Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

Trafficking dynamics of VEGFR1, VEGFR2, and NRP1 in human endothelial cells.

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to 'see' (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells-specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.

Racial differences in the limb skeletal muscle transcriptional programs of patients with critical limb ischemia.

Critical limb ischemia (CLI) is the most severe manifestation of peripheral artery disease (PAD) and is characterized by high rates of morbidity and mortality. As with most severe cardiovascular disease manifestations, Black individuals disproportionately present with CLI. Accordingly, there remains a clear need to better understand the reasons for this discrepancy and to facilitate personalized therapeutic options specific for this population. Gastrocnemius muscle was obtained from White and Black healthy adult volunteers and patients with CLI for whole transcriptome shotgun sequencing (WTSS) and enrichment analysis was performed to identify alterations in specific Reactome pathways. When compared to their race-matched healthy controls, both White and Black patients with CLI demonstrated similar reductions in nuclear and mitochondrial encoded genes and mitochondrial oxygen consumption across multiple substrates, indicating a common bioenergetic paradigm associated with amputation outcomes regardless of race. Direct comparisons between tissues of White and Black patients with CLI revealed hemostasis, extracellular matrix organization, platelet regulation, and vascular wall interactions to be uniquely altered in limb muscles of Black individuals. Among traditional vascular growth factor signaling targets, WTSS revealed only Tie1 to be significantly altered from White levels in Black limb muscle tissues. Quantitative reverse transcription polymerase chain reaction validation of select identified targets verified WTSS directional changes and supports reductions in MMP9 and increases in NUDT4P1 and GRIK2 as unique to limb muscles of Black patients with CLI. This represents a critical first step in better understanding the transcriptional program similarities and differences between Black and White patients in the setting of amputations related to CLI and provides a promising start for therapeutic development in this population.

Tumor and endothelial cells collaborate via transcellular receptor complexes.

Many cellular signaling pathways are initiated by cell-surface ligand-sensing complexes that incorporate not just one but multiple receptors. Most studies focus on receptors coexpressed on a single cell (cis interactions), but complexes containing receptors on adjacent cells (trans interactions) are also possible. Recent work by Morin et al published in this journal provides critical evidence for such trans interactions between Neuropilin-1 (NRP1) expressed on human tumor cells and vascular endothelial growth factor receptor 2 (VEGFR2) expressed on adjacent endothelial cells, with the ligand VEGFA binding and bridging the two receptors. They show that the formation of these complexes is correlated with reduced tumor proliferation and increased patient survival. They also observe trans NRP1-VEGFA-VEGFR2 repressing angiogenesis and cis NRP1-VEGFA-VEGFR2 increasing angiogenesis in selected cancers. The distinct molecular signature of each tumor and each patient will determine which type of complexes dominate and will influence prognosis and treatment. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Arterialized collateral capillaries progress from nonreactive to capable of increasing perfusion in an ischemic arteriolar tree.

OBJECTIVE: CCA, outward remodeling of capillaries that anastomose 2 arteriolar trees with different parent feed arteries, may represent a therapeutic target for patients who lack collaterals. ACCs can reperfuse an ischemic tree, but their functional capacity is unknown. Therefore, we determined whether ACCs mature into resistance vessels that regulate blood flow following arterial occlusion. METHODS: We ligated the lateral spinotrapezius feed artery in Balb/C mice, which induces CCA. At days 7 and 21 following occlusion, we measured vasodilation of ACCs using intravital microscopy and blood flow in the ischemic tree using LSF. We determined the presence of ACCs and neurovascular alignment with immunofluorescence. RESULTS: At day 7, ACCs do not vasodilate following muscle contraction and have reduced responses to endothelial- and smooth muscle-dependent agents. By day 21, ACCs exhibit normal vasodilation, accompanied by normalized increases in relative blood flow to the ischemic zone. Although functioning as resistance vessels by regulating blood flow, ACCs do not appear to be innervated. CONCLUSIONS: ACCs mature into resistance vessels that regulate blood flow to the downstream tissue. Therefore, induction of mature ACCs may be a target for reducing ischemia in patients who lack collateral networks.

A computational analysis of in vivo VEGFR activation by multiple co-expressed ligands.

