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1.  Pre-treatment of mice with tumor-conditioned media accelerates metastasis to lymph nodes and lungs: a new spontaneous breast cancer metastasis model 
Current spontaneous metastasis models require a long period of observation after establishment of primary tumors to see significant metastatic progression. The degree of metastasis is not consistent among animals: this is problematic since it requires the use of a large number of animals to obtain reliable statistics. Here we report that pre-treatment of animals with tumor-conditioned media (TCM) consistently accelerates spontaneous metastasis in breast cancer. An inguinal breast tumor model facilitated by TCM showed robust anterior metastasis to the axillary and brachial lymph nodes (LN), and the lungs compared to the serum-free media (SFM) treated group. The LN in TCM-treated animals showed enhanced angiogenesis and lymphangiogenesis. Primary tumors and lungs in TCM-treated animals showed enhanced lymphangiogenesis with no significant change in angiogenesis. TCM-treated animals also showed metastatic dissemination to abdomen from the primary injection site: this would generally enhance metastasis to other organs. In sum, the addition of TCM pre-treatment to current metastasis models results in accelerated and robust metastasis which would enable more efficient evaluation of anti-metastatic agents.
doi:10.1007/s10585-013-9610-9
PMCID: PMC3947147  PMID: 23963763
Breast cancer; spontaneous metastasis; accelerated metastasis model; triple negative breast cancer; MDA-MB-231; SUM-149
2.  A Bioimage Informatics Based Reconstruction of Breast Tumor Microvasculature with Computational Blood Flow Predictions 
Microvascular research  2013;91:8-21.
Induction of tumor angiogenesis is among the hallmarks of cancer and a driver of metastatic cascade initiation. Recent advances in high-resolution imaging enable highly detailed three-dimensional geometrical representation of the whole-tumor microvascular architecture. This enormous increase in complexity of image-based data necessitates the application of informatics methods for the analysis, mining and reconstruction of these spatial graph data structures. We present a novel methodology that combines ex-vivo high-resolution micro-computed tomography imaging data with a bioimage informatics algorithm to track and reconstruct the whole-tumor vasculature of a human breast cancer model. The reconstructed tumor vascular network is used as an input of a computational model that estimates blood flow in each segment of the tumor microvascular network. This formulation involves a well-established biophysical model and an optimization algorithm that ensures mass balance and detailed monitoring of all the vessels that feed and drain blood from the tumor microvascular network. Perfusion maps for the whole-tumor microvascular network are computed. Morphological and hemodynamic indices from different regions are compared to infer their role in overall tumor perfusion.
doi:10.1016/j.mvr.2013.12.003
PMCID: PMC3977934  PMID: 24342178
3.  Computational Model of VEGFR2 pathway to ERK activation and modulation through receptor trafficking 
Cellular signalling  2013;25(12):10.1016/j.cellsig.2013.08.015.
Vascular Endothelial Growth Factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. We constructed and validated a computational model of VEGFR2 trafficking and signaling, to study the role of receptor trafficking kinetics in modulating ERK phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized and validated against four previously published in vitro experiments. Based on these parameters, model simulations demonstrated interesting behaviors that may be highly relevant to understanding VEGF signaling in endothelial cells. First, at moderate VEGF doses, VEGFR2 phosphorylation and ERK phosphorylation are related in a log-linear fashion, with a stable duration of ERK activation; but with higher VEGF stimulation, phosphoERK becomes saturated, and its duration increases. Second, a large endosomal fraction of VEGFR2 makes the ERK activation reaction network less sensitive to perturbations in VEGF dosage. Third, extracellular-matrix-bound VEGF binds and activates VEGFR2, but by internalizing at a slower rate, matrix-bound VEGF-induced intracellular ERK phosphorylation is predicted to be greater in magnitude and more sustained, in agreement with experimental evidence. Fourth, different endothelial cell types appear to have different trafficking rates, which result in different levels of endosomal receptor localization and different ERK response profiles.
doi:10.1016/j.cellsig.2013.08.015
PMCID: PMC3865527  PMID: 23993967
Angiogenesis; mathematical model; receptor tyrosine kinase; systems biology; endothelial signaling; trafficking
4.  Inhibition of breast cancer growth and metastasis by a biomimetic peptide 
Scientific Reports  2014;4:7139.
