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IL-8 (CXCL8) is a potent chemoattractant and pro-angiogenic factor that is involved in maintaining homeostasis and is implicated in a wide range of inflammatory disorders. IL-8 was initially understood to be produced by monocytes to induce neutrophil migration through binding surface receptors IL-8RA (CXCR1) and IL-8RB (CXCR2). Although neutrophil secretion of IL-8 has been reported as ranging from 0--10 molecules/cell/second [1,2], it is unclear how this may affect neutrophil activation. In this study, we create and parameterize a mechanistic model of IL-8 signaling using data from in vitro cell culture experiments. We use this model to estimate receptor internalization rates which had not been previously reported. Through sensitivity analyses and additional simulations, we find that neutrophil secretion regulates the level of IL-8RB available, especially in pM-range environments.

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.

We built a whole-body computational model to study the role of the poorly understood vascular endothelial growth factor (VEGF)165b splice isoform in peripheral artery disease (PAD). This model was built and validated using published and new experimental data from cells, mice, and humans, and explicitly accounts for known properties of VEGF165b : lack of extracellular matrix (ECM)-binding and weak phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR2) in vitro. The resulting model captures all known information about VEGF165b distribution and signaling in human PAD, and provides novel, nonintuitive insight into VEGF165b mechanism of action in vivo. Although VEGF165a and VEGF165b compete for VEGFR2 in vitro, simulations show that these isoforms do not compete for VEGFR2 at much lower physiological concentrations. Instead, reduced VEGF165a may drive impaired VEGFR2 signaling. The model predicts that VEGF165b does compete for binding to VEGFR1, supporting a VEGFR1-mediated response to anti-VEGF165b . The model predicts a key role for VEGF165b in PAD, but in a different way than previously hypothesized.

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.

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.

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Inducing therapeutic angiogenesis to effectively form hierarchical, non-leaky networks of perfused vessels in tissue engineering applications and ischemic disease remains an unmet challenge, despite extensive research and multiple clinical trials. Here, we use a previously-developed, multi-scale, computational systems pharmacology model of human peripheral artery disease to screen a diverse array of promising pro-angiogenic strategies, including gene therapy, biomaterials, and antibodies. Our previously-validated model explicitly accounts for VEGF immobilization, Neuropilin-1 binding, and weak activation of VEGF receptor 2 (VEGFR2) by the “VEGF(xxxb)” isoforms. First, we examine biomaterial-based delivery of VEGF engineered for increased affinity to the extracellular matrix. We show that these constructs maintain VEGF close to physiological levels and extend the duration of VEGFR2 activation. We demonstrate the importance of sub-saturating VEGF dosing to prevent angioma formation. Second, we examine the potential of ligand- or receptor-based gene therapy to normalize VEGF receptor signaling. Third, we explore the potential for antibody-based pro-angiogenic therapy. Our model supports recent observations that improvement in perfusion following treatment with anti-VEGF(165b) in mice is mediated by VEGF-receptor 1, not VEGFR2. Surprisingly, the model predicts that the approved anti-VEGF cancer drug, bevacizumab, may actually improve signaling of both VEGFR1 and VEGFR2 via a novel ‘antibody swapping’ effect that we demonstrate here. Altogether, this model provides insight into the mechanisms of action of several classes of pro-angiogenic strategies within the context of the complex molecular and physiological processes occurring in vivo. We identify molecular signaling similarities between promising approaches and key differences between promising and ineffective strategies. INSIGHT STATEMENT: Inducing angiogenesis to form hierarchical networks of perfused vessels remains an unmet challenge. We use a computational systems pharmacology model to screen multiple promising pro-angiogenic strategies, highlighting the dependence of therapy action on the complex underlying molecular and physiological processes occurring in vivo. We show that biomaterial-based delivery of VEGF engineered for increased affinity to the extracellular matrix maintains VEGF close to physiological levels and extends the duration of VEGFR2 activation without saturating VEGFR2. We also explore antibody-based pro-angiogenic therapy, demonstrating accurate model prediction of signaling in mice following treatment with anti-VEGF(165b). Intriguingly, the model predicts that an approved anti-VEGF drug, bevacizumab, may improve signaling of both VEGFR1 and VEGFR2 via a novel ‘antibody swapping’ effect that we demonstrate here.

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.

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 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.

