1997 — 1999 |
Huard, Johnny |
P60Activity Code Description: To support a multipurpose unit designed to bring together into a common focus divergent but related facilities within a given community. It may be based in a university or may involve other locally available resources, such as hospitals, computer facilities, regional centers, and primate colonies. It may include specialized centers, program projects and projects as integral components. Regardless of the facilities available to a program, it usually includes the following objectives: to foster biomedical research and development at both the fundamental and clinical levels; to initiate and expand community education, screening, and counseling programs; and to educate medical and allied health professionals concerning the problems of diagnosis and treatment of a specific disease. |
Myoblast Mediated Gene Transfer to the Joint @ University of Pittsburgh At Pittsburgh
Several genetic and acquired pathologic conditions of the musculoskeletal system, such as, arthritis, damage to ligament, cartilage, and meniscus may be amenable to gene therapy. Event through ex vivo gene transfer using synovial cells has been shown to deliver genes encoding for anti-arthritic proteins into the rabbit knee joint, its success has been limited due mainly to a transient transgene expression. Although the cause of this transient expression is unknown, the use of a cell that becomes post-mitotic with differentiation such as myoblasts may allow high level of gene transfer and persistent transgene expression. In fact, we have observed that myoblasts can be transduced with a higher efficiency than synovial cells using the same adenoviral solution in vitro. In addition we have observed that engineered myoblasts are capable of adhering as well as differentiating into post-mitotic myotubes and myofibers into several structures in the joint including the ligament, meniscus, and synovium. The presence of post-mitotic myofibers in the knee joint opens up the possibility for long term expression of the secreted protein. Here we present a series of experiments aimed at validating the hypothesis that autologuous myoblast transfer using retrovirally-transduced primary myoblasts may achieve a high level of gene transfer and a persistent transgene expression into the joint. We then plan to investigate whether myoblast can deliver a persistently high level of IL1-Ra production into an arthritic knee to eventually alleviate the symptoms of arthritis.
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1 |
1999 — 2002 |
Huard, Johnny |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Maturation Dependent Adenoviral Infectivity of Myofibers @ University of Pittsburgh At Pittsburgh
Increasing attention has been focused on skeletal muscle as a target tissue for gene transfer both for the production of proteins that may be therapeutic for muscle disorders and the systemic delivery of non-muscle proteins. Although the engineering of new mutant muscle proteins and the systemic delivery of non-muscle proteins. Although the engineering of new mutant vectors has reduced the problems associated with viral cytotoxicity and immune rejection, the inability of viral vectors to efficiently transduce mature muscle fibers has remained a major barrier to the application of gene transfer to skeletal muscle. Results from our laboratory and others have shown that adenovirus efficiently transduces neonatal muscle; however, within a few days of mouse development, the muscle is largely refracted to adenoviral transduction. Here we present a series of preliminary results and proposed research protocols aimed at defining the barriers to adenoviral transduction of mature muscle. We then plan to investigate methods by which these barriers can be overcome. The proposed research should define and eliminate a major hurdle facing the application of viral gene delivery to skeletal muscle to enable efficient application of muscle-based gene therapy for both inherited and acquired diseases.
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1 |
2001 — 2004 |
Huard, Johnny |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Improving Muscle Healing Through Prevention of Fibrosis @ Children's Hosp Pittsburgh/Upmc Hlth Sys
Muscle injuries, especially pulls and strains, present a challenging problem in traumatology and are among the most common and most often disabling injuries in athletes. The injured muscles are capable of healing, although very slowly and often with incomplete functional recovery. The injured muscle can promptly initiate regeneration for the healing process, but that process in inefficient and is hindered by fibrosis ie, scar tissue formation. More importantly, the scar tissue that replaces the damaged myofibers is a potential contributing factor in the tendency of strains to recur. We have identified various growth factors capable of enhancing myoblast proliferation and differentiation, and their delivery within injured muscle improves muscle regeneration, but the development of fibrosis still limits recovery. On the other hand, it has been reported that the over expression of transforming growth factor (TGF-) in various injured tissues is the major cause of fibrosis in animals and humans. Indeed, we have observed that TGF- plays a central role in skeletal muscle fibrosis and, more importantly, that the use of antifibrosis agents, such as decorin, that inactivate the effect of this molecule can reduce muscle fibrosis and consequently improve muscle healing to a near complete recovery after injuries. Our recent observation that decor in can also enhance muscle regeneration makes this molecule more than ideal to improve muscle healing after injury. We therefore propose to investigate the kinetics of TGF- expression, muscle regeneration, and fibrosis after strain and to delineate the mechanism by which this molecule initiates the fibrosis cascade in skeletal muscle. We will consequently develop biological approaches based on decor in to efficiently prevent the scarring process by blocking the action of TGF- and activate muscle regeneration at the adequate time period post-injury. We finally propose to characterize efficient way to deliver therapeutic and lasting levels of decor in into the injured muscle through the following strategies: (1) direct intramuscularly injection of the recombinant proteins and (2) in vivo gene delivery by gene vectors. These studies should further our understanding of the muscle healing process, expedite the methodology to promote efficient muscle healing, and contribute to the development of innovative therapies for other muscle diseases, such as dystrophies.