Metastasis is the main cause of mortality in cancer patients. Though there are many anti-cancer drugs targeting primary tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression, particularly, lymphangiogenesis is pivotal for metastasis in breast cancer. Here we report that a novel collagen IV derived biomimetic peptide inhibits breast cancer growth and metastasis by blocking angiogenesis and lymphangiogenesis. The peptide inhibits blood and lymphatic endothelial cell viability, migration, adhesion, and tube formation by targeting IGF1R and Met signals. The peptide blocks MDA-MB-231 tumor growth by inhibiting tumor angiogenesis in vivo. Moreover, the peptide inhibits lymphangiogenesis in primary tumors. MDA-MB-231 tumor conditioned media (TCM) was employed to accelerate spontaneous metastasis in tumor xenografts, and the anti-metastatic activity of the peptide was tested in this model. The peptide prevents metastasis to the lungs and lymph nodes by inhibiting TCM-induced lymphangiogenesis and angiogenesis in the pre-metastatic organs. In summary, a novel biomimetic peptide inhibits breast cancer growth and metastasis by blocking angiogenesis and lymphangiogenesis in the pre-metastatic organs as well as primary tumors.
doi:10.1038/srep07139
PMCID: PMC4238022  PMID: 25409905
5.  A Biomimetic Collagen Derived Peptide Exhibits Anti-Angiogenic Activity in Triple Negative Breast Cancer 
PLoS ONE  2014;9(11):e111901.
We investigated the application of a mimetic 20 amino acid peptide derived from type IV collagen for treatment of breast cancer. We showed that the peptide induced a decrease of proliferation, adhesion, and migration of endothelial and tumor cells in vitro. We also observed an inhibition of triple negative MDA-MB-231 xenograft growth by 75% relative to control when administered intraperitoneally for 27 days at 10 mg/kg. We monitored in vivo the changes in vascular properties throughout the treatment using MRI and found that the vascular volume and permeability surface area product decreased significantly. The treatment also resulted in an increase of caspase-3 activity and in a reduction of microvascular density. The multiple mode of action of this peptide, i.e., anti-angiogenic, and anti-tumorigenic, makes it a viable candidate as a therapeutic agent as a monotherapy or in combination with other compounds.
doi:10.1371/journal.pone.0111901
PMCID: PMC4226498  PMID: 25384034
6.  Angiogenesis Interactome and Time Course Microarray Data Reveal the Distinct Activation Patterns in Endothelial Cells 
PLoS ONE  2014;9(10):e110871.
Angiogenesis involves stimulation of endothelial cells (EC) by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the “angiome”) could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A). We used the Short Time-series Expression Miner (STEM) to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME) show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC) and human microvascular EC (MEC). The results show that VEGFR1–VEGFR2 levels are more closely coupled than VEGFR1–VEGFR3 or VEGFR2–VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.
doi:10.1371/journal.pone.0110871
PMCID: PMC4199761  PMID: 25329517
7.  Long-Term Suppression of Ocular Neovascularization by Intraocular Injection of Biodegradable Polymeric Particles Containing a Serpin-Derived Peptide 
Biomaterials  2013;34(30):10.1016/j.biomaterials.2013.06.044.
Aberrant angiogenesis can cause or contribute to a number of diseases such as neovascular age-related macular degeneration (NVAMD). While current NVAMD treatments target angiogenesis, these treatments are not effective for all patients and also require frequent intravitreal injections. New agents and delivery systems to treat NVAMD could be beneficial to many patients. We have recently developed a serpin-derived peptide as an anti-angiogenic agent. Here, this peptide is investigated for activity in human retinal endothelial cells in vitro and for reducing angiogenesis in a laser-induced choroidal neovascularization mouse model of NVAMD in vivo. While frequent intravitreal injections can be tolerated clinically, reducing the number of injections can improve patient compliance, safety, and outcomes. To achieve this goal, and to maximize the in vivo activity of injected peptide, we have developed biodegradable polymers and controlled release particle formulations to extend anti-angiogenic therapy. To create these devices, the anionic peptides are first self-assembled into nanoparticles using a biodegradable cationic polymer and then as a second step, these nanoparticles are encapsulated into biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles. In situ, these particles show approximately zero-order, linear release of the anionic peptide over 200 days. These particles are made of safe, hydrolytically degradable polymers and have low endotoxin. Long-term in vivo experiments in the laser-induced neovascularization model for NVAMD show that these peptide-releasing particles decrease angiogenesis for at least fourteen weeks in vivo following a single particle dose and therefore are a promising treatment strategy for NVAMD.
doi:10.1016/j.biomaterials.2013.06.044
PMCID: PMC3838902  PMID: 23849876
Angiogenesis; controlled drug release; drug delivery; microsphere; ophthalmology; peptide
8.  Antiangiogenic cancer drug sunitinib exhibits unexpected proangiogenic effects on endothelial cells 
OncoTargets and therapy  2014;7:1571-1582.
