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Constitutively secreted by endothelial cells, soluble FLT1 (sFLT1 or sVEGFR1) binds and sequesters extracellular vascular endothelial growth factors (VEGF), thereby reducing VEGF binding to VEGF receptor tyrosine kinases and their downstream signaling. In doing so, sFLT1 plays an important role in vascular development and in the patterning of new blood vessels in angiogenesis. Here, we develop multiple mechanistic models of sFLT1 secretion and identify a minimal mechanistic model that recapitulates key qualitative and quantitative features of temporal experimental datasets of sFLT1 secretion from multiple studies. We show that the experimental data on sFLT1 secretion is best represented by a delay differential equation (DDE) system including a maturation term, reflecting the time required between synthesis and secretion. Using optimization to identify appropriate values for the key mechanistic parameters in the model, we show that two model parameters (extracellular degradation rate constant and maturation time) are very strongly constrained by the experimental data, and that the remaining parameters are related by two strongly constrained constants. Thus, only one degree of freedom remains, and measurements of the intracellular levels of sFLT1 would fix the remaining parameters. Comparison between simulation predictions and additional experimental data of the outcomes of chemical inhibitors and genetic perturbations suggest that intermediate values of the secretion rate constant best match the simulation with experiments, which would completely constrain the model. However, some of the inhibitors tested produce results that cannot be reproduced by the model simulations, suggesting that additional mechanisms not included here are required to explain those inhibitors. Overall, the model reproduces most available experimental data and suggests targets for further quantitative investigation of the sFLT1 system.

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.

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.

The vascular endothelial growth factor receptors (VEGFRs) bind to cognate ligands to facilitate signaling pathways critical for angiogenesis, the growth of new capillaries from existing vasculature. Intracellular trafficking regulates the availability of receptors on the cell surface to bind ligands, which regulate activation, and the movement of activated receptors between the surface and intracellular pools, where they can initiate different signaling pathways. Using experimental data and computational modeling, we recently demonstrated and quantified the differential trafficking of three VEGF receptors, VEGFR1, VEGFR2, and coreceptor Neuropilin-1 (NRP1). Here, we expand that approach to quantify how the binding of different VEGF ligands alters the trafficking of these VEGF receptors and demonstrate the consequences of receptor localization and ligand binding on the localization and dynamics of signal initiation complexes. We include simulations of four different splice isoforms of VEGF-A and PLGF, each of which binds to different combinations of the VEGF receptors, and we use new experimental data for two of these ligands to parameterize and validate our model. We show that VEGFR2 trafficking is altered in response to ligand binding, but that trafficking of VEGFR1 is not; we also show that the altered trafficking can be explained by a single mechanistic process, increased internalization of the VEGFR2 receptor when bound to ligand; other processes are unaffected. We further show that even though the canonical view of receptor tyrosine kinases is of activation on the cell surface, most of the ligand-receptor complexes for both VEGFR1 and VEGFR2 are intracellular. We also explore the competition between the receptors for ligand binding, the so-called ‘decoy effect’, and show that while in vitro on the cell surface minimal such effect would be observed, inside the cell the effect can be substantial and may influence signaling. We term this location dependence the ‘reservoir effect’ as the size of the local ligand reservoir (large outside the cell, small inside the cell) plays an integral role in the receptor-receptor competition. These results expand our understanding of receptor-ligand trafficking dynamics and are critical for the design of therapeutic agents to regulate ligand availability to VEGFR1 and hence VEGF receptor signaling in angiogenesis.

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.