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., Author summary Angiogenesis, the growth of new blood vessels from the existing vasculature, is critical for maintenance of health and response to injury. In ischemic disease, this process is impaired, but therapies targeting a key family of proteins, the vascular endothelial growth factors (VEGF), have failed to translate clinically. This suggests a need for deeper understanding of the complex regulation underlying angiogenic signaling. Here, we translate a previously developed and validated model of VEGF family signaling into a human, whole-body framework. The different splice isoforms of VEGF and the related PlGF proteins have different affinities for the extracellular matrix (ECM) and the co-receptor Neuropilin-1. Using our model, we examine the effect of these different binding properties on the distribution of each isoform in tissue, and subsequent receptor signaling. The model predicts isoform-specific receptor activation that is consistent with observed vascular phenotypes in mice expressing a single VEGF isoform; non-ECM-binding isoforms lead to signaling that promotes cell proliferation, while strong ECM-binding promotes migratory signaling and increased vessel branching. This understanding is critical for design of biomaterials that manipulate VEGF-ECM binding to control growth factor delivery, and for understanding of splicing-induced changes in VEGF family signaling in different tissues and in disease.

We summarize recent experimental and computational studies that investigate molecular and cellular mechanisms of sprouting angiogenesis. We discuss how experimental tools have unveiled new opportunities for computational modeling by providing detailed phenomenological descriptions and conceptual models of cell-level behaviors underpinned by high-quality molecular data. Using recent examples, we show how new understanding results from bridging computational and experimental approaches.Experimental data extends beyond the tip cell vs. stalk cell paradigm, and involves numerous molecular inputs such as vascular endothelial growth factor and Notch. This data is being used to generate and validate computational models, which can then be used to predict the results of hypothetical experiments that are difficult to perform in the laboratory, and to generate new hypotheses that account for system-wide interactions. As a result of this integration, descriptions of critical gradients of growth factor-receptor complexes have been generated, and new modulators of cell behavior have been described.We suggest that the recent emphasis on the different stages of sprouting angiogenesis, and integration of experimental and computational approaches, should provide a way to manage the complexity of this process and help identify new regulatory paradigms and therapeutic targets.

Several cytokine families have roles in the development, maintenance, and remodeling of the microcirculation. Of these, the vascular endothelial growth factor (VEGF) family is one of the best studied and one of the most complex. Five VEGF ligand genes and five cell-surface receptor genes are known in the human, and each of these may be transcribed as multiple splice isoforms to generate an extensive family of proteins, many of which are subject to further proteolytic processing. Using the VEGF family as an example, we describe the current knowledge of growth-factor expression, processing, and transport in vivo. Experimental studies and computational simulations are being used to measure and predict the activity of these molecules, and we describe avenues of research that seek to fill the remaining gaps in our understanding of VEGF family behavior.

Extensive experimental studies have identified vascular endothelial growth factor (VEGF) concentrations and concentration gradients as major factors in angiogenesis; however, localized in vivo measurements of these parameters have not been possible. We developed a three-dimensional computational model of skeletal muscle fibers, blood vessels, and interstitial space. Here it is applied to rat extensor digitorum longus. VEGF isoforms are secreted by myocytes, diffuse through extracellular matrix and basement membranes, and bind endothelial cell surface receptors on blood vessels. In addition, one isoform, VEGF164, binds to proteoglycans in the interstitial space. VEGF secretion rate is determined from the predicted tissue oxygen level through its effect on the hypoxia inducible factor-1α transcription factor. We estimate VEGF secretion and its concentrations and gradients in resting muscle and for different levels of exercise. The effects of low levels of inspired oxygen are also studied. We predict that the high spatial heterogeneity of muscle fiber VEGF secretion in hypoxic tissue leads to significant gradients of VEGF concentration and VEGF receptor activation. VEGF concentration gradients are predicted to be significant in both resting and exercising muscle (4% and 6–8% change in VEGF over 10 μm, respectively), sufficient for chemotactic guidance of 50-μm-long sprout tip cells. VEGF gradients also result in heterogeneity in VEGF receptor activation—a possible explanation for the stochasticity of sprout location. In the absence of interstitial flow, gradients are 10-fold steeper in the transverse direction (i.e., perpendicular to the muscle fibers) than in the longitudinal direction. This may explain observed perpendicular anastomoses in skeletal muscle.