Angiogenesis, the formation of new blood vessels, is an essential step for cancer progression, but antiangiogenic therapies have shown limited success. Therefore, a better understanding of the effects of antiangiogenic treatments on endothelial cells is necessary. In this study, we evaluate the changes in cell surface vascular endothelial growth factor receptor (VEGFR) expression on endothelial cells in culture treated with the antiangiogenic tyrosine kinase inhibitor drug sunitinib, using quantitative flow cytometry. We find that proangiogenic VEGFR2 cell surface receptor numbers are increased with sunitinib treatment. This proangiogenic effect might account for the limited effects of sunitinib as a cancer therapy. We also find that this increase is inhibited by brefeldin A, an inhibitor of protein transport from the endoplasmic reticulum to the Golgi apparatus. The complex dynamics of cell surface VEGFRs may be important for successful treatment of cancer with antiangiogenic therapeutics.
doi:10.2147/OTT.S65055
PMCID: PMC4164292  PMID: 25228815
flow cytometry; VEGFRs; sunitinib; angiogenesis; VEGFA
9.  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.
doi:10.1111/jcmm.12164
PMCID: PMC4190897  PMID: 24237862
angiogenesis; computational model; mathematical model; systems biology; multi-scale modelling
10.  Effect of Tumor Microenvironment on Tumor VEGF During Anti-VEGF Treatment: Systems Biology Predictions 
Background
Vascular endothelial growth factor (VEGF) is known to be a potent promoter of angiogenesis under both physiological and pathological conditions. Given its role in regulating tumor vascularization, VEGF has been targeted in various cancer treatments, and anti-VEGF therapy has been used clinically for treatment of several types of cancer. Systems biology approaches, particularly computational models, provide insight into the complexity of tumor angiogenesis. These models complement experimental studies and aid in the development of effective therapies targeting angiogenesis.
Methods
We developed an experiment-based, molecular-detailed compartment model of VEGF kinetics and transport to investigate the distribution of two major VEGF isoforms (VEGF121 and VEGF165) in the body. The model is applied to predict the dynamics of tumor VEGF and, importantly, to gain insight into how tumor VEGF responds to an intravenous injection of an anti-VEGF agent.
Results
The model predicts that free VEGF in the tumor interstitium is seven to 13 times higher than plasma VEGF and is predominantly in the form of VEGF121 (>70%), predictions that are validated by experimental data. The model also predicts that tumor VEGF can increase or decrease with anti-VEGF treatment depending on tumor microenvironment, pointing to the importance of personalized medicine.
Conclusions
This computational study suggests that the rate of VEGF secretion by tumor cells may serve as a biomarker to predict the patient population that is likely to respond to anti-VEGF treatment. Thus, the model predictions have important clinical relevance and may aid clinicians and clinical researchers seeking interpretation of pharmacokinetic and pharmacodynamic observations and optimization of anti-VEGF therapies.
doi:10.1093/jnci/djt093
PMCID: PMC3672077  PMID: 23670728
11.  Quantitative fluorescent profiling of VEGFRs reveals tumor cell and endothelial cell heterogeneity in breast cancer xenografts 
Cancer Medicine  2014;3(2):225-244.
Plasma membrane-localized vascular endothelial growth factor receptors (VEGFR) play a critical role in transducing VEGF signaling toward pro and antiangiogenic outcomes and quantitative characterization of these receptors is critical toward identifying biomarkers for antiangiogenic therapies, understanding mechanisms of action of antiangiogenic drugs, and advancing predictive computational models. While in vitro analysis of cell surface-VEGFRs has been performed, little is known about the levels of cell surface-VEGFR on tumor cells. Therefore, we inoculate nude mice with the human triple-negative breast cancer, MDA-MB-231, cell line; isolate human tumor cells and mouse tumor endothelial cells from xenografts; and quantitatively characterize the VEGFR localization on these cells. We observe 15,000 surface-VEGFR1/tumor endothelial cell versus 8200 surface-VEGFR1/tumor endothelial cell at 3 and 6 weeks of tumor growth, respectively; and we quantify 1200–1700 surface-VEGFR2/tumor endothelial cell. The tumor cell levels of VEGFR1 and VEGFR2 are relatively constant between 3 and 6 weeks: 2000–2200 surface-VEGFR1/tumor cell and ∼1000 surface-VEGFR2/tumor cell. Cell-by-cell analysis provides additional insight into tumor heterogeneity by identifying four cellular subpopulations based on size and levels of cell membrane-localized VEGFR. Furthermore, when these ex vivo data are compared to in vitro data, we observe little to no VEGFRs on MDA-MB-231 cells, and the MDA-MB-231 VEGFR surface levels are not regulated by a saturating dose of VEGF. Overall, the quantification of these dissimilarities for the first time in tumor provides insight into the balance of modulatory (VEGFR1) and proangiogenic (VEGFR2) receptors.