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0.939 |
2001 — 2005 |
Huard, Johnny |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Muscle Based Tissue Engineering to Improve Bone Healing @ Children's Hosp Pittsburgh/Upmc Hlth Sys
DESCRIPTION (provided by applicant): Segmental bone defects and nonunions are relatively common in the craniofacial skeleton. Osteogenic proteins, including bone morphogenetic protein-2 and 4 (BMP-2,4), can promote bone healing in segmental bone defects, but the osteogenic proteins' short half-lives and rapid clearance by the bloodstream limit the success of this technology. Gene therapy and tissue engineering approaches that can achieve high expression levels of these proteins may help to further improve craniofacial bone healing. Our recently isolated clonal population of muscle-derived stem cells (mcl3 cells) that can express stem cell markers, differentiate into both myogenic and osteogenic lineages, and, more importantly, improve bone healing in a calvarial bone defect may be an ideal cell population to mediate gene transfer of osteogenic proteins. The long-term goal of this proposed project is the development of gene therapy approaches based on this novel population of muscle-derived stem cells to efficiently deliver the osteogenic proteins and improve craniofacial bone healing. The mechanism by which these muscle-derived stem cells differentiate into osteogenic lineages under the influence of BMP-2 and BMP-4 will be tested and compared. In addition, we propose to characterize designated approaches of muscle-based tissue engineering using the muscle-derived stem cells in an ex vivo gene transfer of osteogenic proteins in combination with a scaffold to improve bone healing in a mouse calvarial defect. We will investigate the persistence of osteogenic protein expression, the presence of immune response and undesirable side effects related to the overexpression of these proteins, and the biological effects on fracture healing. The use of vascular endothelial growth factor (VEGF), a well-known angiogenic factor, to further improve bone healing will be also characterized. Although this proposed research will focus on muscle-based tissue engineering to regenerate a calvarial defect, this technology ultimately will be applied to other craniofacial sites, as well as appendicular bones. The proposed research will enhance and expand our knowledge of bone healing and develop a clinically relevant treatment based on new molecular therapeutics to treat osseous deficiencies.
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0.939 |
2002 — 2006 |
Huard, Johnny |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Muscle Regeneration Through Stem Cell Transplantation @ Children's Hosp Pittsburgh/Upmc Hlth Sys
DESCRIPTION (provided by applicant): We recently have used a modified preplate technique to isolate 3 populations of myogenic cells from normal mouse skeletal muscle based on their adhesion characteristics and proliferation behaviors. Although 2 of these populations display satellite cell characteristics, the third population consists of long_term proliferating (LTP) cells expressing hematopoietic stem cell markers. The stem cell characteristics of the LTP cells include the abilities to retain their phenotype for more than 30 passages in culture (self-renewal) and to differentiate into various lineages, including muscle, neural, osteogenic, and endothelial. The transplantation of the LTP cells, in contrast to satellite cell transplantation, significantly improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle. The overall goal of our proposed project is to further investigate important features of the LTP cells (i.e., sources and mechanisms involved with the improved transplantation capacity) that are paramount to their potential use in transplantation to improve muscle regeneration and deliver dystrophin to dystrophic muscle. In this project we will investigate potential sources of the LTP cells by determining their relationship to satellite cells and to the Side Population (SP) stem cells derived from skeletal muscle (Aim #1). We subsequently will investigate mechanisms by which the LTP cells display an improved transplantation capacity in skeletal muscle. We will evaluate the relative effects of the proliferation and differentiation kinetics of the cells (Aim #2) and determine the possibility that immune_privileged cell behavior also plays a role in the increased regenerative capacity of LTP cells in skeletal muscle (Aim #3). Analysis of the relative importance of the LTP cells' self-renewal and multipotent capabilities will shed light on the contribution of stem cell characteristics to the improved transplantation capacity in skeletal muscle (Aim #4). This project might increase our understanding of the basic biology of myogenic cell populations that display stem cell characteristics. This in turn may unveil new techniques to improve muscle regeneration in dystrophic or injured skeletal muscle via the transplantation of muscle-derived stem cells.
