Endothelial Cell Capture of Heparin-Binding Growth Factors under Flow

Circulation is an important delivery method for both natural and synthetic molecules, but microenvironment interactions, regulated by endothelial cells and critical to the molecule's fate, are difficult to interpret using traditional approaches. In this work, we analyzed and predicted growth factor capture under flow using computer modeling and a three-dimensional experimental approach that includes pertinent circulation characteristics such as pulsatile flow, competing binding interactions, and limited bioavailability. An understanding of the controlling features of this process was desired. The experimental module consisted of a bioreactor with synthetic endothelial-lined hollow fibers under flow. The physical design of the system was incorporated into the model parameters. The heparin-binding growth factor fibroblast growth factor-2 (FGF-2) was used for both the experiments and simulations. Our computational model was composed of three parts: (1) media flow equations, (2) mass transport equations and (3) cell surface reaction equations. The model is based on the flow and reactions within a single hollow fiber and was scaled linearly by the total number of fibers for comparison with experimental results. Our model predicted, and experiments confirmed, that removal of heparan sulfate (HS) from the system would result in a dramatic loss of binding by heparin-binding proteins, but not by proteins that do not bind heparin. The model further predicted a significant loss of bound protein at flow rates only slightly higher than average capillary flow rates, corroborated experimentally, suggesting that the probability of capture in a single pass at high flow rates is extremely low. Several other key parameters were investigated with the coupling between receptors and proteoglycans shown to have a critical impact on successful capture. The combined system offers opportunities to examine circulation capture in a straightforward quantitative manner that should prove advantageous for biologicals or drug delivery investigations

Fibroblast growth factor-2 interaction with vascular cells and basement membrane under physiological fluid flow and diabetic hyperglycemia

Diabetes is a debilitating disease with a significant impact on global health. People with diabetes experience early and accelerated atherosclerosis and are at increased risk of restenosis. Though the pathogenesis of atherosclerosis and restenosis are complex, endothelial cells (EC), smooth muscle cells (SMC), and basement membrane (BM) play important roles in lesion development. Healthy EC regulate SMC function, but EC become dysfunctional in hyperglycemia or on glycated collagen, which may alter SMC regulation. Fibroblast growth factor-2 (FGF2), which is produced and released by EC and binds to heparan sulfate proteoglycans in the endothelial BM, may be critical to loss of vascular homeostasis.Experimental and computational models of BM-FGF2 binding kinetics under static conditions are well established in the literature but remain largely unexplored under flow. To investigate BM-FGF2 binding kinetics under fluid flow, BM-FGF2 equilibrium and associative binding were measured at various shear stresses. Surprisingly, BM-bound FGF2 increased up to a physiological arterial shear stress of 25 dynes/cm2, after which it decreased. These data suggest that FGF2 binding varies with shear stress possibly by a catch-slip mechanism, where applied force changes the dissociation constant to either promote (―catch‖) or reduce (―slip‖) binding. A computational model of BM-FGF2 interaction incorporating convective-diffusive transport and a surface binding reaction was also created.BM-bound FGF2 is released over time, after which FGF2 affects both EC and SMC. EC and SMC proliferation and intracellular signaling in response to FGF2 were investigated under hyperglycemic conditions, including for cells grown on glycated collagen or in high glucose media. Glycated collagen did not change EC proliferation or pERK signaling, however pAkt signaling decreased on glycated collagen. This suggests that glycated collagen may decrease FGF2 promotion of EC survival. SMC proliferation decreased on glycated collagen, while SMC cultured with FGF2 showed no difference in growth.Improved understanding of BM-FGF2 binding with flow serves as an initial step in a comprehensive model of FGF2 binding to BM and cells under fluid flow. Together with the observed altered FGF2 effects on vascular cells, these data motivate the development of improved growth factor treatments under physiological and disease conditions.M.S., Biomedical Engineering -- Drexel University, 201