doi:10.1002/cam4.188
PMCID: PMC3987073  PMID: 24449499
Biomarker; endothelial cells; heterogeneity; personalized medicine; proteomics; quantitative flow cytometry; receptor localization; vascular endothelial growth factor receptors; xenograft
12.  Synergy between a collagen IV mimetic peptide and a somatotropin-domain derived peptide as angiogenesis and lymphangiogenesis inhibitors 
Angiogenesis  2012;16(1):10.1007/s10456-012-9308-7.
Angiogenesis is central to many physiological and pathological processes. Here we show two potent bioinformatically-identified peptides, one derived from collagen IV and translationally optimized, and one from a somatotropin domain-containing protein, synergize in angiogenesis and lymphangiogenesis assays including cell adhesion, migration and in vivo Matrigel plugs. Peptide-peptide combination therapies have recently been applied to diseases such as human immunodeficiency virus (HIV), but remain uncommon thus far in cancer, age-related macular degeneration and other angiogenesis-dependent diseases. Previous work from our group has shown that the collagen IV-derived peptide primarily binds β1 integrins, while the receptor for the somatotropin-derived peptide remains unknown. We investigate these peptides’ mechanisms of action and find both peptides affect the vascular endothelial growth factor (VEGF) pathway as well as focal adhesion kinase (FAK) by changes in phosphorylation level and total protein content. Blocking of FAK both through binding of β1 integrins and through inhibition of VEGFR2 accounts for the synergy we observe. Since resistance through activation of multiple signaling pathways is a central problem of anti-angiogenic therapies in diseases such as cancer, we suggest that peptide combinations such as these are an approach that should be considered as a means to sustain anti-angiogenic and anti-lymphangiogenic therapy and improve efficacy of treatment.
doi:10.1007/s10456-012-9308-7
PMCID: PMC3867130  PMID: 23053781
Angiogenesis; Synergy; Combination therapy; Peptide; Inhibitor
13.  Theoretical models for coronary vascular biomechanics: Progress & challenges 
Progress in biophysics and molecular biology  2010;104(0):10.1016/j.pbiomolbio.2010.10.001.
A key aim of the cardiac Physiome Project is to develop theoretical models to simulate the functional behaviour of the heart under physiological and pathophysiological conditions. Heart function is critically dependent on the delivery of an adequate blood supply to the myocardium via the coronary vasculature. Key to this critical function of the coronary vasculature is system dynamics that emerge via the interactions of the numerous constituent components at a range of spatial and temporal scales. Here, we focus on several components for which theoretical approaches can be applied, including vascular structure and mechanics, blood flow and mass transport, flow regulation, angiogenesis and vascular remodelling, and vascular cellular mechanics. For each component, we summarise the current state of the art in model development, and discuss areas requiring further research. We highlight the major challenges associated with integrating the component models to develop a computational tool that can ultimately be used to simulate the responses of the coronary vascular system to changing demands and to diseases and therapies.
doi:10.1016/j.pbiomolbio.2010.10.001
PMCID: PMC3817728  PMID: 21040741
Vascular structure; Mechanics; Haemodynamics; Mass transport; Regulation; Adaptation; Mathematical and computational model; Multi-scale; Cellular mechanics; Integration
14.  Multiscale Imaging and Computational Modeling of Blood Flow in the Tumor Vasculature 
Annals of biomedical engineering  2012;40(11):10.1007/s10439-012-0585-5.