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0.939 |
2003 — 2008 |
Huard, Johnny |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Muscle Stem Cell-Based Therapies For Cardiomyopathy @ University of Pittsburgh At Pittsburgh |
1 |
2006 — 2010 |
Huard, Johnny |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Muscle-Based Tissue Engineering to Improve Bone Healing @ University of Pittsburgh At Pittsburgh
Incomplete healing of bone defects ih the craniofacia.l skeleton is common. Osteogenic proteins, including bone morphogenetic protein 2 and 4 (BMP2, BMP4), promote healing in bone defects, but the proteins'short half-lives and rapid clearance by the bloodstream limit their utility. The main goal of our initial R01 and the first competitive renewal project was the development of tissue engineering approach'es, based on muscle-derived stem cells (MDSCs), to efficiently deliver osteogenic proteins and improve craniofacial bone healing. In brief, during this funding period, we demonstrated that MDSCs genetically engineered to express BMP2 and BMp4 differentiate toward an osteogenic lineage and can improve bone healing in calvarial and long bone defects. We also found that concomitant expression Of vasCular endothelial growth factor (VEGF) improves the bone healing observed after implantation Of BMP2 and 13MP4 expressing MDSCs. Additionally, we have demonstrated that donor sex influences the in vitro osteogenic potential and in vivo bone regeneration potential of murine MDSCs and also identified wa.ys, such as genetic engineering and manipulation of the BMP signaling pathways, to improve the osteogenic potential of MDSCs. Finally, we have isolated the human equivalents of the murine MDSCs and determine their osteogenic potential in vitro. We would like to thank NIDCR for their support during the prior funding period. We met and exceeded all the key objectives in the original R01 application as well as the first competitive renewal, and our results formed the basis for 33 + papers and 117+ abstracts. This DE013420 second cornpetitive renewal application outlines experiments designed to extend these initial findings and lead to. possible future clinical applications of MDSCs to improve bone healing. We will focus this second competitive renewal on human equivalents to murine MDSCs and optimization of their use for bone regeneration. Since after implantation of MDSCs into injured musculoskeletal tissues, including bone, the repair process is often mediated by chemoattraction of host cells, we plan to determine the influence of host cells(especially blood vessel wall progenitors) chemoattraCted by donor human cells during the bone healing process (Aim 1). We plan to examine the effect of age and sexotdonor patient on the number and osteogenic potential of hMDCs derived from that patient. We then will investigate ways to optimize bone formation and healing by using hMDC-based tissue engineering, including the modulation of BMP signaling through inhibition of ERK1/2, pj8 MAPK and PI3K pathwa.ys and mechanical stim'ulation of hMDCs prior to implantation (Aim 2). The proposed experiments will provide important information regarding the basic biology of hMDCs and their use for bone healing and further the development of clinical treatments for osseous deficiencies.
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1 |
2009 — 2010 |
Huard, Johnny |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Muscle Stem Cell Therapy in a Mouse Model of Premature Aging @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): It is well-known that aged individuals, an expanding demographic in the United States, have a dramatically increased risk of numerous debilitating diseases including bone fractures, cardiovascular disease, cognitive impairment, diabetes and cancer. Although the molecular basis of the progressive loss of homeostatic reserve with aging is controversial, there are several lines of evidence that implicate accumulated DNA damage as a major determinant in the progression of age-related pathology. In particular, the majority of human progerias (or syndromes of accelerated aging) are caused by inherited mutations in genes required for genome repair and maintenance, including XPF. ERCC1-XPF protein complex is a highly conserved endonuclease that is required for at least two DNA repair mechanisms: nucleotide excision repair (NER) and DNA interstrand crosslink repair. We have several progeroid mouse models of ERCC1-XPF deficiency including Ercc1-/- and Ercc1-/ , which express levels of ERCC1-XPF at 0% and 10% of normal, respectively. The average life span of the Ercc1-/- mice is 21 days and that of the Ercc1-/ mice is 7 months. Both mice develop age-related pathologies including ataxia, kyphosis, cachexia, disc degeneration, osteoporosis, incontinence, epidermal atrophy, sarcopenia, bone marrow degeneration and liver as well as kidney dysfunction. We previously isolated and characterized a population of muscle-derived stem cells (MDSCs) that displays a high regenerative capacity in various tissues of the musculoskeletal system. Our preliminary results suggest that MDSCs isolated from progeroid ERCC1-XPF deficient mice have proliferation and differentiation defects. Furthermore, injection of wild type (wt) MDSCs into Ercc1-/- mice results in their engraftment into multiple tissues and significantly extends lifespan. Thus we hypothesize that a defect in the adult stem cell compartment in ERCC1-XPF deficient mice is involved in their dramatically accelerated aging and that stem cell therapy may represent a potential strategy to prevent or delay age-associated debilitating changes. The focus of this proposal will be on documenting and characterizing the defect in MDSCs in our unique progeroid mouse models and on demonstrating the ability of transplantation of functional MDSCs to delay the onset of age-related pathologies. PUBLIC HEALTH RELEVANCE: Aging is characterized by the progressive erosion of all organ systems which places the elderly at an increased risk of numerous debilitating diseases and organ system failures including cardiovascular disease, dementia, bone fractures, sarcopenia, and cancer. Demographic studies indicate that the number of individuals aged greater than 65 will double in the next 25 years and impose an unprecedented burden on the U.S. health care system. This research proposal is highly significant in that it has the potential to not only reveal a biological mechanism(s) of aging (defect in stem cell compartment), but could also lead to the development of stem cell therapies which could delay or ameliorate the pathologies associated with aging;therefore, identifying strategies, such as stem cell transplantation, is essential for maintaining the health of our aging population.
