cytostatic and cytotoxic responses. RESULTS: Our approach revealed that most single-agent anti-cancer compounds that showed activity for the viability readout had no or little cytotoxic effects. Major compound classes that exhibited this type of response included anti-mitotics, mTOR, CDK, and metabolic inhibitors, as well as many agents selectively inhibiting oncogene-activated pathways. However, within the broad viability-acting classes of compounds, there were often subsets of cell lines that responded by cell death, suggesting that these cells are particularly vulnerable to the tested substance. In those cases we could identify differential levels of protein markers associated with cytotoxic responses. For example, PAI-1, MAPK phosphatase and Notch-3 levels associated with cytotoxic responses to mitotic and proteasome inhibitors, suggesting that these might serve as markers of response also in clinical settings. Furthermore, the cytotoxicity readout highlighted selective synergistic and synthetic lethal drug combinations that were missed by the cell viability readouts. For instance, the MEK inhibitor trametinib synergized with PARP inhibitors. Similarly, combination of two non-cytotoxic compounds, the rapamycin analog everolimus and an ATP-competitive mTOR inhibitor dactolisib, showed synthetic lethality in several mTOR-addicted cell lines. CONCLUSIONS: Taken together, by studying the combination of cytotoxic and cytostatic drug responses, we identified a deeper spectrum of cellular responses both to single agents and combinations that may be highly relevant for identifying precision medicine approaches in TNBC as well as in other types of cancers.

Systematic Mapping of Kinase Addiction Combinations in Breast Cancer Cells by Integrating Drug Sensitivity and Selectivity Profiles.

Chemical perturbation screens offer the possibility to identify actionable sets of cancer-specific vulnerabilities. However, most inhibitors of kinases or other cancer targets result in polypharmacological effects, which complicate the identification of target dependencies directly from the drug-response phenotypes. In this study, we developed a chemical systems biology approach that integrates comprehensive drug sensitivity and selectivity profiling to provide functional insights into both single and multi-target oncogenic signal addictions. When applied to 21 breast cancer cell lines, perturbed with 40 kinase inhibitors, the subtype-specific addiction patterns clustered in agreement with patient-derived subtypes, while showing considerable variability between the heterogeneous breast cancers. Experimental validation of the top predictions revealed a number of co-dependencies between kinase targets that led to unexpected synergistic combinations between their inhibitors, such as dasatinib and axitinib in the triple-negative basal-like HCC1937 cell line.

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.