The evolution in our understanding of tumor angiogenesis has been the result of pioneering imaging and computational modeling studies spanning the endothelial cell, microvasculature and tissue levels. Many of these primary data on the tumor vasculature are in the form of images from pre-clinical tumor models that provide a wealth of qualitative and quantitative information in many dimensions and across different spatial scales. However, until recently, the visualization of changes in the tumor vasculature across spatial scales remained a challenge due to a lack of techniques for integrating micro- and macroscopic imaging data. Furthermore, the paucity of three-dimensional (3-D) tumor vascular data in conjunction with the challenges in obtaining such data from patients presents a serious hurdle for the development and validation of predictive, multiscale computational models of tumor angiogenesis. In this review, we discuss the development of multiscale models of tumor angiogenesis, new imaging techniques capable of reproducing the 3-D tumor vascular architecture with high fidelity, and the emergence of “image-based models”of tumor blood flow and molecular transport. Collectively, these developments are helping us gain a fundamental understanding of the cellular and molecular regulation of tumor angiogenesis that will benefit the development of new cancer therapies. Eventually, we expect this exciting integration of multiscale imaging and mathematical modeling to have widespread application beyond the tumor vasculature to other diseases involving a pathological vasculature, such as stroke and spinal cord injury.
doi:10.1007/s10439-012-0585-5
PMCID: PMC3809908  PMID: 22565817
Angiogenesis; Tumor; Vasculature; Multiscale; Imaging; Mathematical modeling; Computational modeling; Cancer; Tumor microenvironment; Systems biology
15.  Blood-plasma separation in Y-shaped bifurcating microfluidic channels: A dissipative particle dynamics simulation study 
Physical biology  2012;9(2):10.1088/1478-3975/9/2/026010.
The motion of a suspension of red blood cells (RBCs) flowing in a Y-shaped bifurcating microfluidic channel is investigated using a validated low-dimensional RBC (LD-RBC) model based on dissipative particle dynamics (DPD). Specifically, the RBC is represented as a closed torus-like ring of ten colloidal particles, which leads to efficient simulations of blood flow in microcirculation over a wide range of hematocrits. Adaptive no-slip wall boundary conditions were implemented to model hydrodynamic flow within a specific wall structure of diverging 3D microfluidic channels, paying attention to controlling density fluctuations. Plasma skimming and the all-or-nothing phenomenon of RBCs in a bifurcating microfluidic channel have been investigated in our simulations for healthy and diseased blood, including the size of cell-free layer on the daughter branches. The feed hematocrit level in the parent channel has considerable influence on blood-plasma separation. Compared to the blood-plasma separation efficiencies of healthy RBCs, malaria-infected stiff RBCs (iRBCs) have a tendency to travel into the low flowrate daughter branch because of their different initial distribution in the parent channel. Our simulation results are consistent with previously published experimental results and theoretical predictions.
doi:10.1088/1478-3975/9/2/026010
PMCID: PMC3419813  PMID: 22476709
plasma skimming; Zweifach-Fung effect; cell-free layer; malaria; DPD
16.  Collagen IV and CXC chemokine derived anti-angiogenic peptides suppress glioma xenograft growth 
Anti-Cancer Drugs  2012;23(7):706-712.
Peptides are receiving increased attention as therapeutic agents, due to their high binding specificity and versatility to be modified as targeting or carrier molecules. Particularly, peptides with anti-angiogenic activity are of high interest due to their applicability to a wide range of cancers. In this study we investigate the biological activity of two novel antiangiogenic peptides in pre-clinical glioma models. One peptide SP2000 is derived from collagen IV and the other peptide SP3019 belongs to the CXC family. We previously characterized the capacity of SP2000 and SP3019 to inhibit multiple biological endpoints linked to angiogenesis in human endothelial cells in several assays. Here we report additional studies using endothelial cells and focus on the activity of these peptides against human glioma cell growth, migration and adhesion in vitro and growth as tumor xenografts in vivo. We found that SP2000 completely inhibits migration of the glioma cells at 50 μM and SP3019 produced 50% inhibition at 100 μM. Their relative anti-adhesion activities were similar with SP2000 and SP3019 generating 50% adhesion inhibition at 4.9 ± 0.82 μM and 21.3 ± 5.92 μM respectively. In vivo glioma growth inhibition was 63 % for SP2000 and 76% for SP3019 after 2 weeks of administration at daily doses of 10mg/kg and 20 mg/kg, respectively. The direct activity of these peptides against glioma cells in conjunction with their anti-angiogenic activities warrants their further development as either stand-alone agents or in combination with standard cytotoxic or emerging targeted therapies in malignant brain tumors.
doi:10.1097/CAD.0b013e3283531041
PMCID: PMC3384759  PMID: 22495619
Angiogenesis; cancer therapy; endothelial cell; glioblastoma; proliferation; migration; adhesion
17.  Structure-activity relationship study of collagen derived anti-angiogenic biomimetic peptides 
Chemical biology & drug design  2012;80(1):27-37.