Background The vascular endothelial growth factor (VEGF) family is central to cancer angiogenesis. However, targeting VEGF as an anti-cancer therapeutic approach has shown success for some tumor types but not others. Here we examine the expression of the expanded VEGF family in prostate cancer, including the Semaphorin (Sema) family members that compete with VEGFs for Neuropilin binding and can themselves have pro- or anti-angiogenic activity. Results First, we used multivariate statistical methods, including partial least squares and clustering, to examine VEGF/Sema gene expression variability in previously published prostate cancer microarray datasets. We show that unlike some cancers, such as kidney cancer, primary prostate cancer is characterized by both a down-regulation of the pro-angiogenic members of the VEGF family and a down-regulation of anti-angiogenic members of the Sema family. We found pro-lymphangiogenic signatures, including the genes encoding VEGFC and VEGFD, associated with primary tumors that ultimately became aggressive. In contrast to primary prostate tumors, prostate cancer metastases showed increased expression of key pro-angiogenic VEGF family members and further repression of anti-angiogenic class III Sema family members. Given the lack of success of VEGF-targeting molecules so far in prostate cancer, this suggests that the reduction in anti-angiogenic Sema signaling may potentiate VEGF signaling and even promote resistance to VEGF-targeting therapies. Inhibition of the VEGF ‘accelerator’ may need to be accompanied by promotion of the Sema ‘brake’ to block cancer angiogenesis. To leverage our mechanistic understanding, and to link multigene expression changes to outcomes, we performed individualized computational simulations of competitive VEGF and Sema receptor binding across many tumor samples. The simulations suggest that loss of Sema expression promotes angiogenesis by lowering plexin signaling, not by potentiating VEGF signaling via relaxation of competition. Conclusions The combined analysis of bioinformatic data with computational modeling of ligand-receptor interactions demonstrated that enhancement of angiogenesis in prostate cancer metastases may occur through two different routes: elevation of VEGFA and reduction of class 3 Semaphorins. Therapeutic inhibition of angiogenesis in metastatic prostate cancer should account for both of these routes. Electronic supplementary material The online version of this article (doi:10.1186/s12918-015-0201-z) contains supplementary material, which is available to authorized users.

The vascular network carries blood throughout the body, delivering oxygen to tissues and providing a pathway for communication between distant organs. The network is hierarchical and structured, but also dynamic, especially at the smaller scales. Remodeling of the microvasculature occurs in response to local changes in oxygen, gene expression, cell–cell communication, and chemical and mechanical stimuli from the microenvironment. These local changes occur as a result of physiological processes such as growth and exercise, as well as acute and chronic diseases including stroke, cancer, and diabetes, and pharmacological intervention. While the vasculature is an important therapeutic target in many diseases, drugs designed to inhibit vascular growth have achieved only limited success, and no drug has yet been approved to promote therapeutic vascular remodeling. This highlights the challenges involved in identifying appropriate therapeutic targets in a system as complex as the vasculature. Systems biology approaches provide a means to bridge current understanding of the vascular system, from detailed signaling dynamics measured in vitro and pre-clinical animal models of vascular disease, to a more complete picture of vascular regulation in vivo. This will translate to an improved ability to identify multi-component biomarkers for diagnosis, prognosis, and monitoring of therapy that are easy to measure in vivo, as well as better drug targets for specific disease states. In this review, we summarize systems biology approaches that have advanced our understanding of vascular function and dysfunction in vivo, with a focus on computational modeling.

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.

The regulation of vascular endothelial growth factor A (VEGF) is critical to neovascularization in numerous tissues under physiological and pathological conditions. VEGF has multiple isoforms, created by alternative splicing or proteolytic cleavage, and characterized by different receptor-binding and matrix-binding properties. These isoforms are known to give rise to a spectrum of angiogenesis patterns marked by differences in branching, which has functional implications for tissues. In this review, we detail the extensive extracellular regulation of VEGF and the ability of VEGF to dictate the vascular phenotype. We explore the role of VEGF-releasing proteases and soluble carrier molecules on VEGF activity. While proteases such as MMP9 can 'release' matrix-bound VEGF and promote angiogenesis, for example as a key step in carcinogenesis, proteases can also suppress VEGF's angiogenic effects. We explore what dictates pro- or anti-angiogenic behavior. We also seek to understand the phenomenon of VEGF gradient formation. Strong VEGF gradients are thought to be due to decreased rates of diffusion from reversible matrix binding, however theoretical studies show that this scenario cannot give rise to lasting VEGF gradients in vivo. We propose that gradients are formed through degradation of sequestered VEGF. Finally, we review how different aspects of the VEGF signal, such as its concentration, gradient, matrix-binding, and NRP1-binding can differentially affect angiogenesis. We explore how this allows VEGF to regulate the formation of vascular networks across a spectrum of high to low branching densities, and from normal to pathological angiogenesis. A better understanding of the control of angiogenesis is necessary to improve upon limitations of current angiogenic therapies.

