Keith L. March - US grants

DESCRIPTION (Adapted from Investigator's Abstract): Smooth muscle cell (SMC) proliferation plays a major role in vascular diseases. This cell cycle re-entry may involve modulation of cell cycle-regulatory protein expression. The SV40 large T antigen (TAg) is a viral oncoprotein able to transform cells in part due to its ability to complex with cellular proteins which behave as negative growth regulators; this suggested it expression in vascular smooth muscle cells as an approach to capture and identify negative regulators of smooth muscle cell cycling. Transgenic mice targeting a temperature-sensitive mutant SV40 TAg (tsA58) to SMC have been generated and have served as a source of SMC populations, which have in turn been the subject of initial studies with regard to cell cycle, differentiation, and the expression of TAg-associated proteins. The objectives of this proposal are the isolation of clonal populations of smooth muscle cells from adult and embryonic mice which are reversibly transformed by the association of the tsA58-TAg with a spectrum of negative growth regulatory proteins; and the use of these cells to study their range of phenotypes in relation to the spatiotemporal origin of the cells. The specific aims are: 1.) Isolation and characterization of clonal vascular SMC lines of neurocrest and mesodermal origin rom adult mice expression the tsA58-TAg antigen in smooth muscle cells; 2.) Isolation and characterization of clonal vascular smooth muscle cell lines from these progenitors from mice at various developmental stages; and 3.) Characterization of negative cell cycle regulatory proteins associating with T-antigen in adult and embryonic vascular smooth cells in vitro, and expressed during normal development of vascular smooth muscle tissue in vivo. These transgenic SMC exhibit a relatively differentiated phenotype in culture; and some isolates have shown temperature-dependent differentiation. This supports the concept that clonal SMC lines derived from adult and embryonic mice will provide a range of phenotypes for study. Proteins typical of adult as well as earlier developmental stages will be detected by co-immunoprecipitation with TAg. Initial studies have detected three phosphoproteins of unknown identity (pp130, pp160, pp170) associated with TAg in SMC. These and other such proteins will be evaluated for identity with known cell cycle regulatory proteins. Experiments will then be extended to assess the in vivo expression of negative growth regulators. These studies are expected to yield molecular information concerning the development spectrum of proteins regulating cycling within vascular SMC.

DESCRIPTION: (Applicant's Description) Coronary vessel occlusion leading to myocardial ischemia is one of the leading causes of morbidity and mortality in the Western hemisphere. Several methods have been developed to achieve revascularization of ischeniie regions of myocardium. Mechanical approaches include surgical bypass and balloon angioplasty to circumvent or open occluded vessels, and approaches such as transmyocardial laser revascularization (TMR) to directly enhance myocardial blood flow by the provision of new intramyocardial vascular channels. TMR, which has been shown to lead to significant anginal relief, was initially thought to create persistent laser-drilled vascular channels. It is now hypothesized that the beneficial results of TMR are associated with the induction of intraynyocardial angiogenesis in the context of a tissue bearing response. Proton beam radiation is based on the characteristic of proton beams to deposit energy at prescribed, adjustable tissue depths while minimizing exposure of overlying skin and superficial structures and sparing structures deep to the desired target tissue volume. The unique ability of proton beams to deposit specific doses of ionizing radiation to subsurface tissues suggests the possibility that proton beams specifically directed to myocardial tissues could induce a localized angiogenic response. This myocardial angiogenesis could supply blood flow capable of preserving myocardial function in the context of native arterial occlusion. This exploratory study will examine the feasibility of using external proton radiation to induce myocardial revascularization.