Structure-activity relationship (SAR) studies are essential in the generation of peptides with enhanced activity and efficacy as therapeutic agents. In this study we report a SAR study for a family of mimetic peptides derived from type IV collagen with potent anti-angiogenic properties. The SAR study was conducted using a number of validated in vitro assays including cell proliferation, adhesion, migration and tubule formation. We report a critical sequence (NINNV) within this peptide series which is required for the potent anti-angiogenic activity. Detailed amino acid substitutions resulted in peptides with superior efficacy. Specifically, substitutions with Isoleucine at positions twelve and eighteen along with the substitution of the Methionine at position ten with the non-natural amino acid d-Alanine led to an increase in potency by two orders of magnitude over the parent peptide. Several mimetic peptides in this series exhibit a significant improvement of activity over the parent peptide. This improved in vitro activity is expected to correlate with an increase in in vivo activity leading to effective peptides for anti-angiogenic therapy for different disease applications including cancer and age-related macular degeneration.
doi:10.1111/j.1747-0285.2012.01376.x
PMCID: PMC3371172  PMID: 22405100
angiogenesis; peptidomimetics; endothelial cell; cancer; age-related macular degeneration; lymphangiogenesis
18.  Computational Model of Gab1/2-Dependent VEGFR2 Pathway to Akt Activation 
PLoS ONE  2013;8(6):e67438.
Vascular endothelial growth factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. However, no detailed mass-action models of VEGF receptor signaling have been developed. We constructed and validated the first computational model of VEGFR2 trafficking and signaling, to study the opposing roles of Gab1 and Gab2 in regulation of Akt phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized against 5 previously published in vitro experiments, and the model was validated against six independent published datasets. The model showed agreement at several key nodes, involving scaffolding proteins Gab1, Gab2 and their complexes with Shp2. VEGFR2 recruitment of Gab1 is greater in magnitude, slower, and more sustained than that of Gab2. As Gab2 binds VEGFR2 complexes more transiently than Gab1, VEGFR2 complexes can recycle and continue to participate in other signaling pathways. Correspondingly, the simulation results show a log-linear relationship between a decrease in Akt phosphorylation and Gab1 knockdown while a linear relationship was observed between an increase in Akt phosphorylation and Gab2 knockdown. Global sensitivity analysis demonstrated the importance of initial-concentration ratios of antagonistic molecular species (Gab1/Gab2 and PI3K/Shp2) in determining Akt phosphorylation profiles. It also showed that kinetic parameters responsible for transient Gab2 binding affect the system at specific nodes. This model can be expanded to study multiple signaling contexts and receptor crosstalk and can form a basis for investigation of therapeutic approaches, such as tyrosine kinase inhibitors (TKIs), overexpression of key signaling proteins or knockdown experiments.
doi:10.1371/journal.pone.0067438
PMCID: PMC3689841  PMID: 23805312
19.  Predicting the Effects of Anti-angiogenic Agents Targeting Specific VEGF Isoforms 
The AAPS Journal  2012;14(3):500-509.
Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis, whose effect on cancer growth and development is well characterized. Alternative splicing of VEGF leads to several different isoforms, which are differentially expressed in various tumor types and have distinct functions in tumor blood vessel formation. Many cancer therapies aim to inhibit angiogenesis by targeting VEGF and preventing intracellular signaling leading to tumor vascularization; however, the effects of targeting specific VEGF isoforms have received little attention in the clinical setting. In this work, we investigate the effects of selectively targeting a single VEGF isoform, as compared with inhibiting all isoforms. We utilize a molecular-detailed whole-body compartment model of VEGF transport and kinetics in the presence of breast tumor. The model includes two major VEGF isoforms, VEGF121 and VEGF165, receptors VEGFR1 and VEGFR2, and co-receptors Neuropilin-1 and Neuropilin-2. We utilize the model to predict the concentrations of free VEGF, the number of VEGF/VEGFR2 complexes (considered to be pro-angiogenic), and the receptor occupancy profiles following inhibition of VEGF using isoform-specific anti-VEGF agents. We predict that targeting VEGF121 leads to a 54% and 84% reduction in free VEGF in tumors that secrete both VEGF isoforms or tumors that overexpress VEGF121, respectively. Additionally, 21% of the VEGFR2 molecules in the blood are ligated following inhibition of VEGF121, compared with 88% when both isoforms are targeted. Targeting VEGF121 reduces tumor free VEGF and is an effective treatment strategy. Our results provide a basis for clinical investigation of isoform-specific anti-VEGF agents.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9363-4) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9363-4
PMCID: PMC3385824  PMID: 22547351
angiogenesis; cancer drug target; computational model; pharmacokinetic model; systems biology
20.  Computational models of VEGF-associated angiogenic processes in cancer 
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.