The vascular endothelial growth factor (VEGF) family of cytokines promotes vascularization, tumorigenesis and metastasis in many cancers. Our goal is to develop computational models that combine mechanistic topological data on the VEGF protein interaction network with gene expression datasets for a large population of prostate cancers. We have assembled databases of prostate cancer gene expression data, and analyzed the data using bioinformatic techniques, identifying key VEGF-based subgroups of prostate cancer plus biomarkers that identify these groups. This year, we have submitted and revised a manuscript based on this analysis, we expect it to be accepted soon. We have also created new computational models to simulate prostate cancer, based on the individualized gene expression data. We have used these models to simulate therapies that target the VEGF pathway, including anti-ligands such as bevacizumab and anti-receptors. Among are predictions are estimates of the high degree of variability in response to drugs that is predicted by the model, which is reflected in the results of clinical trials targeting VEGF in prostate cancer. This progress will continue into Year 3, where we will expand the VEGF family members included in our models, and extract key biomarkers indicative of likely success of VEGF- or VEGFR-targeting therapies.

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 murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published(1-3). For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 μm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

Triple negative breast cancers (TNBC) are difficult to treat due to a lack of targets and heterogeneity. Inhibition of angiogenesis is a promising therapeutic strategy, but has had limited effectiveness so far in breast cancer. To quantify heterogeneity in angiogenesis-related gene expression in breast cancer, we focused on two families – VEGFs and semaphorins – that compete for neuropilin co-receptors on endothelial cells. We compiled microarray data for over 2,600 patient tumor samples and analyzed the expression of VEGF- and semaphorin-related ligands and receptors. We used principal component analysis to identify patterns of gene expression, and clustering to group samples according to these patterns. We used available survival data to determine whether these clusters had prognostic as well as therapeutic relevance. TNBC was highly associated with dysregulation of VEGF- and semaphorin-related genes; in particular, it appeared that expression of both VEGF and semaphorin genes were altered in a pro-angiogenesis direction. A pattern of high VEGFA expression with low expression of secreted semaphorins was associated with 60% of triple-negative breast tumors. While all TNBC groups demonstrated poor prognosis, this signature also correlated with lower 5-year survival rates in non-TNBC samples. A second TNBC pattern, including high VEGFC expression, was also identified. These pro-angiogenesis signatures may identify cancers that are more susceptible to VEGF inhibition.

The interplay between the innate immune system restriction factor APOBEC3G and the HIV protein Vif is a key host-retrovirus interaction. APOBEC3G can counteract HIV infection in at least two ways: by inducing lethal mutations on the viral cDNA; and by blocking steps in reverse transcription and viral integration into the host genome. HIV-Vif blocks these antiviral functions of APOBEC3G by impeding its encapsulation. Nonetheless, it has been shown that overexpression of APOBEC3G, or interfering with APOBEC3G-Vif binding, can efficiently block in vitro HIV replication. Some clinical studies have also suggested that high levels of APOBEC3G expression in HIV patients are correlated with increased CD4+ T cell count and low levels of viral load; however, other studies have reported contradictory results and challenged this observation. Stem cell therapy to replace a patient’s immune cells with cells that are more HIV-resistant is a promising approach. Pre-implantation gene transfection of these stem cells can augment the HIV-resistance of progeny CD4+ T cells. As a protein, APOBEC3G has the advantage that it can be genetically encoded, while small molecules cannot. We have developed a mathematical model to quantitatively study the effects on in vivo HIV replication of therapeutic delivery of CD34+ stem cells transfected to overexpress APOBEC3G. Our model suggests that stem cell therapy resulting in a high fraction of APOBEC3G-overexpressing CD4+ T cells can effectively inhibit in vivo HIV replication. We extended our model to simulate the combination of APOBEC3G therapy with other biological activities, to estimate the likelihood of improved outcomes.

Adipose-derived stromal/stem cells (ASCs) are a promising cell source for vascular-based approaches to clinical therapeutics, as they have been shown to give rise to both endothelial and perivascular cells. While it is well known that ASCs can present a heterogeneous phenotypic profile, spontaneous interactions among these subpopulations that result in the formation of complex tissue structures have not been rigorously demonstrated. Our study reports the novel finding that ASCs grown in monolayers in the presence of angiogenic cues are capable of self-assembling into complex, three-dimensional vascular structures. This phenomenon is only apparent when the ASCs are seeded at a high density (20,000 cells/cm(2)) and occur through orchestrated interactions among three distinct subpopulations: CD31-positive cells (CD31+), α-smooth muscle actin-positive cells (αSMA+), and cells that are unstained for both these markers (CD31-/αSMA-). Investigations into the kinetics of the process revealed that endothelial vessel-like structures initially arose from individual CD31+ cells through proliferation and their interactions with CD31-/αSMA- cells. During this period, αSMA+ cells proliferated and appeared to migrate toward the vessel structures, eventually engaging in cell-cell contact with them after 1 week. By 2 weeks, the lumen-containing CD31+ vessels grew greater than a millimeter in length, were lined with vascular basement membrane proteins, and were encased within a dense, three-dimensional cluster of αSMA+ and CD31-/αSMA- cells. The recruitment of αSMA+ cells was largely due to platelet-derived growth factor (PDGF) signaling, as the inhibition of PDGF receptors substantially reduced αSMA+ cell growth and vessel coverage. Additionally, we found that while hypoxia increased endothelial gene expression and vessel width, it also inhibited the growth of the αSMA+ population. Together, these findings underscore the potential use of ASCs in forming mature vessels in vitro as well as the need for a further understanding of the heterotypic interactions among ASC subpopulations.