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1 |
2012 — 2013 |
Huard, Johnny |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Nerve Repair Through Muscle Progenitor Cell Transplantation @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Muscle Progenitor Cells (MPCs) isolated from skeletal muscle have been shown to be both multipotent and of significant therapeutic value for the repair of various tissues of the musculoskeletal system, including skeletal and cardiac muscles, bone and articular cartilage, yet their ability to undergo neurogenic differentiation has not been fully investigated. We have recently observed that mice and human MPCs are capable of undergoing in vitro and in vivo neurogenic and glial cell differentiation, and inducing functionl healing of sciatic nerve defects in mice. The repair process, determined by histological and immunohistochemical analyses, were also validated by a functional assay (sciatic functional index). Although we have reported that both murine MPCs (mMPCs) and human MPCs (hMPCs) promoted axonal in-growth through their differentiation into glial cells, it is likely that the use of a scaffold to immobilize the injected cells at the injury site could further promote nerve repair. Since we have recently shown that the use of cell sheets as a stem cell delivery vehicle, which immobilizes the cells at the site of injury, further improved the beneficial effect imparted by the stem cells on the injured cardiac and ligamentous tissues, we are proposing to characterize whether the nerve repair process could be enhanced, through the use of a cell sheet comprised of MPCs (Aim #1). Since we have observed that the regenerative potential of MPCs in various tissues correlates with the ability of the cells to induce angiogenesis, we posit that a similar mechanism may explain the beneficial effect imparted by the MPCs in nerve repair. Indeed, it has been observed that VEGF administration can support the growth of regenerating nerve fibers through the process of angiogenesis; moreover, the beneficial effect imparted by bone marrow transplantation on nerve repair appears to occur through the secretion of various trophic factors that promote axon angiogenesis. We are proposing to determine the influence that angiogenesis plays in the nerve healing process (Aim #2) through gain and loss of function experiments using VEGF and sFlt-1 expressing MPCs. Successful completion of these aims will not only provide exciting results for quantitative evidence of the efficacy of MPC transplantation to improve nerve healing, but could also shed light on the mechanism(s) of action by which progenitor cells interact with the microenvironment (especially via angiogenesis) at the site of nerve injury.