[unreadable] DESCRIPTION (provided by applicant): The discovery that pluripotent cells reside in the stromal cell fraction of subcutaneous adipose tissue has revealed a novel source of cells for autologous cell therapy. These adipose stromal cells (ASCs) have been shown to differentiate into myocytes, chondrocytes and neural cells; however, little is known about their ability to differentiate into cells of vascular and hematopoietic lineages. Our preliminary data suggests that many human and murine ASCs express surface markers that are typically found on either hematopoietic or endothelial stem and progenitor cells. We will therefore attempt to clarify whether ASCs have the multipotentiality of incorporating into nascent or reparative vasculature in the context of ischemic angiogenesis or atherosclerotic intimal formation; and to contribute to hematopoiesis as well as circulating vascular progenitors following bone marrow ablation. We will also determine the particular markers that are associated with these potentials. Specific Aim 1 will examine whether ASCs can differentiate into endothelial and vascular smooth muscle cells in vitro, enhance angiogenesis in vivo, and incorporate into the nascent vascular network in the setting of ischemia. Specific Aim 2 will investigate whether ASCs have the potential to differentiate into hematopoietic cell lineages in vitro, and engraft into bone marrow as either hematopoietic stem cells or hemangioblasts, which can subsequently give rise to blood lineages as well as circulating vascular progenitors. Specific Aim 3 will determine whether ASCs are capable of homing to sites of vascular injury, and modulating the local injury response, perhaps by providing endothelial cells or supporting those which pre-exist. Together, these studies will help define the multipotency and biology of ASCs, and delineate how ASCs could be used for autologous cell therapy in the setting of either cardiovascular or hematologic disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a renewal application for an institutional National Research Service Award entitled Training in Vascular Biology and Medicine at the Indiana University School of Medicine under the direction of Keith L. March, MD, PhD, the Director of the Indiana Center for Vascular Biology and Medicine (ICVBM). The primary focus of this training grant is to provide vascular biology training to fellows from multiple participating Divisions and Departments, from an interdisciplinary perspective with a particular emphasis on translation of basic findings into clinical research and practice. The importance, as well as rarity, of an integrated program in vascular biology and medicine is recognized by both scientific and clinical communities, and by national organizations such as the American Heart Association, and National Institutes of Health. At Indiana University, over the past 7 years, we have developed a multidisciplinary program in Vascular Biology and Medicine, comprised of 30 investigators from basic as well as clinical departments. We have created a highly interactive environment for collaboration among these investigators. Importantly, we have developed several group activities which lend themselves to a robust training environment: a vascular biology seminar series, a vascular journal club, and two vascular research discussion groups, each of which was originally targeted toward the eventual submission of a PPG or UOl multi-investigator grant. Thus, a key strength of the training program is its provision of a structure linking scientists and trainees from multiple disciplines into a unified program, focused on providing the training necessary for emerging junior faculty to develop a cohesive view of vascular biology, and, ultimately, to be successful in establishing independent research careers. The ICVBM T32 has been able to provide research fellowships for 8 post-doctoral fellows since its funding 4 years ago. These trainees have been highly productive, and their work has resulted in 25 publications in peer-reviewed journals. Furthermore, of the 5 trainees who have completed their T32 support, 2 have accepted academic faculty appointments, and 1 has progressed to a second fellowship in Pediatric Cardiology in preparation for an academic career in this field.

DESCRIPTION (provided by applicant): Cellular therapy is a strategy aimed at repairing, enhancing, and replacing the biological function of a damaged tissue or system by means of autologous or allogeneic cells. Until very recently, most work involving cellular therapy had involved either the replacement (rescue) of hematopoietic tissue following myeloablative doses of chemotherapy, or the use of immune cells as therapy for cancer. Recent advances in understanding of stem and progenitor cell biology, cell delivery methods, and a range of animal models of human disease have also paved the way for exploring novel cellular strategies to address non-malignant diseases of multiple organs and tissues, including ischemic limbs, poorly healing wounds, ischemic hearts, and several others. In developing this proposal, we have built upon the synergistic expertise of leaders in complementary areas of stem and progenitor cell biology, who have each played key roles in defining a strongly collaborative environment at Indiana University in basic understanding as well as translation of adult stem cell research for diseases focusing on both the hematopoietic and cardiovascular systems. The central themes of this proposal are: 1) Defining synergistic interactions among progenitor cells of distinct lineages;2) Evaluation of progenitor cell types with regard to mechanisms of defective tissue repair and regeneration in the context of aging and acquired disease;and 3) study of cells from each of the three selected lineages (hematopoietic, mesenchymal, and endothelial) from three readily available tissue sources that facilitate direct translation into human disease (cord blood;adipose tissue;and vascular structures including umbilical cord and saphenous veins). Each of these themes will be consistently pursued using cells that have routinely been well-defined and purified using multi-marker cell analysis tools and in vivo functional evaluation. These themes will be developed in 4 "full scale" projects which will continue throughout the operational time of the Consortium, complemented by 2 "small scale" pilot proposals which will be submitted for additional consideration as described in detail below.