The human APOBEC3G is an innate restriction factor that, in the absence of Vif, restricts HIV-1 replication by inducing excessive deamination of cytidine residues in nascent reverse transcripts and inhibiting reverse transcription and integration. To shed light on impact of A3G-Vif interactions on HIV replication, we developed a multi-scale computational system consisting of intracellular (single-cell), cellular and extracellular (multicellular) events by using ordinary differential equations. The single-cell model describes molecular-level events within individual cells (such as production and degradation of host and viral proteins, and assembly and release of new virions), whereas the multicellular model describes the viral dynamics and multiple cycles of infection within a population of cells. We estimated the model parameters either directly from previously published experimental data or by running simulations to find the optimum values. We validated our integrated model by reproducing the results of in vitro T cell culture experiments. Crucially, both downstream effects of A3G (hypermutation and reduction of viral burst size) were necessary to replicate the experimental results in silico. We also used the model to study anti-HIV capability of several possible therapeutic strategies including: an antibody to Vif; upregulation of A3G; and mutated forms of A3G. According to our simulations, A3G with a mutated Vif binding site is predicted to be significantly more effective than other molecules at the same dose. Ultimately, we performed sensitivity analysis to identify important model parameters. The results showed that the timing of particle formation and virus release had the highest impacts on HIV replication. The model also predicted that the degradation of A3G by Vif is not a crucial step in HIV pathogenesis., Author Summary According to UNAIDS (The Joint UN Programme on HIV/AIDS) and WHO, HIV/AIDS has killed more than 25 million people worldwide since its recognition in 1981. Recently, APOBEC3G, a member of the APOBEC family, has been shown to be a potent inhibitor of HIV infection. In contrast, a viral protein, called Vif, is known to protect the virus by binding to APOBEC3G and causing the degradation of this enzyme. We have developed a computational model to simulate in vitro experiments that include A3G-Vif interactions at the intracellular level and T cell-HIV dynamics at the multicellular level. Experimental data were used to establish system parameters and also to validate predictions of our models. We studied various drugs targeting APOBEC3G and Vif pathways to find the optimum therapeutic approach against HIV replication. Our model predicted that a mutated form of APOBEC3G that does not bind to Vif performs significantly better at suppressing HIV replication compared to other drugs. We also found that the drug should be administered shortly after infection and it must be available to all cells in order to be effective.

The process of oxygen delivery from capillary to muscle fiber is essential for a tissue with variable oxygen demand, such as skeletal muscle. Oxygen distribution in exercising skeletal muscle is regulated by convective oxygen transport in the blood vessels, oxygen diffusion and consumption in the tissue. Spatial heterogeneities in oxygen supply, such as microvascular architecture and hemodynamic variables, had been observed experimentally and their marked effects on oxygen exchange had been confirmed using mathematical models. In this study, we investigate the effects of heterogeneities in oxygen demand on tissue oxygenation distribution using a multiscale oxygen transport model. Muscles are composed of different ratios of the various fiber types. Each fiber type has characteristic values of several parameters, including fiber size, oxygen consumption, myoglobin concentration, and oxygen diffusivity. Using experimentally measured parameters for different fiber types and applying them to the rat extensor digitorum longus muscle, we evaluated the effects of heterogeneous fiber size and fiber type properties on the oxygen distribution profile. Our simulation results suggest a marked increase in spatial heterogeneity of oxygen due to fiber size distribution in a mixed muscle. Our simulations also suggest that the combined effects of fiber type properties, except size, do not contribute significantly to the tissue oxygen spatial heterogeneity. However, the incorporation of the difference in oxygen consumption rates of different fiber types alone causes higher oxygen heterogeneity compared to control cases with uniform fiber properties. In contrast, incorporating variation in other fiber type-specific properties, such as myoglobin concentration, causes little change in spatial tissue oxygenation profiles.