The splice isoforms of vascular endothelial growth A (VEGF) each have different affinities for the extracellular matrix (ECM) and the coreceptor NRP1, which leads to distinct vascular phenotypes in model systems expressing only a single VEGF isoform. ECM-immobilized VEGF can bind to and activate VEGF receptor 2 (VEGFR2) directly, with a different pattern of site-specific phosphorylation than diffusible VEGF. To date, the way in which ECM binding alters the distribution of isoforms of VEGF and of the related placental growth factor (PlGF) in the body and resulting angiogenic signaling is not well-understood. Here, we extend our previous validated cell-level computational model of VEGFR2 ligation, intracellular trafficking, and site-specific phosphorylation, which captured differences in signaling by soluble and immobilized VEGF, to a multi-scale whole-body framework. This computational systems pharmacology model captures the ability of the ECM to regulate isoform-specific growth factor distribution distinctly for VEGF and PlGF, and to buffer free VEGF and PlGF levels in tissue. We show that binding of immobilized growth factor to VEGF receptors, both on endothelial cells and soluble VEGFR1, is likely important to signaling in vivo. Additionally, our model predicts that VEGF isoform-specific properties lead to distinct profiles of VEGFR1 and VEGFR2 binding and VEGFR2 site-specific phosphorylation in vivo, mediated by Neuropilin-1. These predicted signaling changes mirror those observed in murine systems expressing single VEGF isoforms. Simulations predict that, contrary to the 'ligand-shifting hypothesis,' VEGF and PlGF do not compete for receptor binding at physiological concentrations, though PlGF is predicted to slightly increase VEGFR2 phosphorylation when over-expressed by 10-fold. These results are critical to design of appropriate therapeutic strategies to control VEGF availability and signaling in regenerative medicine applications.

Further Support for ECM Control of Receptor Trafficking and Signaling.

Recently, Sack et al. (2016) presented an interesting, novel data set in Journal of Cellular Physiology examining the effect of substrate stiffness on VEGF processing and signaling. The data represent a clear contribution to the field. However, the authors' conclusion that "extracellular matrix binding is essential for VEGF internalization" conflicts with other knowledge in the field, and is not supported by their data. Instead, their data demonstrate the effect of heparin addition and changing ECM stiffness on both VEGF binding to fibronectin and VEGF binding to endothelial receptors. This is consistent with other work showing that matrix binding reduces VEGF-VEGFR internalization, shifting downstream signaling. J. Cell. Physiol. 232: 36-37, 2017. © 2016 Wiley Periodicals, Inc.

Agent-based computational model of retinal angiogenesis simulates microvascular network morphology as a function of pericyte coverage.

OBJECTIVE: Define a role for perivascular cells during developmental retinal angiogenesis in the context of EC Notch1-DLL4 signaling at the multicellular network level. METHODS: The retinal vasculature is highly sensitive to growth factor-mediated intercellular signaling. Although EC signaling has been explored in detail, it remains unclear how PC function to modulate these signals that lead to a diverse set of vascular network patterns in health and disease. We have developed an ABM of retinal angiogenesis that incorporates both ECs and PCs to investigate the formation of vascular network patterns as a function of pericyte coverage. We use our model to test the hypothesis that PC modulate Notch1-DLL4 signaling in endothelial cell-endothelial cell interactions. RESULTS: Agent-based model (ABM) simulations that include PCs more accurately predict experimentally observed vascular network morphologies than simulations that lack PCs, suggesting that PCs may influence sprouting behaviors through physical blockade of endothelial intercellular connections. CONCLUSIONS: This study supports a role for PCs as a physical buffer to signal propagation during vascular network formation-a barrier that may be important for generating healthy microvascular network patterns.

Site-Specific Phosphorylation of VEGFR2 Is Mediated by Receptor Trafficking: Insights from a Computational Model.