Multiplexed angiogenic biomarker quantification on single cells

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multi-cell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell (EC) characteristics and response in tumor microenvironments. However, data on other angiogenic receptors, such as platelet derived growth factor receptors (PDGFRs), Tie receptors, are also necessary for the development of an accurate angiogenesis model. To gain insights on the involvement of these angiogenic receptors in angiogenesis, I develop a method to quantify receptor concentrations as well as the cell-by-cell heterogeneity. I establish protocols to measure cell membrane VEGFR, NRP1, Tie2, and PDGFR concentration on several cell and tissue models including human dermal fibroblasts (HDFs) in vitro, a 2D endothelial/fibroblast co-culture model in vitro, and a patient-derived xenograft (PDX) model of glioblastoma (GBM). I demonstrate VEGF-A165-mediated downregulation of membrane PDGFRα (~25%) and PDGFRβ (~30%) on HDFs, following a 24-hour treatment. This supports the idea that VEGF-A165 acts independently of VEGFRs to signal through PDGFRα and PDGFRβ. I uncover high intratumoral heterogeneity within the GBM PDX model, with tumor EC-like subpopulations having high concentrations of membrane VEGFR1, VEGFR2, EGFR, IGFR, and PDGFRs. To gain greater insights into cell heterogeneity and examine angiogenic signaling pathways as a whole, I utilize the unique spectral properties of quantum dots (Qdots), and combines Qdots with qFlow cytometry, to dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, I reduce nonspecific binding between Qdot-conjugated antibodies and cells, identify optimal labeling conditions, and establish that 800 – 20,000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations we demonstrate measurement of 1,100 VEGFR1 and 6,900 VEGFR2 per HUVEC. 24 h VEGF-A165 treatment induce ~90% upregulation of VEGFR1 and ~30% downregulation of VEGFR2 concentration. We further analyze HUVEC heterogeneity and observe that 24 h VEGF-A165 treatment induces ~15% decrease in VEGFR2 heterogeneity. Overall, we demonstrate experimental and analysis strategies for quantifying two or more RTKs at single-level using Qdots, which will provide new insights into biological systems

VEGF immobilization and VEGFR2 trafficking and phosphorylation: in vitro and in vivo implications

Modern drug development is marked by high failure rates in translation to the clinic. Further, many drugs that succeed in clinical trials work for only a fraction of patients. Systems pharmacology attempts to address these challenges by improving our understanding of the disease-therapy system, integrating detailed molecular interactions, cellular signaling, tissue architecture, and whole body physiology. I built cutting-edge, molecularly-detailed, multi-scale computational models to study the effects of immobilization of growth factors on signaling in angiogenesis, focusing in particular on the binding of vascular endothelial growth factor (VEGF) family members to the ECM. While most studies of VEGF signaling use only VEGF presented in solution, there is evidence that a large potion of VEGF may be ECM-bound in vivo, and relative expression of isoforms binding to ECM vs. found only in solution varies by tissue and changes in disease, motivating further study of this question. Starting at the in vitro level, we showed that differential signaling of VEGF-receptor 2 (VEGFR2) in response to soluble vs. immobilized VEGF can be explained by reduced internalization of ECM-VEGF-VEGFR2 complexes. Moving in vivo, we predicted differences in both growth factor distribution and receptor activation by VEGF family ligands, as a function of their ECM-binding properties. These predictions are consistent with observed vascular phenotypes in mice expressing single VEGF isoforms. Next, we explored how VEGF splicing changes in peripheral artery disease lead to impaired angiogenic responses to ischemia. Our model showed that the VEGF165b isoform, which does not bind to ECM or to the coreceptor NRP1, is a weak activator of VEGFR2 in vivo, and competes for binding to VEGF-receptor 1, but not VEGF-receptor 2. Finally, we used this model to screen potential therapeutic strategies designed to promote VEGF-mediated revascularization in ischemic disease and tissue engineering applications. Within a single system, we compared failed and promising biomaterial-based VEGF delivery systems, antibody-based therapeutics, and gene therapy strategies to identify key rules for design, optimization, and translation of these pro-angiogenic therapies