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1 |
2014 — 2015 |
Huard, Johnny |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Biomimetic Coacervate Delivery of Muscle Stem Cell to Improve Cardiac Repair @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Cellular cardiomyoplasty (CCM), which involves the transplantation of exogenous cells into the heart, is a promising approach to repair injured myocardium and improve cardiac function. We have isolated a population of muscle-derived stem cells (MDSCs) from the skeletal muscle of mice and humans, that when compared with myoblasts, display a significantly improved capacity for cardiac regeneration in a mouse model of acute myocardial infarction (AMI). Transplanted MDSCs survive significantly better than skeletal myoblasts due to their high expression of cellular antioxidants, which confers the cells with an increased resistance to stress, and through a paracrine effect which reduces myocardial fibrosis, promotes angiogenesis, and ameliorates left ventricular (LV) remodeling. We have successfully expanded human MDSCs, to clinically relevant numbers in culture and more importantly, human MDSCs have already entered the clinical arena for the treatment of bladder dysfunction & myocardial infarction, confirming that MDSCs represent a viable therapeutic cell source for CCM. However, several limitations, such as a poor delivery approach of the cells (direct intramyocardial injection in PBS) that leads to limited cell retention and survival as well as the low cardiomyogenic potential of the MDSCs, may still limit the cardiac regenerative potential of the MDSCs (Primary focus of the application). The use of FGF2-coacervate, as a novel delivery vehicle for the MDSCs, represents a new area of research that could not only promote cell retention, survival, and the cardiac regenerative potential of the MDSCs, but also synergistically enhance angiogenesis through the release of FGF2. We have shown that coacervate loaded with FGF2 was capable of enhancing cardiac repair and regeneration through the promotion of angiogenesis and supporting the survival of residual cardiomyocytes (preliminary data). Therefore, the focus of Aim 1 of this proposal will be to combine the new FGF2-coacervate technology with MDSCs to further improve cardiac repair. We will compare this combinatorial therapy to MDSCs and FGF2- coacervate therapies separately. Moreover, we have observed that the viral transduction of MDSCs with Wnt- 11, a molecule required for cardiogenesis, enhances the cardiomyogenic differentiation of the MDSCs in vitro and cardiac repair in vivo when injected directly into injured myocardium. In a second set of experiments (Aim 2), we will determine whether the intramyocardial injection of Wnt-11 transduced MDSCs (Wnt-11 MDSCs) in combination with FGF2 coacervate, can further enhance the cardiac regenerative potential of the Wnt-11 MDSCs when compared to non-transduced MDSCs and Inducible Pluripotent Stem Cell (iPSC)- derived cardiomyocytes delivered with FGF2-coacervate. The successful completion of these aims will increase our understanding of the basic biology of muscle-derived progenitor cell populations with enhanced cardiomyogenic potential for cardiac repair and facilitate the development of new therapeutic technologies that merge the merits of stem cell therapy with biomimetic coacervate to improve cardiac repair and regeneration.
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1 |
2014 — 2015 |
Huard, Johnny |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
The Use of Coacervate Technology as a New Drug Delivery System For Musculoskeleta @ University of Pittsburgh At Pittsburgh
DESCRIPTION (provided by applicant): Incomplete healing of critical size bone defects, including defects of the craniofacial skeleton, are common. Osteogenic proteins, including bone morphogenetic protein 2 and 4 (BMP2, BMP4), promote bone healing, but the proteins have short half-lives and are rapidly cleared by the bloodstream, which limits their utility. The goals f our previous work were to develop gene therapy and tissue engineering approaches to efficiently deliver osteogenic proteins and improve bone healing using muscle-derived stem cells (MDSCs). We have shown that murine and human MDSCs (hMDSCs) genetically engineered to express BMP2 or 4 could differentiate toward an osteogenic lineage and improve bone healing in calvarial and long bone defects. We also found that concomitant gene delivery of vascular endothelial growth factor (VEGF) improves bone healing after the implantation of BMP2 or 4 expressing MDSCs. Similarly, we have reported that MDSCs isolated from mouse and human skeletal muscle were also capable of chondrogenic differentiation and could be used to promote articular cartilage repair after acute injury (osteochondral defects) and disease (osteoarthritis [OA]), especially when genetically modified to express bone morphogenetic protein 4 (BMP4-MDSC). However, in contrast to bone, our findings also suggested that genetic modification of MDSCs to express both BMP4 and the angiogenic antagonist sFlt-1, could accelerate the AC repair capacity of the cells supporting the fact that blocking angiogenesis is beneficial for AC repair. Although we have made substantial progress in muscle stem cell based therapy for bone and AC healing over the past number of years, most of our previous work involved genetic modification of the stem cells prior to transplantation, a step that limits te clinical translation of the work. We therefore propose a new set of experiments which will aim to circumvent the necessity of using viral transduction via the use of a novel heparin-polycation coacervate delivery system capable of slowly releasing the required therapeutic proteins including BMP2, VEGF and/or sFlt-1 to promote bone and AC repair in conjunction with non- transduced MDSCs. In the first set of experiments we will utilize the heparin-polycation coacervate delivery system to deliver BMP2 and VEGF to enhance hMDSC mediated bone repair. The second set of experiment will aim to promote AC repair after the induction of OA utilizing the heparin-polycation coacervate delivery system to slowly release BMP2 and sFlt-1, in combination with hMDSCs, to enhance AC repair. The efficiency of bone and AC repair with the coacervate-hMDSCs technology will be compared to both hMDSC based gene therapy and hMDSC based free protein therapy (i.e. without the use of the heparin-polycation coacervate). This application outlines a new area of research designed to offer valuable clinically relevant approaches based on novel bioengineering concepts for the treatment of musculoskeletal tissues following injury and disease.