In recent years, Judah Folkman’s vision of anticancer therapeutics based on inhibiting angiogenesis pathways has begun to be realized. In particular, antibodies sequestering VEGF, and small molecule tyrosine kinase inhibitors targeting VEGF receptors, are now approved for various cancers, alone or in combination with other therapies. More are in the development pipeline, with similar or distinct mechanisms of action. The VEGF system is complex, involving multiple ligands and receptors, along with transport throughout the body via the bloodstream. Predicting outcomes of perturbing this system, either by a single agent or combinations, can be aided by in silico models. Here we discuss the ways in which the above drugs target the VEGF pathway in cancer, and describe the development and implementation of multiscale mathematical models to simulate the action of these drugs in order to predict and compare their likely efficacy for various types of tumors.

We have created an experimentally-based computational model describing spatial transport of vascular endothelial growth factor (VEGF) and its receptors to quantitatively understand how guidance cues may modulate blood vessel sprout growth. VEGF binds to endothelial cells and initiates angiogenesis. Both VEGF concentration and VEGF gradients may control sprout formation. Soluble VEGF receptor 1 (sFlt1) can bind and sequester VEGF. Based on observations in developing vasculature, we hypothesize that a local reduction in sFlt1 expression can increase locally available VEGF and thus control angiogenesis. However, the complex VEGF interaction network makes it difficult to isolate how individual proteins contribute to the spatial distribution of the growth factor using experiments alone. Our computational model represents the local environment of a single blood vessel and nearby tissue and directly incorporates the network of VEGF interactions. In the model, parenchymal cells secrete VEGF, which diffuses through interstitial space and binds extracellular matrix (ECM) and sFlt1. VEGF binds endothelial cells via membrane-bound receptors Flt1 and Flk1, and endothelial cells secrete sFlt1. Additionally, the model accounts for degradation of VEGF and sFlt1 as well as internalization of receptor-bound ligands. Using partial differential equations, we simulate this system, which is constrained by experimentally-derived parameters. Our simulations show that when a sprout-leading tip cell secretes less sFlt1 than neighboring cells, there is decreased local sFlt1 sequestration of VEGF, thus resulting in augmented VEGF-Flk1 levels on the surface of the low-sFlt1 secreting tip cell. This could lead to sprout generation. We also show how variations in sFlt1 secretion and tip cell configuration may affect the gradients of guidance cues and directionality of sprout growth.

Tumour angiogenesis allows a growing mass of cancer cells to overcome oxygen diffusion limitation and to increase cell survival. The growth of capillaries from pre-existing blood vessels is the result of numerous signalling cascades involving different molecules and of cellular events involving multiple cell and tissue types. Computational models offer insight into the mechanisms governing angiogenesis and provide quantitative information on parameters difficult to assess by experiments alone. In this article, we summarize results from computational models of tumour angiogenic processes with a focus on the molecular-detailed vascular endothelial growth factor-associated models that have been developed in our laboratory, spanning multiple scales from the molecular to whole body.

Vascular endothelial growth factor (VEGF) is one of the most potent cytokines targeted in anti-angiogenic therapies. Bevacizumab, a recombinant humanized monoclonal antibody to VEGF, is being used clinically in combination with chemotherapy for colorectal, non-small cell lung and breast cancers, and as a single agent for glioblastoma, and is being tested for other types of cancer in numerous clinical trials. It has been reported that the intravenous injection of bevacizumab leads to an increase of plasma VEGF concentration in cancer patients. The mechanism responsible for this counterintuitive increase has not been elucidated, although several hypotheses have been proposed. We use a multiscale systems biology approach to address this problem. We have constructed a whole-body pharmacokinetic model comprising three compartments: blood, normal tissue and tumor tissue. Molecular interactions between VEGF-A family members, their major receptors, the extracellular matrix, and an anti-VEGF ligand are considered for each compartment. Diffusible molecules extravasate, intravasate, are removed from the healthy tissue through the lymphatics, and are cleared from the blood. Our model reproduces the experimentally-observed increase of plasma VEGF following intravenous administration of bevacizumab, and predicts this increase to be a consequence of inter-compartmental exchange of VEGF, the anti-VEGF agent and the VEGF/anti-VEGF complex. Our results suggest that a fraction of the anti-VEGF drug extravasates, allowing the agent to bind the interstitial VEGF. When the complex intravasates (via a combination of lymphatic drainage and microvascular transport of macromolecules) and dissociates in the blood, VEGF is released and the VEGF concentration increases in the plasma. These results provide a new hypothesis on the kinetics of VEGF and on the VEGF distribution in the body caused by anti-angiogenic therapies, as well as their mechanisms of action and could help in designing anti-angiogenic therapies.