doi:10.1093/imammb/dqq025
PMCID: PMC4104688  PMID: 21266494
computational biology; systems biology; angiogenesis
21.  Inhibition of Lymphangiogenesis and Angiogenesis in Breast Tumor Xenografts and Lymph Nodes by a Peptide Derived from Transmembrane Protein 45A12 
Neoplasia (New York, N.Y.)  2013;15(2):112-124.
Angiogenesis, the formation of new blood vessels from preexisting blood vessels, is a process that supports tumor growth and metastatic dissemination. Lymphangiogenesis also facilitates metastasis by increasing dissemination through the lymphatic vessels (LVs). Even after treatment with antiangiogenic agents, breast cancer patients are vulnerable to LV-mediated metastasis. We report that a 14-amino acid peptide derived from transmembrane protein 45A shows multimodal inhibition of lymphangiogenesis and angiogenesis in breast cancer. The peptide blocks lymphangiogenic and angiogenic phenotypes of lymphatic and blood endothelial cells induced by tumor-conditioned media prepared from MDA-MB-231 breast cancer cells. The peptide delays growth of MDA-MB-231 tumor xenografts and normalizes tumor-conditioned lymph nodes (LNs). These studies demonstrate the antilymphangiogenic and antiangiogenic potential of the peptide against primary tumors and premetastatic, tumor-conditioned regional LNs. Mechanistically, the peptide blocks vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) and downstream proteins by binding to neuropilin 1/2 (NRP1/2) and inhibiting VEGFR2/3 and NRP1/2 complex formation in the presence of VEGFA/C.
PMCID: PMC3579314  PMID: 23441126
22.  Compartment Model Predicts VEGF Secretion and Investigates the Effects of VEGF Trap in Tumor-Bearing Mice 
Frontiers in Oncology  2013;3:196.
Angiogenesis, the formation of new blood vessels from existing vasculature, is important in tumor growth and metastasis. A key regulator of angiogenesis is vascular endothelial growth factor (VEGF), which has been targeted in numerous anti-angiogenic therapies aimed at inhibiting tumor angiogenesis. Systems biology approaches, including computational modeling, are useful for understanding this complex biological process and can aid in the development of novel and effective therapeutics that target the VEGF family of proteins and receptors. We have developed a computational model of VEGF transport and kinetics in the tumor-bearing mouse, which includes three-compartments: normal tissue, blood, and tumor. The model simulates human tumor xenografts and includes human (VEGF121 and VEGF165) and mouse (VEGF120 and VEGF164) isoforms. The model incorporates molecular interactions between these VEGF isoforms and receptors (VEGFR1 and VEGFR2), as well as co-receptors (NRP1 and NRP2). We also include important soluble factors: soluble VEGFR1 (sFlt-1) and α-2-macroglobulin. The model accounts for transport via macromolecular transendothelial permeability, lymphatic flow, and plasma clearance. We have fit the model to available in vivo experimental data on the plasma concentration of free VEGF Trap and VEGF Trap bound to mouse and human VEGF in order to estimate the rates at which parenchymal cells (myocytes and tumor cells) and endothelial cells secrete VEGF. Interestingly, the predicted tumor VEGF secretion rates are significantly lower (0.007–0.023 molecules/cell/s, depending on the tumor microenvironment) than most reported in vitro measurements (0.03–2.65 molecules/cell/s). The optimized model is used to investigate the interstitial and plasma VEGF concentrations and the effect of the VEGF-neutralizing agent, VEGF Trap (aflibercept). This work complements experimental studies performed in mice and provides a framework with which to examine the effects of anti-VEGF agents, aiding in the optimization of such anti-angiogenic therapeutics as well as analysis of clinical data. The model predictions also have implications for biomarker discovery with anti-angiogenic therapies.