Matrix-binding isoforms and non-matrix-binding isoforms of vascular endothelial growth factor (VEGF) are both capable of stimulating vascular remodeling, but the resulting blood vessel networks are structurally and functionally different. Here, we develop and validate a computational model of the binding of soluble and immobilized ligands to VEGF receptor 2 (VEGFR2), the endosomal trafficking of VEGFR2, and site-specific VEGFR2 tyrosine phosphorylation to study differences in induced signaling between these VEGF isoforms. In capturing essential features of VEGFR2 signaling and trafficking, our model suggests that VEGFR2 trafficking parameters are largely consistent across multiple endothelial cell lines. Simulations demonstrate distinct localization of VEGFR2 phosphorylated on Y1175 and Y1214. This is the first model to clearly show that differences in site-specific VEGFR2 activation when stimulated with immobilized VEGF compared to soluble VEGF can be accounted for by altered trafficking of VEGFR2 without an intrinsic difference in receptor activation. The model predicts that Neuropilin-1 can induce differences in the surface-to-internal distribution of VEGFR2. Simulations also show that ligated VEGFR2 and phosphorylated VEGFR2 levels diverge over time following stimulation. Using this model, we identify multiple key levers that alter how VEGF binding to VEGFR2 results in different coordinated patterns of multiple downstream signaling pathways. Specifically, simulations predict that VEGF immobilization, interactions with Neuropilin-1, perturbations of VEGFR2 trafficking, and changes in expression or activity of phosphatases acting on VEGFR2 all affect the magnitude, duration, and relative strength of VEGFR2 phosphorylation on tyrosines 1175 and 1214, and they do so predictably within our single consistent model framework.

Endothelial cell-dependent regulation of arteriogenesis.

RATIONALE: Arteriogenesis is the process of formation of arterial conduits. Its promotion is an attractive therapeutic strategy in occlusive atherosclerotic diseases. Despite the functional and clinical importance of arteriogenesis, the biology of the process is poorly understood. Synectin, a gene previously implicated in the regulation of vascular endothelial cell growth factor signaling, offers a unique opportunity to determine relative contributions of various cell types to arteriogenesis. OBJECTIVE: We investigated the cell-autonomous effects of a synectin knockout in arterial morphogenesis. METHODS AND RESULTS: A floxed synectin knockin mouse line was crossbred with endothelial-specific (Tie2, Cdh5, Pdgfb) and smooth muscle myosin heavy chain-specific Cre driver mouse lines to produce cell type-specific deletions. Ablation of synectin expression in endothelial, but not smooth muscle cells resulted in the presence of developmental arterial morphogenetic defects (smaller size of the arterial tree, reduced number of arterial branches and collaterals) and impaired arteriogenesis in adult mice. CONCLUSIONS: Synectin modulates developmental and adult arteriogenesis in an endothelial cell-autonomous fashion. These findings show for the first time that endothelial cells are central to both developmental and adult arteriogenesis and provide a model for future studies of factors involved in this process.

Molecular mechanism matters: Benefits of mechanistic computational models for drug development.

Making drug development a more efficient and cost-effective process will have a transformative effect on human health. A key, yet underutilized, tool to aid in this transformation is mechanistic computational modeling. By incorporating decades of hard-won prior knowledge of molecular interactions, cellular signaling, and cellular behavior, mechanistic models can achieve a level of predictiveness that is not feasible using solely empirical characterization of drug pharmacodynamics. These models can integrate diverse types of data from cell culture and animal experiments, including high-throughput systems biology experiments, and translate the results into the context of human disease. This provides a framework for identification of new drug targets, measurable biomarkers for drug action in target tissues, and patient populations for which a drug is likely to be effective or ineffective. Additionally, mechanistic models are valuable in virtual screening of new therapeutic strategies, such as gene or cell therapy and tissue regeneration, identifying the key requirements for these approaches to succeed in a heterogeneous patient population. These capabilities, which are distinct from and complementary to those of existing drug development strategies, demonstrate the opportunity to improve success rates in the drug development pipeline through the use of mechanistic computational models.

A systems biology view of blood vessel growth and remodelling.

Blood travels throughout the body in an extensive network of vessels - arteries, veins and capillaries. This vascular network is not static, but instead dynamically remodels in response to stimuli from cells in the nearby tissue. In particular, the smallest vessels - arterioles, venules and capillaries - can be extended, expanded or pruned, in response to exercise, ischaemic events, pharmacological interventions, or other physiological and pathophysiological events. In this review, we describe the multi-step morphogenic process of angiogenesis - the sprouting of new blood vessels - and the stability of vascular networks in vivo. In particular, we review the known interactions between endothelial cells and the various blood cells and plasma components they convey. We describe progress that has been made in applying computational modelling, quantitative biology and high-throughput experimentation to the angiogenesis process.