Microcirculation (2010) 17, 333–347. doi: 10.1111/j.1549-8719.2010.00034.x Abstract Objective: Chronic and acute ischemic diseases—peripheral artery disease, coronary artery disease, stroke—result in tissue damage unless blood flow is maintained or restored in a timely manner. Mice of different strains recover from arteriolar ligation (by increasing collateral blood flow) at different speeds. We quantify the spatio-temporal patterns of microvascular network remodeling following arteriolar ligation in different mouse strains to better understand inter-individual variability. Methods: Whole-muscle spinotrapezius microvascular networks of mouse strains C57Bl/6, Balb/c and CD1 were imaged using confocal microscopy following ligation of feeding arterioles. Results: Baseline arteriolar structures of C57Bl/6 and Balb/c mice feature heavily ramified arcades and unconnected dendritic trees, respectively. This network angioarchitecture identifies ischemia-protected and ischemia-vulnerable tissues; unlike C57Bl/6, downstream capillary perfusion in Balb/c spinotrapezius is lost following ligation. Perfusion recovery requires arterialization (expansion and investment of mural cells) of a subset of capillaries forming a new low-resistance collateral pathway between arteriolar trees. Outbred CD1 exhibit either Balb/c-like or C57Bl/6-like spinotrapezius angioarchitecture, predictive of response to arteriolar ligation. Conclusions: This collateral capillary arterialization process may explain the reported longer time required for blood flow recovery in Balb/c hindlimb ischemia, as low-resistance blood flow pathways along capillary conduits must be formed (“arterialization”) before reperfusion.

Background VEGF proteolysis by plasmin or matrix metalloproteinases (MMPs) is believed to play an important role in regulating vascular patterning in vivo by releasing VEGF from the extracellular matrix (ECM). However, a quantitative understanding of the kinetics of VEGF cleavage and the efficiency of cell-mediated VEGF release is currently lacking. To address these uncertainties, we develop a molecular-detailed quantitative model of VEGF proteolysis, used here in the context of an endothelial sprout. Methodology and Findings To study a cell's ability to cleave VEGF, the model captures MMP secretion, VEGF-ECM binding, VEGF proteolysis from VEGF165 to VEGF114 (the expected MMP cleavage product of VEGF165) and VEGF receptor-mediated recapture. Using experimental data, we estimated the effective bimolecular rate constant of VEGF165 cleavage by plasmin to be 328 M−1s−1 at 25°C, which is relatively slow compared to typical MMP-ECM proteolysis reactions. While previous studies have implicated cellular proteolysis in growth factor processing, we show that single cells do not individually have the capacity to cleave VEGF to any appreciable extent (less than 0.1% conversion). In addition, we find that a tip cell's receptor system will not efficiently recapture the cleaved VEGF due to an inability of cleaved VEGF to associate with Neuropilin-1. Conclusions Overall, VEGF165 cleavage in vivo is likely to be mediated by the combined effect of numerous cells, instead of behaving in a single-cell-directed, autocrine manner. We show that heparan sulfate proteoglycans (HSPGs) potentiate VEGF cleavage by increasing the VEGF clearance time in tissues. In addition, we find that the VEGF-HSPG complex is more sensitive to proteases than is soluble VEGF, which may imply its potential relevance in receptor signaling. Finally, according to our calculations, experimentally measured soluble protease levels are approximately two orders of magnitude lower than that needed to reconcile levels of VEGF cleavage seen in pathological situations.

Vascular endothelial growth factor (VEGF) is a family of cytokines for which the dysregulation of expression is involved in many diseases; for some, excess VEGF causes pathological hypervascularization, while for others VEGF-induced vascular remodeling may alleviate ischemia and/or hypoxia. Anti-angiogenic therapies attacking the VEGF pathway have begun to live up to their promise for treatment of certain cancers and of age-related macular degeneration. However, the corollary is not yet true: in coronary artery disease and peripheral artery disease, clinical trials of pro-angiogenic VEGF delivery have not, so far, proven successful. The VEGF and VEGF-receptor system is complex, with at least five ligand genes, some encoding multiple protein isoforms and five receptor genes. A systems biology approach for designing pro-angiogenic therapies, using a combination of quantitative experimental approaches and detailed computational models, is essential to deal with this complexity and to understand the effects of drugs targeting the system. This approach allows us to learn from unsuccessful clinical trials and to design and test novel single therapeutics or combinations of therapeutics. Among the parameters that can be varied in order to determine optimal strategy are dosage, timing of multiple doses, route of administration, and the molecular target.

