Nicanor Moldovan - US grants

(Applicant's Description) Therapeutic angiogenesis attempts to change the course of diseases by altering the microvascular blood supply to the organs, either by reducing it in tumors. or increasing it in ischemic tissues. A new arsenal of methods is being developed including gene therapy for localized administration of angiogenic factors, efficient monoclonal antibodies against adhesion molecules; or cloned peptides mimicking the natural angiostatic mechanisms. However, the basic mechanisms governing the efficiency of these approaches are poorly understood. Here we suggest that a potent system for controlling angiogenesis is the monocyte/macrophage activity, which is essential for the progression of angiogenesis and tissue remodeling. We discovered that, when present in ischemic tissues, these cells produce long-lasting channels in the tissues they infiltrate. Our hypothesis is that these channels represent a prerequisite for angiogenesis in vivo, therefore are an ideal therapeutic target. In order to prove this new concept, and to bring it to practical applicability, the following specific aims will be pursued: 1) Model in vitro the formation of channels by monocytes/macrophages, and find the molecular factors on which this process depends. 2) Determine the influence that specific physiologic and pathologic conditions, thought to be associated with angiogenesis, have on the formation of channels by monocytes/macrophages in vitro. 3) Test the role the channel formation has in progression of angiogenesis in vitro. 4) reproduce in vivo and analyze the formation of channels in matrices of controlled composition. 5) test the possibility for therapeutic manipulation of angiogenesis in selected transgenic animals, by either using inhibitors of channel formation, or modifying tissue concentrations and/or distribution of monocytes (changing the distribution of chemotactic factors, or injecting concentrates of monocytes). To this end, we developed assays which will be used in vitro and in vivo for: a) characterization of molecular mechanisms of channel formation; b) the impact of channels system on angiogenesis; c) identification of compounds targeting the monocytes and macrophages, likely to influence the course of angiogenesis. The new approach for management of angiogenesis we suggest here, complements the current efforts in the field, bringing a broader understanding of basic mechanisms of angiogenesis and expending the spectrum of available therapeutic options.

DESCRIPTION (provided by applicant): This proposal is responsive to the Area of Scientific Priority "Development of tools to facilitate research on the basic biology of aging" of the National Institute on Aging. Accurate, fast and unbiased methods to detect rare cells in cell suspensions for regenerative medicine are in much need and in short supply. Rapid detection of rare cells in blood and other body fluids is critical for the diagnosis of a variety of age-dependent pathological conditions, and for screening potential donors for cell therapy. Current methods rely on sophisticated, multi- parameter analysis of cell surface markers of uncertain relevance, making them impractical for reliable diagnosis. Long-term goal. Using a combination of advanced protein engineering, materials science, molecular biology of cell signaling and bioengineering, we propose a novel platform for a faster, unbiased and efficient method for rare cell capture, analysis and expansion. The principles of this platform are generally applicable to circulating progenitor cells (for endothelial, smooth muscle, or fibroblast progenitors), or tumor cells, etc. For proof of principle we will focus on 'endothelial progenitor cells'(EPC), a class of circulating adult stem cells with a central role in maintaining a functional endothelial layer and in adult neovascularization, both deteriorating with age. Using a phage display technology, our team has identified a number of peptides with specific binding to EPC. These peptides were incorporated in a terpolymer while still maintaining their affinity for EPC. Moreover, using rational peptide design based on crystallographic data, our team also synthesized a biotinylated peptide binding to the endothelial-type VEGF receptor 2, also present on EPC. Specific Aim 1. Platform assembly. In this step we will synthesize the capture peptides with affinity for the cell surface, and will deposit the peptide-enriched attachment matrix in tissue culture wells. Specific Aim 2. Assay optimization. In order to obtain the best discrimination between target cells and bystanders, we will test the number and composition of cell colonies derived from suspensions of known composition, as dependent on layer thickness, fiber density, polymer and peptide composition of the scaffold, as well as on the cell attachment time, washing strength and duration. Specific Aim 3. Assay validation. We will validate our assay on a human population stratified by age, by comparing the results obtained by this method with the traditional endothelial cell colony assay, and with flow cytometry. Our proposal has both short and long term economic and educational benefits. Its implementation will immediately create several research positions, and help maintaining more others. Moreover, the proprietary technology developed may enable the formation of a start-up company, creating additional employment opportunities relating to biotechnology, material sciences and administration. PUBLIC HEALTH RELEVANCE: This application is in response to the Area of Scientific Priority "Development of tools to facilitate research on the basic biology of aging" of the National Institute on Aging. Based on multiple collaborations and on solid preliminary data, we propose to develop a novel assay, and the corresponding instrument, for collecting from blood, analyzing and multiplying circulating stem/progenitor cells (for endothelium, vascular muscle, neurons, cardiomyocytes, etc., tumor cells). Among these of outmost importance are the endothelial progenitor cells (EPC), the focus of our proposal. Circulating levels of EPC and their endothelial differentiation capacity were associated with aging, gender, and with multiple cardiovascular pathologies and risk factors, such as atherosclerosis, heart failure, diabetes, hypertension, obesity, smoking, etc. EPC are also a major cell type intended to be used for cell therapy for heart, lung and blood diseases. Therefore, both diagnosis and cell therapy protocols will substantially benefit from this improved tool of progenitor cells detection in circulation, in bone marrow aspirates, or after multiplication in tissue culture. The project will have both short term economic impact and long term sustainability by creation of a company for commercialization of the assay kit.

