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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  Development of a biomimetic peptide derived from collagen IV with anti-angiogenic activity in breast cancer 
Cancer Biology & Therapy  2011;12(9):808-817.
Breast cancer is one of the most commonly diagnosed malignancies in women. Despite the remarkable success of mammography screening and use of adjuvant systemic therapy, it is estimated that approximately 200,000 new diagnoses will be made this year and 40,000 deaths will occur due to this disease (American Cancer Society). Angiogenesis, the growth of vessels from pre-existing microvasculature, is an essential component of tumor progression and has emerged as a therapeutic modality for anti-angiogenic therapies in cancer.
Here we report in vitro and in vivo findings with a 20 amino acid peptide belonging to the collagen IV family, modified to facilitate possible translation to clinical applications. The two cysteines in its natural peptide progenitor were replaced by L-α-amino-n-butyric acid, a non-natural amino acid. The modified peptide was tested in vitro using endothelial cells and in vivo using mouse orthotopic breast cancer xenograft model with MDA-MB-231 human breast cancer cells. This modified peptide demonstrated no significant changes in activity from the parent peptide; however, because it lacks cysteines, it is more suitable for clinical translation. We also investigated its efficacy in combination with a commonly used chemotherapeutic agent paclitaxel; the inhibition of tumor growth by the peptide was similar to that of paclitaxel alone, but the combination did not exhibit any additional inhibition. We have performed further characterization of the mechanism of action (MOA) for this peptide to identify its target receptors, enhancing its translation potential as an antiangiogenic, non-vascular endothelial growth factor (VEGF) targeting agent for therapy in breast cancer.
doi:10.4161/cbt.12.9.17677
PMCID: PMC3225757  PMID: 21878750
angiogenesis; cancer therapy; drug development; integrins; triple-negative; xenograft
17.  Novel Peptide-specific QSAR Analysis Applied to Collagen IV Peptides with Antiangiogenic Activity 
Journal of medicinal chemistry  2011;54(19):6492-6500.
Angiogenesis is the growth of new blood vessels from existing vasculature. Excessive vascularization is associated with a number of diseases including cancer. Anti-angiogenic therapies have the potential to stunt cancer progression. Peptides derived from type IV collagen are potent inhibitors of angiogenesis. We wanted to gain a better understanding of collagen IV structure-activity relationships using a ligand-based approach. We developed novel peptide-specific QSAR models to study the activity of the peptides in endothelial cell proliferation, migration, and adhesion inhibition assays. We found that the models produced quantitatively accurate predictions of activity and provided insight into collagen IV derived peptide structure-activity relationships.
doi:10.1021/jm200114f
PMCID: PMC3195544  PMID: 21866962
18.  Effects of Fiber Type and Size on the Heterogeneity of Oxygen Distribution in Exercising Skeletal Muscle 
PLoS ONE  2012;7(9):e44375.
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.
doi:10.1371/journal.pone.0044375
PMCID: PMC3445540  PMID: 23028531
19.  Expression of VEGF Receptors on Endothelial Cells in Mouse Skeletal Muscle 
PLoS ONE  2012;7(9):e44791.
VEGFR surface localization plays a critical role in converting extracellular VEGF signaling towards angiogenic outcomes, and the quantitative characterization of these parameters is critical for advancing computational models; however the levels of these receptors on blood vessels is currently unknown. Therefore our aim is to quantitatively determine the VEGFR localization on endothelial cells from mouse hindlimb skeletal muscles. We contextualize this VEGFR quantification through comparison to VEGFR-levels on cells in vitro. Using quantitative fluorescence we measure and compare the levels of VEGFR1 and VEGFR2 on endothelial cells isolated from C57BL/6 and BALB/c gastrocnemius and tibialis anterior hindlimb muscles. Fluorescence measurements are calibrated using beads with known numbers of phycoerythrin molecules. The data show a 2-fold higher VEGFR1 surface localization relative to VEGFR2 with 2,000–3,700 VEGFR1/endothelial cell and 1,300–2,000 VEGFR2/endothelial cell. We determine that endothelial cells from the highly glycolytic muscle, tibialis anterior, contain 30% higher number of VEGFR1 surface receptors than gastrocnemius; BALB/c mice display ∼17% higher number of VEGFR1 than C57BL/6. When we compare these results to mouse fibroblasts in vitro, we observe high levels of VEGFR1 (35,800/cell) and very low levels of VEGFR2 (700/cell), while in human endothelial cells in vitro, we observe that the balance of VEGFRs is inverted, with higher levels VEGFR2 (5,800/cell) and lower levels of VEGFR1 (1,800/cell). Our studies also reveal significant cell-to-cell heterogeneity in receptor expression, and the quantification of these dissimilarities ex vivo for the first time provides insight into the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) signaling.
doi:10.1371/journal.pone.0044791
PMCID: PMC3440347  PMID: 22984559
20.  Serpin-Derived Peptides Are Antiangiogenic and Suppress Breast Tumor Xenograft Growth1 
Translational Oncology  2012;5(2):92-97.