doi:10.3389/fonc.2013.00196
PMCID: PMC3727077  PMID: 23908970
systems biology; mathematical model; computational model; angiogenesis; tumor xenograft model; anti-angiogenic therapy; cancer
23.  Experimental and theoretical studies of oxygen gradients in rat pial microvessels 
Using modified oxygen needle microelectrodes and intravital videomicroscopy, measurements were made of tissue oxygen tension (PO2) profiles near cortical arterioles and transmural PO2 gradients in the pial arterioles of the rat. Under control conditions, the transmural PO2 gradient averaged 1.17 ± 0.06 mm Hg/μm (mean ± s.e., n = 40). Local arteriolar dilation resulted in a marked decrease in the transmural PO2 gradient to 0.68 ± 0.04 mm Hg/μm (P < 0.001, n = 38). The major finding of this study is a dependence of the transmural PO2 gradient on the vascular tone of the pial arterioles. Using a model of oxygen transport in an arteriole and experimental PO2 profiles, values of radial perivascular and intravascular O2 fluxes were estimated. Our theoretical estimates show that oxygen flux values at the outer surface of the arteriolar wall are approximately 10−5 mL O2/cm2 per sec, independent of the values of the arteriolar wall O2 consumption within a wide range of consumption values. This also means that PO2 transmural gradients for cerebral arterioles are within the limits of 1 to 2 mm Hg/μm. The data lead to the conclusion that O2 consumption of the arteriolar wall is within the range for the surrounding tissue and O2 consumption of the endothelial layer appears to have no substantial impact on the transmural PO2 gradient.
doi:10.1038/jcbfm.2008.51
PMCID: PMC3529159  PMID: 18506196
cortical microvessels; O2 transport model; oxygen microelectrodes; tissue PO2 profiles; transmural PO2 gradient
24.  Blood Flow and Cell-Free Layer in Microvessels 
Blood is modeled as a suspension of red blood cells using the dissipative particle dynamics method. The red blood cell membrane is coarse-grained for efficient simulations of multiple cells, yet accurately describes its viscoelastic properties. Blood flow in microtubes ranging from 10 to 40 μm in diameter is simulated in three dimensions for values of hematocrit in the range of 0.15–0.45 and carefully compared with available experimental data. Velocity profiles for different hematocrit values show an increase in bluntness with an increase in hematocrit. Red blood cell center-of-mass distributions demonstrate cell migration away from the wall to the tube center. This results in the formation of a cell-free layer next to the tube wall corresponding to the experimentally observed Fahraeus and Fahraeus–Lindqvist effects. The predicted cell-free layer widths are in agreement with those found in in vitro experiments; the results are also in qualitative agreement with in vivo experiments. However, additional features have to be taken into account for simulating microvascular flow, e.g., the endothelial glycocalyx. The developed model is able to capture blood flow properties and provides a computational framework at the mesoscopic level for obtaining realistic predictions of blood flow in microcirculation under normal and pathological conditions.
doi:10.1111/j.1549-8719.2010.00056.x
PMCID: PMC3529161  PMID: 21044216
apparent viscosity; red blood cell; blood flow resistance; dissipative particle dynamics
25.  Small peptides derived from somatotropin domain-containing proteins inhibit blood and lymphatic endothelial cell proliferation, migration, adhesion and tube formation 
Angiogenesis is thoroughly balanced and regulated in health; however, it is dysregulated in many diseases including cancer, age-related macular degeneration, cardiovascular diseases such as coronary and peripheral artery diseases and stroke, abnormal embryonic development, and abnormal wound healing. In addition to angiogenesis, lymphangiogenesis is pivotal for maintaining the immune system, homeostasis of body fluids and lymphoid organs; dysregulated lymphangiogenesis may cause inflammatory diseases and lymph node mediated tumor metastasis. Anti-angiogenic or anti-lymphangiogenic small peptides may play an important role as therapeutic agents normalizing angiogenesis or lymphangiogenesis in disease conditions. Several novel endogenous peptides derived from proteins containing a conserved somatotropin domain have been previously identified with the help of our bioinformatics-based methodology. These somatotropin peptides were screened for inhibition of angiogenesis and lymphangiogenesis using in vitro proliferation, migration, adhesion and tube formation assays with blood and lymphatic endothelial cells. We found that the peptides have the potential for inhibiting both angiogenesis and lymphangiogenesis. Focusing the study on the inhibition of lymphangiogenesis, we found that a peptide derived from the somatotropin conserved domain of transmembrane protein 45A human was the most potent lymphangiogenesis inhibitor, blocking lymphatic endothelial cell migration, adhesion, and tube formation.
doi:10.1016/j.biocel.2011.08.020
PMCID: PMC3206162  PMID: 21920451
Lymphatic endothelial cell; Blood endothelial cell; Endogenous somatotropin peptides; Transmembrane protein 45A human

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