DESCRIPTION (provided by applicant): Long term monitoring of oxygen concentrations at the level of biomedical implants, and optimizing its availability are key issues in diagnostics, tissue engineering and biotechnology. Recent advances in the use of EPR detectable, oxygen-sensitive probes with excellent sensitivity, accuracy and remote accessibility, opened a new era of opportunity for real-time monitoring of oxygenation in vivo. However, for many applications these probes need to be encapsulated behind a semi-permeable membrane. We developed a nanofilter-limited, implantable model device with dual EPR-based oxygen sensor and drug delivery capabilities, and we tested its capacity to track oxygenation in a tissue engineered construct containing bone marrow progenitor cells. Using a quantitative model of oxygen diffusion in the vicinity of implants, we also demonstrated that our oxygen sensor provides a proportional relationship between the reported local pO2 and microvascular density, supported by in situ observations at the end of the experiment. Furthermore, we synthesized a novel class of biomaterials, incorporating by electrospinning the EPR sensitive nano-crystals directly into a poly-caprolactone microfibrillar scaffold. Using EPR imaging, we showed the in vivo distribution of oxygen within this scaffold and found that the determined average pO2 was compatible with its subcutaneous location. In addition, we demonstrated the capacity of this scaffold to support proliferation and endothelial differentiation of bone marrow progenitor cells. Here we propose to further validate, improve the design and expand the applications of this device. This project will take into consideration, besides the development of the device and the logistics of its use, also the design of its interface with the tissue, with the goal to develop a tool for studying and optimizing implant oxygenation as dependent on nearby neovascularization. Specific Aims: 1. Determine oxygenation within tissue engineering constructs as dependent on their vascularization. 2. Test the hypothesis that oxygenation within a filter-limited implant is sensitive to pharmacological modulation of nearby angiogenesis. 3. Demonstrate that stimulation of neovascularization in peri-implant space using tissue engineering methods could also improve implant oxygenation. The progress in the use of implanted oxygen probes will be readily translated into novel clinical applications such as monitoring available oxygen in tissue engineering constructs, with improved perfusion, optimized cell encapsulation, or better functioning of oxygen or glucose sensors. PUBLIC HEALTH RELEVANCE: We propose to develop a method and an implantable device for minimally invasive, in vivo monitoring of local oxygen concentrations in biomedical implants. These will be useful for monitoring oxygenation in oxygen- dependent sensors, in encapsulated cells, in tissue engineering constructs, and for other branches of regenerative medicine.