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Research Projects Projects accomplished in the previous five years of funding
Projects (ongoing): Mehrdad Abedi, MD – Hematopoietic origin of fibroblasts at sites of injury Tissue fibroblasts play a key role in growth factor secretion, matrix deposition and matrix degradation, giving them an important role in many physiologic and pathologic processes. Fibroblasts are critical to the process of wound repair, and with their conversion to myofibroblasts they exert contractile forces to reduce the size of the wound. Conversely, uncontrolled proliferation and/or activation of fibroblasts results in tissue fibrosis, a hallmark of many pathologic processes. Despite these key roles of fibroblasts, there is relatively poor understanding about their origin and their relationship to other cells at the site of injury, such as macrophages, pericytes, and smooth muscle cells. In injured tissue, fibroblasts are generally thought to arise from the local connective tissue. Recently, however, a number of in vivo transplantation studies have revived the notion of bone marrow as a major source of tissue fibroblasts, in that up to 40% of fibroblasts in some experiments come from bone marrow. Mesenchymal cells from the marrow have been expected to be the source of these marrow derived fibroblasts. However, recent experiments in our COBRE laboratory have found that mainly CD45+, Sca+ and cKit+ cells from the marrow are capable of producing fibroblasts in the injured skin. The hypothesis is that the origin of marrow derived fibroblasts is the hematopoietic component of bone marrow. Given the extensive literature and techniques available for studying hematopoietic cells, these findings provide the COBRE with the opportunity to determine novel markers/determinants in the development of the dermal fibroblast population after injury. The following specific aims are proposed: 1) identify the marrow cells involved in the generation of tissue fibroblasts. Specifically, we will use the well defined model of bone marrow differentiation to identify the intermediary cells between marrow stem cells and tissue fibroblasts. We will pay particular attention to fibrocytes as one of the most studied marrow derived cells that can differentiate into fibroblasts; 2) determine the nature of marrow derived fibroblasts in comparison to their endogenous counterparts. We will determine whether marrow derived cells are both fully functional and synthetically active, and therefore can have a significant role in wound healing; 3) Harvest marrow cells that have homed to the injury area or after their topical delivery to wound in a novel fibrin construct we have developed. At different time points after engraftment, we will test for known markers as well as identify differentially expressed genes that can potentially determine the developmental steps leading to the formation of mature fibroblasts. In summary, this proposal focuses on the developmental biology of fibroblasts present at sites of injury and will apply well established hematopoietic models of cell differentiation to determine their origin.
Leslie Cousens, PhD - Development of novel bispecific antibodies for facilitating site-specific tissue repair Bispecific antibodies (BiAbs) have drawn considerable attention from the research community. Their unique structure contains two distinct antigen-binding specificities, so they can be used for any application where it is desirable to juxtapose two molecules or cells within a distance of a few nanometers. For instance, a therapeutic agent can be placed on one arm, while the other is available to specifically target a diseased or injured tissue. This therapeutic agent can take many forms, including that of a toxin, drug, prodrug, enzyme, DNA, anti-vascular agent, gene therapy vector, radionucleotide or even a functional cell. Indeed, bispecific antibodies have been used in both research and clinical efforts to target cytotoxic T and NK cells to kill tumor cells. Considering recent advances in the identification of tissue-, disease-, and injury-specific antigens together with a better understanding in the fields of immunology and stem cell biology, BiAb technology holds great promise for addressing a number of therapeutic needs. The goal of this proposal is to take what we have learned from the application of BiAbs in targeting killer cells to tumors and demonstrate their broader potential in targeting a variety of cells to different injured tissues. These studies could have great therapeutic relevance to problems encountered in tissue repair. Moreover, establishing useful BiAb that can help recruit cells to the site of injury could increase our understanding of cell trafficking and the role of certain cell types, for example macrophages and bone marrow derived cells. The hypothesis is that specific antibodies will direct trafficking of specific cell populations in different states of differentiation to sites of injury, where they can facilitate tissue repair and reconstitution. To test this hypothesis, we propose 1) to combine (chemically heteroconjugate) one mAb directed at an injury-associated antigen (i.e. fibrinopeptide A, VCAM-1, etc.) with a second mAb specific for an “effector” cell population (i.e. F4/80 expressed by macrophages, c-kit expressed by bone marrow-derived stem cells, etc.) to produce a series of bispecific antibodies (BiAb), 2) to use these BiAbs to target these selected cell populations to injured skin and skeletal muscle tissues after experimentally-induced wounds in the respective mouse models (i.e. a full thickness tail-wound and a mechanical crush injury); and 3) to evaluate the effects of bispecific-antibody targeted cells on tissue repair and reconstitution. These studies will provide critical proof of the principle that the BiAbs can be used as a platform technology to specifically target cells to particular tissues. Based on these studies, a variety of mechanistic approaches and clinical applications in tissue repair are envisioned, limited only by the identification targeted cell- and injured tissue-specific antigens.