DESCRIPTION (provided by applicant): Pulmonary emphysema is a prevalent lung disease defined by permanent enlargement of airspaces, but also associated with systemic effects on organs that include the bone marrow and the cardiovascular system. Little is known about the mechanisms of systemic illness in emphysema and their impact, if any, on the lung disease. The lung destruction, clinically apparent after years of cigarette smoking has been attributed to protease-antiprotease imbalance, chronic inflammation, oxidative stress, and excessive alveolar cell death with loss of pulmonary capillaries that support the alveolar unit. We demonstrated that pluripotent cells contained in adipose stroma, called adipose stem or stromal cells promote capillary growth and limit ischemic tissue damage in models of acute skeletal muscle, myocardial, and cerebral ischemia and these salutary effects are mediated by angiogenic and anti-apoptotic paracrine factors. Given the feasibility of obtaining these stem cells, requiring no or limited ex vivo expansion, we tested the effect of adipose stem cells on cigarette smoke-induced murine emphysema. This form of regenerative treatment resulted in preservation of alveolar surface area and marked protection of the bone marrow from the suppressive effects of smoke on the number and cycling of multiple lineages of progenitor cells. We therefore generated the novel hypothesis that treatment with adipose stem cells will ameliorate the alveolar structural loss induced by cigarette smoking by decreasing lung structural cell death and reducing the loss of bone marrow-derived progenitor cells. To test this novel promising therapy for emphysema and to advance understanding of the crosstalk between the lung and bone marrow in emphysema development, we focused on 3 specific aims: 1. to determine the efficacy of adipose stem cells to limit cigarette smoke-induced murine emphysema; 2. to determine the effect of adipose stem cell treatment on the cigarette smoke-induced bone marrow-derived progenitor cell loss and to establish its importance to the development of emphysema; 3. to establish the role of adipose stem cell-secreted paracrine factors VEGF, HGF, and TSG-6 in the inhibition of lung destruction and of bone marrow hypoplasia induced by adipose stem cell exposure. We have assembled a multidisciplinary team with expertise in emphysema pathobiology, adipose stem cell-, vascular-, and bone marrow stem cell-biology to investigate the proposed comprehensive research plan. If completed, this work will determine the effectiveness, optimal approach, and mechanisms of adult, thus generally ethically accepted, adipose stem cell therapy in a relevant emphysema model, and will accelerate its implementation as a potential therapeutic approach in COPD.

DESCRIPTION (provided by applicant): The Indiana Regional Cardiovascular Cell Therapy Center (IRCCTC) will extend the work of the Cardiovascular Cell Therapy Research Network, particularly in the area of peripheral arterial disease (PAD). Critical limb ischemia (CLI) results in at least 50,000 amputations annually, with a cost estimated at $4.3 billion/year. In a recent Phase l/ll trial we have demonstrated safety and feasibility of intra-muscular injection of autologous bone marrow mononuclear cells (ABMNC) in patients with CLI, and provided initial evidence that this treatment improves amputation-free survival at one year. Although these results are promising, there is an opportunity to improve the effectiveness of cell therapy for CLI by identifying more potent sources of progenitor cells and by evaluating functional characteristics of transplanted cells. While many cardiovascular cell-based trials have focused on ABMNC, adipose stromal cells (ASCs) have demonstrated qualities particularly suitable for promoting limb salvage in CLI. In addition, cord blood mononuclear cells (CBMNC) have been shown to include vasculogenic endothelial progenitor cells, to augment perfusion in ischemic limbs, and to be tolerated by an immunocompetent host. CLI presents an excellent opportunity to examine these two readily accessible cell populations with regard to suitability for cardiovascular cell therapy, in that there exist no other options fo salvage of the index limb, potential adverse events are not immediately life-threatening, and tissue can be obtained for analysis in the event of amputation. Also, mechanisms promoting limb salvage in CLI have relevance to myocardial ischemic syndromes. In this application for a new Regional Center, we propose two protocols, respectively evaluating allogeneic cord blood mononuclear cells and adipose stromal cells as potential therapies for CLI. Aim 1 will evaluate whether CBMNC are superior to placebo in promoting amputation-free survival at 1 year in subjects with critical limb ischemia of Rutherford Category 4, and no /high-risk options for standard revascularization. Aim 2 will test the hypothesis that adipose stromal cells (ASCs) are safe and may increase time to amputation in subjects with critical limb ischemia and ulcers / tissue loss (Rutherford Category 5-6).