A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and anatomical sites harboring virus that contribute to viral rebound upon treatment interruption. Despite antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are reported in HIV-infected individuals on ART. Biomarkers for macrophage activation and neuronal damage in cerebrospinal fluid (CSF) of HIV-infected individuals demonstrate continued effects of HIV in brain and suggest that the central nervous system (CNS) may serve as a viral reservoir. Using a simian immunodeficiency virus (SIV)/macaque model for HIV encephalitis and AIDS, we evaluated whether infected cells persist in brain despite ART. Eight SIV-infected pig-tailed macaques were virally suppressed with ART, and plasma and CSF viremia levels were analyzed longitudinally. To assess whether virus persisted in brain macrophages (BrMΦ) in these macaques, we used a macrophage quantitative viral outgrowth assay (MΦ-QVOA), PCR, and in situ hybridization (ISH) to measure the frequency of infected cells and the levels of viral RNA and DNA in brain. Viral RNA in brain tissue of suppressed macaques was undetectable, although viral DNA was detected in all animals. The MΦ-QVOA demonstrated that the majority of suppressed animals contained latently infected BrMΦ. We also showed that virus produced in the MΦ-QVOAs was replication competent, suggesting that latently infected BrMΦ are capable of reestablishing productive infection upon treatment interruption. This report provides the first confirmation of the presence of replication-competent SIV in BrMΦ of ART-suppressed macaques and suggests that the highly debated issue of viral latency in macrophages, at least in brain, has been addressed in SIV-infected macaques treated with ART. IMPORTANCE Resting CD4 + T cells are currently the only cells that fit the definition of a latent reservoir. However, recent evidence suggests that HIV/SIV-infected macrophages persist despite ART. Markers of macrophage activation and neuronal damage are observed in the CSF of HIV-infected individuals and of SIV-infected macaques on suppressive ART regimens, suggesting that the CNS has continued virus infection and latent infection. A controversy exists as to whether brain macrophages represent a latent source of replication-competent virus capable of reestablishing infection upon treatment interruption. In this study, we demonstrated the presence of the latent macrophage reservoir in brains of SIV-infected ART-treated macaques and analyzed the reservoir using our established outgrowth assay to quantitate macrophages harboring replication-competent SIV genomes. Our results support the idea of the existence of other latent reservoirs in addition to resting CD4 + T cells and underscore the importance of macrophages in developing strategies to eradicate HIV. Resting CD4 + T cells are currently the only cells that fit the definition of a latent reservoir. However, recent evidence suggests that HIV/SIV-infected macrophages persist despite ART. Markers of macrophage activation and neuronal damage are observed in the CSF of HIV-infected individuals and of SIV-infected macaques on suppressive ART regimens, suggesting that the CNS has continued virus infection and latent infection. A controversy exists as to whether brain macrophages represent a latent source of replication-competent virus capable of reestablishing infection upon treatment interruption. In this study, we demonstrated the presence of the latent macrophage reservoir in brains of SIV-infected ART-treated macaques and analyzed the reservoir using our established outgrowth assay to quantitate macrophages harboring replication-competent SIV genomes. Our results support the idea of the existence of other latent reservoirs in addition to resting CD4 + T cells and underscore the importance of macrophages in developing strategies to eradicate HIV.

Computational models are complex scientific constructs that have become essential for us to better understand the world. Many models are valuable for peers within and beyond disciplinary boundaries. However, there are no widely agreed-upon standards for sharing models. This paper suggests 10 simple rules for you to both (i) ensure you share models in a way that is at least “good enough,” and (ii) enable others to lead the change towards better model-sharing practices.

Methods and Software. Since its founding in 2005, PLOS Computational Biology has been the home of exceptional computational research and cutting-edge methodological advances. [...]techniques that are used to process neuroscience data may be quite different from those used to analyze genomic data. [...]having expertise in those fields—as our Neuroscience and Genomics editors do—is increasingly helpful for the review and handling of these submissions. [...]Methods and Software submissions are now handled by domain-specific editors, as other Research articles are, which both increases the pool of editors handling these submissions and provides each submission with editors that are familiar with the subtleties and background of the domain-specific approaches, applications, and implementations.

This study provides further evidence that the latent reservoir is comprised of both CD4 + T cells and myeloid cells. The data presented here suggest that CD4 + T cells and macrophages found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. Additionally, we have shown that monocytes in blood contain latent virus and, though not considered a reservoir themselves due to their short life span, could contribute to the size of the latent reservoir upon entering the tissue and differentiating into long-lived macrophages. These new insights into the size and location of the SIV reservoir using a model that is heavily studied in the HIV field could have great implications for HIV-infected individuals and should be taken into consideration with the development of future HIV cure strategies. Human immunodeficiency virus (HIV) eradication or long-term suppression in the absence of antiretroviral therapy (ART) requires an understanding of all viral reservoirs that could contribute to viral rebound after ART interruption. CD4 T cells (CD4s) are recognized as the predominant reservoir in HIV type 1 (HIV-1)-infected individuals. However, macrophages are also infected by HIV-1 and simian immunodeficiency virus (SIV) during acute infection and may persist throughout ART, contributing to the size of the latent reservoir. We sought to determine whether tissue macrophages contribute to the SIVmac251 reservoir in suppressed macaques. Using cell-specific quantitative viral outgrowth assays (CD4-QVOA and MΦ-QVOA), we measured functional latent reservoirs in CD4s and macrophages in ART-suppressed SIVmac251-infected macaques. Spleen, lung, and brain in all suppressed animals contained latently infected macrophages, undetectable or low-level SIV RNA, and detectable SIV DNA. Silent viral genomes with potential for reactivation and viral spread were also identified in blood monocytes, although these cells might not be considered reservoirs due to their short life span. Additionally, virus produced in the MΦ-QVOA was capable of infecting healthy activated CD4s. Our results strongly suggest that functional latent reservoirs in CD4s and macrophages can contribute to viral rebound and reestablishment of productive infection after ART interruption. These findings should be considered in the design and implementation of future HIV cure strategies. IMPORTANCE This study provides further evidence that the latent reservoir is comprised of both CD4 + T cells and myeloid cells. The data presented here suggest that CD4 + T cells and macrophages found throughout tissues in the body can contain replication-competent SIV and contribute to rebound of the virus after treatment interruption. Additionally, we have shown that monocytes in blood contain latent virus and, though not considered a reservoir themselves due to their short life span, could contribute to the size of the latent reservoir upon entering the tissue and differentiating into long-lived macrophages. These new insights into the size and location of the SIV reservoir using a model that is heavily studied in the HIV field could have great implications for HIV-infected individuals and should be taken into consideration with the development of future HIV cure strategies.