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1 |
2014 — 2018 |
Huard, Johnny |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Bone Abnormalities & Healing Defect in Muscular Dystrophy @ University of Texas Hlth Sci Ctr Houston
DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is a degenerative muscle disorder characterized by a lack of dystrophin expression that ultimately results in cardiac or respiratory failure. DMD patients also acquire osteopenia, fragility fracture, and scoliosis indicating that a deficiency in skeletal system homeostasis also occurs in DMD patients. It is speculated that these skeletal abnormalities are likely a secondary consequence to muscle loss (sarcopenia); however, it remains unclear if they could be due to a direct intrinsic skeletal defect. Recent evidence has emerged implicating adult stem cell dysfunction in the histopathogenesis of DMD. Muscle derived progenitor cells (MPCs) isolated from dystrophin/utrophin double knock-out (dKO) mice (a severe animal model of DMD) have been found to be defective in their proliferation and differentiation capacities. We, and others, have reported that these dKO mice exhibit a spectrum of degenerative changes in their bone, articular cartilage, and intervertebral discs and experience spinal deformities, heterotopic ossification, cardiomyopathy and a decreased lifespan, all of which support a premature musculoskeletal aging phenotype in this mouse model. A defect in bone healing was also observed in these mice; however, it is still unclear whether this defect is an intrinsic bone healing problem or associated with the secondary effects of sarcopenia (Aim 1). Preliminary evidence supports the existence of an adult stem cell defect in both MPCs and mesenchymal stem cells (MSCs) in these mice, supporting the theory that abnormal bone healing could be the consequence of an autonomous defect in the adult stem cell compartment. Thus the second aim of this project will be to further validate whether the MPCs and MSCs in these mice, analyzed at different ages, are defective in their proliferation and osteogenic differentiation capacities compared to MPCs and MSCs isolated from mdx and wild type (WT) mice. It has recently been shown that reducing fibroblast growth factor-2 (FGF2) activity prevents stem cell depletion/exhaustion; therefore, we also propose to determine whether FGF2 inhibitor-loaded biomimetic coacervate could rescue this autonomous adult stem cell defect and delay the onset of bone related histopathologies in dKO mice (Aim 2). Since there is also evidence that the stem cell niche may also negatively impact adult stem cell function, via a non-autonomous mechanism, we propose experiments to determine if the bone defect observed in dKO mice can be rescued through parabiotic pairing which will rejuvenate the dystrophic microenvironment by creating a shared circulation between a dKO and a young WT animal (Aim 3). We have preliminary data that supports the fact that circulating factors from young animals have a beneficial effect on the bone morphologies and healing capacity of dKO mice. In summary, this innovative grant application will: 1) determine whether the bone abnormalities and healing in dKO mice represent an intrinsic bone defect and 2) characterize whether the progressive bone histopathology observed in the dKO mice, is primarily driven by cell autonomous and/or non-autonomous mechanisms.
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1 |
2018 — 2019 |
Huard, Johnny Lu, Aiping (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Effects of Circulating Factors and Progenitors On Wound Healing During Pregcy @ University of Texas Hlth Sci Ctr Houston
Abstract: Wound healing is a complex biological process requiring growth factors and progenitor cells that act in concert to restore the integrity of the injured tissue. Fibrosis or scarring of tissue results from nonspecific repair as well as aberrant wound healing in response to tissue injury and not only predisposes tissue to a secondary injury but also contributes to 45?50% of deaths in the Western world. Increased age is a major risk factor for impaired wound healing due to tissue fibrosis. Many studies at the cellular and molecular level have examined age-related differences in wound healing and have identified an age-related decline in progenitor cells. It has been reported that exposure to factors present in the serum of young mice restores the regenerative capacity of aged progenitor cells. Also, fetal dermis has been shown to regenerate after injury without scarring, while adult skin wound healing usually results in scar tissue. Pregnancy represents a unique biological model of a naturally-shared circulatory system between young and old organisms. Our preliminary data have demonstrated improved muscle healing after injury in pregnant mice. More importantly, we have observed an improved myogenic differentiation capacity of aged muscle progenitor cells (MPCs) after stimulation with serum from pregnant mice, suggesting that circulating factors may influence the potency of stem cells in the mother during pregnancy. We hypothesize that circulating factors during pregnancy have beneficial effects on maternal wound healing, such as reduction in fibrosis for optimal tissue regeneration. Therefore, we propose, in Aim1, to determine whether pregnancy accelerates wound healing (skeletal muscle and skin) and which circulating factors in pregnancy are responsible for this beneficial effect. We will utilize previously described skin wound and muscle injury mouse models and adapt them to investigate the healing process, including fibrosis and contributing circulating factors, during pregnancy. In addition, we will obtain skin biopsies from pregnant and non-pregnant women after surgery to analyze tissue healing with respect to scarring at the biopsy site. It has been reported that fetal microchimeric cells (FMCs) can be found in the circulation and organs of mammals during pregnancy as a result of bidirectional passage of maternal and fetal cells. The presence of fetal cells in maternal circulation may play a beneficial role in repairing damaged maternal tissues. Little has been done to determine the exact roles of circulating fetal progenitor cells and how they interact with maternal progenitor cells during pregnancy with respect to wound healing. In Aim 2, we propose to determine which circulating fetal and maternal progenitor cells in pregnant mice are responsible for the beneficial effects on wound healing during pregnancy. Green fluorescent protein (GFP)- expressing male mice will be crossed with female mice without GFP, and GFP will be used to track FMCs. The Y chromosome will be used as an additional method to track the FMCs. Results from this study will identify rejuvenating factors and novel progenitor cells within blood circulation during pregnancy with potential for development of novel and clinically relevant therapies to improve wound healing and reduce fibrosis.