Satori Iwamoto, MD, PhD – Use of stem cells in human chronic wounds In recent years, considerable success in the treatment of non-healing chronic wounds has been achieved with some advanced therapies, including growth factors and tissue engineering products. However, up to 50% of difficult to heal wounds, including venous ulcers of long duration, are unresponsive to treatment. In a pilot study, we have recently shown that topically applied cultured autologous mesenchymal stem cells (MSC) may accelerate healing in difficult to heal human wounds. We obtained a single bone marrow aspirate of 35-50 ml from patients with acute and chronic, long-standing, venous ulcers. Marrow cells were grown in vitro under conditions favoring the propagation of MSC. Flow cytometry, immunostaining, and functional inductive studies showed findings highly consistent with published reports of human MSC. We developed a novel modified fibrin spray system to deliver the cells to the wound. We found a significant direct correlation between the application of MSC and healing. These studies are on-going. However, we are also developing ways to use mobilization of bone marrow stem cells to achieve targeting of stem cells to sites of injury. This will first be done in murine studies, optimizing the reagents and the conditions, with the ultimate goal of achieving proper mobilization and targeting in human chronic wounds. The goal of this proposal is to determine the true potential of stem cells in wound healing. To test the overall hypothesis that stem cells can be safely used for tissue repair, we will perform a clinical randomized controlled trial. This trail will be backed by methods and results using murine studies. Within the context of this clinical trial, we propose the following two specific aims:
These studies will determine whether bone marrow-derived stem cells can accelerate closure of difficult to heal wounds, and will prospectively establish the role of promising molecular markers in epidermal migration and healing.
Paul Liu, MD – Augmenting ischemic skin flap survival using AAV-FGF2 and AAV-VEGF 165 This project will develop a novel application for a recent technique within gene therapy in the field of reconstructive surgery. We propose to use adeno-associated viral vectors designed to cause infected cells to elaborate potent blood supply-building proteins, namely VEGF, PDGF, and FGF2. This enhanced vascular network appears to rescue ischemic tissue from death, allowing “flaps” (tissue transferred from one anatomic location to another for the purpose of closing a wound or reconstructing parts of the body) to be constructed of longer length, greater size, or greater reliability. Statistics compiled by the American Society of Plastic Surgeons (www.plasticsurgery.org) tracked over 5.2 million reconstructive surgeries in the US last year alone. In addition, this project is germane to the overall mission of bettering wound healing, and may be applicable to any situation of tissue ischemia. It builds upon earlier, published work of the applicant (P Liu), who, though currently Chairman of Surgery at Roger Williams Hospital, Providence, RI, has never been the recipient of competitive Federal funding except a T32 training grant. It is not mentored, but will rely on the critical input from collaborators at Brown University and Roger Williams skilled in those techniques new to the applicant. The specific hypothesis tested is: Engineering tissue with AAV-delivered angiogenic genes can improve survival of ischemic flaps derived from that tissue via recruitment of endothelial progenitor cells. In addition to testing the effects of each of the transgenes, our approach will take advantage of the greater efficiency of viral-mediated gene transfer to assess the combination of VEGF + FGF2, which, when delivered via liposome in plasmid form, was more effective than single gene therapy delivered the same way. We propose the following specific aims:
We expect the approach to be both efficacious and clinically relevant. Addressing Aim 2 will help answer a controversial issue in vascular biology, namely, where does the new blood supply in injury repair come from? We will utilize siRNA methods of gene silencing to help get at that answer, as well as localization technology (IVIS) and adoptive transfer of endothelial progenitor cell-enriched populations into the ischemic tissue. Lastly, a new portable spectroscopic device, the ViOptix probe, measuring spectral shifts in the near infrared spectrum of oxygenated hemoglobin as a function of perfusion, will help determine real time tissue viability.
LuGuang Luo, MD, PhD – Bone marrow repairs human islet injury and supports its longevity The success of islet transplantation is hampered by the high rate of islet cell death and dysfunction after isolation. Therefore, the repair of islet damage from the isolation process and the opportunity to maintain islets long term in vitro as a new islet resource would represent significant advances and lead to a more widespread use of islet cell transplantation. Successful utilization of bone marrow in repairing skin, neuron, heart, and muscle injury led us to propose that bone marrow could offer a potential solution to these challenges. In our preliminary studies using co-cultures of whole bone marrow with islet, bone marrow was shown to increase islet function/survival (more than six months), stimulate islet growth and generate long-term insulin producing tissue in vitro. We hypothesize that specific subpopulations of marrow cells may be responsible for these findings. We have also hypothesized that extracellular ATP, ATP receptor (purinoreceptor P2XR), and interleukin 1β (IL-1β) are involved in bone marrow-induced repair of islet injury. In this project, we plan to identify whether multiple or single specific lineage marrow cells contribute to islet reconstitution. We will examine whether these reconstituted islets have sufficient function and vascularization in vivo as determined by transplantation into non-obese diabetic severe combined immunodeficiency disease (NOD/SCID) mice. Finally, we will investigate whether bone marrow modulates ATP, its receptor P2XR, IL-1β and its downstream pathways. This project will have benefits for current islet transplantation protocols and will provide insight into the mechanisms of islet cell death and regeneration.
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