Angiogenesis is the growth of new capillaries from pre-existent microvasculature. A wide range of pathological conditions, from atherosclerosis to cancer, can be attributed to either excessive or deficient angiogenesis. Central to the physiological regulation of angiogenesis is the vascular endothelial growth factor (VEGF) system – its ligands and receptors (VEGFRs) are thus prime molecular targets of pro-angiogenic and anti-angiogenic therapies. Of growing interest as a prognostic marker and therapeutic target in angiogenesis-dependent diseases is soluble VEGF receptor-1 (sVEGFR1, also known as sFlt-1) – a truncated version of the cell membrane-spanning VEGFR1. For instance, it is known that sVEGFR1 is involved in the endothelial dysfunction characterizing the pregnancy disorder of pre-eclampsia, and sVEGFR1’s therapeutic potential as an anti-angiogenic agent is being evaluated in pre-clinical models of cancer. This mini review begins with an examination of the protein domain structure and biomolecular interactions of sVEGFR1 in relation to the full-length VEGFR1. A synopsis of known and inferred physiological and pathological roles of sVEGFR1 is then given, with emphasis on the utility of computational systems biology models in deciphering the molecular mechanisms by which sVEGFR1’s purported biological functions occur. Finally, we present the need for a systems biology perspective in interpreting circulating VEGF and sVEGFR1 concentrations as surrogate markers of angiogenic status in angiogenesis-dependent diseases.

Vascular endothelial growth factor (VEGF) is a potent cytokine that binds to specific receptors on the endothelial cells lining blood vessels. The signaling cascade triggered eventually leads to the formation of new capillaries, a process called angiogenesis. Distributions of VEGF receptors and VEGF ligands are therefore crucial determinants of angiogenic events and, to our knowledge, no quantification of abluminal vs. luminal receptors has been performed. We formulate a molecular-based compartment model to investigate the VEGF distribution in blood and tissue in humans and show that such quantification would lead to new insights on angiogenesis and VEGF-dependent diseases. Our multiscale model includes two major isoforms of VEGF (VEGF121 and VEGF165), as well as their receptors (VEGFR1 and VEGFR2) and the non-signaling co-receptor neuropilin-1 (NRP1). VEGF can be transported between tissue and blood via transendothelial permeability and the lymphatics. VEGF receptors are located on both the luminal and abluminal sides of the endothelial cells. In this study, we analyze the effects of the VEGF receptor localization on the endothelial cells as well as of the lymphatic transport. We show that the VEGF distribution is affected by the luminal receptor density. We predict that the receptor signaling occurs mostly on the abluminal endothelial surface, assuming that VEGF is secreted by parenchymal cells. However, for a low abluminal but high luminal receptor density, VEGF binds predominantly to VEGFR1 on the abluminal surface and VEGFR2 on the luminal surface. Such findings would be pertinent to pathological conditions and therapies related to VEGF receptor imbalance and overexpression on the endothelial cells and will hopefully encourage experimental receptor quantification for both luminal and abluminal surfaces on endothelial cells., Author Summary Angiogenesis is the growth of new blood vessels from pre-existing vasculature that occurs in physiological (e.g., exercise) and pathological contexts (e.g., cancer). This process is often triggered by a signaling cascade that occurs upon ligand-receptor binding between vascular endothelial growth factor (VEGF) and its receptors (VEGFR1/Flt-1, VEGFR2/KDR). These receptors are expressed by endothelial cells that line the blood vessels. Little is known about the quantitative proportion of abluminal receptors (facing the tissue) as compared to those on the luminal surface (facing the blood). We have built a compartment model with molecular details from human tissues to investigate why such experimental data would be of importance. We conclude that the receptor distribution on the endothelial cells can significantly alter the VEGF distribution and the VEGF signaling (through its binding to the receptors) and that quantification of luminal vs. abluminal VEGF receptors would shed light on VEGF signaling and VEGF-dependent mechanisms of angiogenesis.

Most physiological processes are subjected to molecular regulation by growth factors, which are secreted proteins that activate chemical signal transduction pathways through binding of specific cell-surface receptors. One particular growth factor system involved in the in vivo regulation of blood vessel growth is called the vascular endothelial growth factor (VEGF) system. Computational and numerical techniques are well suited to handle the molecular complexity (the number of binding partners involved, including ligands, receptors, and inert binding sites) and multiscale nature (intratissue vs. intertissue transport and local vs. systemic effects within an organism) involved in modeling growth factor system interactions and effects. This chapter introduces a variety of in silico models that seek to recapitulate different aspects of VEGF system biology at various spatial and temporal scales: molecular-level kinetic models focus on VEGF ligand–receptor interactions at and near the endothelial cell surface; mesoscale single-tissue 3D models can simulate the effects of multicellular tissue architecture on the spatial variation in VEGF ligand production and receptor activation; compartmental modeling allows efficient prediction of average interstitial VEGF concentrations and cell-surface VEGF signaling intensities across multiple large tissue volumes, permitting the investigation of whole-body intertissue transport (e.g., vascular permeability and lymphatic drainage). The given examples will demonstrate the utility of computational models in aiding both basic science and clinical research on VEGF systems biology.