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0.955 |
2018 — 2019 |
Guilak, Farshid (co-PI) [⬀] Huard, Johnny Lu, Aiping (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Muscle Stem Cells Reprogrammed Through Genome Engineering For Autonomously Regulated Anti-Fibrotic Therapy @ University of Texas Hlth Sci Ctr Houston
Our previous studies indicated that transforming growth factor (TGF)-?1 plays a key role in skeletal muscle fibrosis after injury. Antifibrotic agents that inactivate TGF-?1 can reduce muscle fibrosis and significantly improve muscle regeneration and repair. Furthermore, we have also demonstrated that the transplantation of muscle- derived stem cell (MDSCs) could improve muscle regeneration after injury, but the differentiation of the injected cells into fibrotic cells limits the beneficial effect on muscle repair. In fact, we have observed that MDSCs under the influence of TGF-?1 from the injured muscle microenvironment, not only induce an autocrine expression of TGF-?1 but also promote the MDSCs? differentiation into myofibroblasts that contribute to the development of fibrosis. We have recently reported that combining losartan (anti-fibrotic agent) with MDSC transplantation significantly improved the regenerative potential of MDSCs in skeletal muscle by preventing the MDSC?s differentiation into fibrosis. Although the systemic use of losartan is safe and FDA-approved, it likely leads to widespread blockade of TGF-?1 which might not be desirable. In addition, pharmacological anti-fibrotic therapies are often effective at diminishing fibrosis, but are used at high, unregulated doses and have significant side effects. More recently, using the CRISPR/Cas9 genome editing system, our co-Principal Investigator (Dr. Guilak) created stem cells that can antagonize IL-1? or TNF-?-mediated inflammation in an auto-regulated, feedback- controlled manner for musculoskeletal regenerative medicine applications. They have demonstrated proof-of- concept of the ability to custom-design stem cells that are immune to pro-inflammatory cytokines as a potential cell source for optimal tissue repair. We therefore propose that this novel genome engineering system can also be used to antagonize TGF-?1-mediated fibrosis and further improve muscle healing after injury. We propose to target the TGF-? soluble receptor type II (TSRTII), to antagonize TGF-?1-mediated fibrosis in the application. We are proposing to develop an autoregulatory gene circuit in MDSCs, such that the TGF-?1 gene will be reprogrammed by nuclease-mediated integration of the TSRTII, a TGF-?1 antagonist, immediately downstream of the TGF-?1 signaling pathway. Transgene expression from the endogenous TGF-?1 locus in engineered MDSCs will provide rapid feedback-control to produce TSRTII in response to TGF-?1. We will first test whether the gene-edited MDSCs show the ability to mitigate the fibrotic effects of TGF-?1 in vitro, by examining the differentiation of MDSCs into myofibroblasts and the expression of TSRTII and genes involved in the TGF-?1 pathway. Next, we propose to determine whether muscle regeneration & repair with control MDSCs show significant fibrotic events in response to TGF-?1, whereas muscle repair with genome-edited MDSCs will be protected from fibrosis after injury in vivo.