Angiogenesis is the formation of neovasculature from preexisting microvessels. Several endogenous proteins regulate the balance of vessel formation and regression in the body including pigment epithelium-derived factor (PEDF), which has been shown to be antiangiogenic and to suppress tumor growth. Using sequence homology and bioinformatics, we previously identified several peptide sequences homologous to an active region of PEDF existing in multiple proteins in the human proteome. These short 11-mer peptides are found in a DEAH box helicase protein, CKIP-1 and caspase 10, and show similar activity in altering endothelial cell adhesion, migration and inducing apoptosis.We tested the peptide derived from DEAH box helicase protein in a triple-negative MDA-MB-231 breast orthotopic xenograft model in severe combined immunodeficient mice and show significant tumor suppression.
PMCID: PMC3323930  PMID: 22496925
21.  Increase of plasma VEGF after intravenous administration of bevacizumab is predicted by a pharmacokinetic model 
Cancer research  2010;70(23):9886-9894.
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.
doi:10.1158/0008-5472.CAN-10-1419
PMCID: PMC3058319  PMID: 21118974
angiogenesis; anti-VEGF; bevacizumab; anti-angiogenic therapy; mathematical model
22.  Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies 
BMC Systems Biology  2011;5:193.
Background
Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer.
Results
Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10-5 cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF121 comprises at least 25% of the VEGF secreted by tumor cells.
Conclusions
This study explores the optimal drug characteristics required for an anti-VEGF agent to have a therapeutic effect and the tumor-specific properties that influence the response to therapy. Our model provides a framework for investigating the use of VEGF-neutralizing drugs for personalized medicine treatment strategies.
doi:10.1186/1752-0509-5-193
PMCID: PMC3229549  PMID: 22104283
23.  A Two-Compartment Model of VEGF Distribution in the Mouse 
PLoS ONE  2011;6(11):e27514.
Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis – the growth of new microvessels from existing microvasculature. Angiogenesis is a complex process involving numerous molecular species, and to better understand it, a systems biology approach is necessary. In vivo preclinical experiments in the area of angiogenesis are typically performed in mouse models; this includes drug development targeting VEGF. Thus, to quantitatively interpret such experimental results, a computational model of VEGF distribution in the mouse can be beneficial. In this paper, we present an in silico model of VEGF distribution in mice, determine model parameters from existing experimental data, conduct sensitivity analysis, and test the validity of the model.
The multiscale model is comprised of two compartments: blood and tissue. The model accounts for interactions between two major VEGF isoforms (VEGF120 and VEGF164) and their endothelial cell receptors VEGFR-1, VEGFR-2, and co-receptor neuropilin-1. Neuropilin-1 is also expressed on the surface of parenchymal cells. The model includes transcapillary macromolecular permeability, lymphatic transport, and macromolecular plasma clearance. Simulations predict that the concentration of unbound VEGF in the tissue is approximately 50-fold greater than in the blood. These concentrations are highly dependent on the VEGF secretion rate. Parameter estimation was performed to fit the simulation results to available experimental data, and permitted the estimation of VEGF secretion rate in healthy tissue, which is difficult to measure experimentally. The model can provide quantitative interpretation of preclinical animal data and may be used in conjunction with experimental studies in the development of pro- and anti-angiogenic agents. The model approximates the normal tissue as skeletal muscle and includes endothelial cells to represent the vasculature. As the VEGF system becomes better characterized in other tissues and cell types, the model can be expanded to include additional compartments and vascular elements.
doi:10.1371/journal.pone.0027514
PMCID: PMC3210788  PMID: 22087332
24.  Systems biology of pro-angiogenic therapies targeting the VEGF system 
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 to 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.
doi:10.1002/wsbm.92
PMCID: PMC2990677  PMID: 20890966
Drug efficacy; Drug transport; Physiome; Protein interaction networks; Systems medicine
25.  Gene Therapy from the perspective of Systems Biology 
Gene therapy research has expanded from its original concept of replacing absent or defective DNA with functional DNA for transcription. Genetic material may be delivered via multiple vectors, including naked plasmid DNA, viruses and even cells with the goal of increasing gene expression; and the targeting of specific tissues or cell types is aimed at decreasing risks of systemic or side effects. As with the development of any drug, there is an amount of empiricism in the choice of gene target, route of administration, dosing and in particular the scaling-up from pre-clinical models to clinical trials. Systems Biology, whose arsenal includes high-throughput experimental and computational studies that account for the complexities of host-disease-therapy interactions, holds significant promise in aiding the development and optimization of gene therapies, including personalized therapies and the identification of biomarkers for success of these strategies. In this review we describe some of the obstacles and successes in gene therapy, using the specific example of growth factor gene delivery to promote angiogenesis and blood vessel remodeling in ischemic diseases; we also make references to anti-angiogenic gene therapy in cancer. The opportunities for Systems Biology and in silico modeling to improve on current outcomes are highlighted.
PMCID: PMC3021921  PMID: 20886389
Mathematical model; computational model; bioinformatics; angiogenesis; gene delivery; coronary artery disease; peripheral artery disease

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