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0.955 |
2019 — 2020 |
Huard, Johnny Narkar, Vihang A Tashman, Scott (co-PI) [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Biological Approaches to Enhance Skeletal Muscle Rehabilitation After Anterior Cruciate Ligament Injury @ University of Texas Hlth Sci Ctr Houston
Abstract: Anterior cruciate ligament (ACL) reconstruction is the 6th most common orthopaedic procedure performed in the United States, with more than 130,000 ACL reconstructions performed annually. While ACL reconstruction is often considered successful, many patients do not return to pre-injury functional levels (skeletal muscle), and the procedure does not appear to be effective for preventing osteoarthritis (OA). These muscle strength deficits are associated with changes in muscle physiology and structure, including atrophy, infiltration of senescent cells, reduction in muscle progenitor cells (MPCs) and the development of fibrotic tissue through the activation of fibrogenic-adipogenic progenitor (FAPs). In fact, accumulation/infiltration of senescent cells occurs in aging-associated muscle atrophy/sarcopenia, impairing muscle function. We believe that senescent cell accumulation also occurs after ACL injury/surgery, interfering with muscle recovery. Thus, novel biological interventions may be necessary to supplement conventional rehabilitation approaches in order to specifically address muscle histopathology and fully restore muscle function after ACL injury/surgery. Our laboratory has investigated multiple biological strategies for improving muscle healing after a variety of injuries, diseases and aging. We have shown that promotion of angiogenesis is one of the most efficacious strategies to improve muscle healing after injury. One such approach, muscle-specific over-expression of estrogen-related receptor gamma (ERR-?), recapitulates exercise by transcribing a pro-angiogenic gene program that increases muscle vascularization. We posit that this exercise-mimetic pathway may highlight a new concept and help in the development of novel, non-invasive rehabilitation strategies for restoring muscle function after ACL injury. In Aim 1, we will investigate whether muscle histopathology after ACL injury/surgery is associated with the infiltration of senescent cells, a functional defect in MPCs and over-activation of FAPs, as well as whether ERR- ? activation prevents these muscle histopathological cellular events after ACL surgeries. In Aim 2, we will determine correlations between muscle weakness, reduction in muscle regeneration and angiogenesis, and the development of fibrosis, and investigate whether exercise mimicry via ERR-? activation via the use of our transgenic mice, can create resistance to muscle weakness, using our ACL injury model. Our ERR? transgenic mice, engineered to enhance skeletal muscle vascularization, will be a powerful new tool for investigating the link between muscle vascularity, muscle/fibrotic progenitor cells, senescent cells, and muscle weakness after ACL injury. Muscle atrophy and degeneration are major contributors to poor outcomes for a wide range of musculoskeletal disorders, including joint replacement and rotator cuff tears as well as knee injuries. Pharmacological approaches for stimulating angiogenesis and/or reducing senescent cells (using currently available senolytic drugs) have tremendous potential to improve outcomes in many patients after ACL injury/surgery for whom physical therapy alone fails to restore pre-injury muscle function.
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0.955 |
2019 — 2020 |
Huard, Johnny Kolonin, Mikhail G |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Ablation of Non-Myogenic Progenitor Cells as a New Therapeutic Approach to Duchenne Muscular Dystrophy @ University of Texas Hlth Sci Ctr Houston
Abstract Duchenne muscular dystrophy (DMD) is a deadly genetic disease characterized by a lack of dystrophin expression, resulting in progressive weakening and wasting of skeletal and cardiac muscles. Currently, there is no cure for DMD. The approved hormonal treatments have side effects and delay the disease progression only transiently. The emerging gene correction strategies, although effective in mouse models, are likely to be of limited immediate value to patients due to issues associated with virus-mediated gene therapy. Therefore, new approaches to suppress the disease progression are needed. Myogenic muscle progenitor cells (MPCs), also known as satellite cells, become dysfunctional (reduced proliferation and differentiation capacities) as disease progresses, coincidentally with reduced muscle regeneration, aggravating fatty infiltration, and fibrotic tissue accumulation in skeletal muscle. Mesenchymal stromal cells (MSCs) are non-myogenic progenitors of fibroblasts and adipocytes. We and others have reported that MSCs get activated during the disease progression in DMD and turn into fibroadipogenic progenitors (FAPs) that proliferate, induce MPC dysfunction and contribute to muscle pathology. Our preliminary data indicates that FAPs express markers of adipocyte progenitors, also known as adipose stromal cells (ASCs), the MSCs derived from fat tissue, suggesting ASCs as a source of FAPs. The goal of this application is to test whether DMD pathogenesis can be delayed via depletion of MSC-derived FAPs in the mouse model. In Aim 1, an inducible genetic ablation of proliferating MSC will be performed using a suicide transgene. In Aim 2, pharmacological ablation will be performed with a hunter-killer peptide targeting ASCs. We will test if DMD progression, measured as MPC dysfunction, fatty infiltration, fibrotic tissue accumulation, and the resulting loss of skeletal muscle function, can be suppressed by these experimental treatments. We predict that ablation of FAPs derived from MSCs/ASCs from the dystrophic milieu will delay MPC depletion and DMD progression. Information obtained from these studies will help develop new therapeutic approaches for treating muscular dystrophy.
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0.955 |