Susan W. Volk

Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs.

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2–3 days of age with a retroviral vector (RV) expressing canine β-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5–60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6–17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal.

Smad-Runx interactions during chondrocyte maturation.

Background: Intracellular signaling triggered by bone morphogenetic proteins (BMPs) results in activated Smad complexes that regulate transcription of BMP-responsive genes. However, the low specificity of Smad binding to regulatory sequences implies that additional tissue-specific transcription factors are also needed. Runx2 (Cbfa1) is a transcription factor required for bone formation. We have examined the role of Smads and Runx2 in BMP induction of type X collagen, which is a marker of chondrocyte hypertrophy leading to endochondral bone formation. Methods: Pre-hypertrophic chondrocytes from the cephalic portion of the chick embryo sternum were placed in culture in the presence or absence of rhBMP-2. Cultures were transiently transfected with DNA containing the BMP-responsive type X collagen promoter upstream of the luciferase gene. The cultures were also transfected with plasmids, causing over-expression of Smads or Runx2, or both. After 24-48 hours, cell extracts were examined for levels of luciferase expression. Results: In the presence of BMP-2, chondrocytes over-expressing BMP-activated Smad1 or Smad5 showed significant enhancement of luciferase production compared with that seen with BMP alone. This enhancement was not observed with over-expression of Smad2, a transforming growth factor beta (TGF-&bgr;)-activated Smad. Over-expression of Runx2 in BMP-treated cultures increased transcriptional activity to levels similar to those seen with Smads 1 or 5. When chondrocytes were simultaneously transfected with both Runx2 and Smad 1 or 5, promoter activity was further increased, indicating that BMP-stimulated Smad activity can be augmented by increasing the levels of Runx2. Conclusions: These results implicate the skeletal tissue transcription factor Runx2 in regulation of chondrocyte hypertrophy and suggest that maximal transcription of the type X collagen gene in pre-hypertrophic chondrocytes involves interaction of BMP-stimulated Smads with Runx2. Clinical Relevance: Many skeletal abnormalities are associated with impaired regulation of chondrocyte hypertrophy in growth plates. These studies demonstrate that both BMP-activated Smads and Runx2 levels can modulate chondrocyte transition to hypertrophy.

A BMP responsive transcriptional region in the chicken type X collagen gene.

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promote both chondrogenesis and chondrocyte hypertrophy. BMPs have recently been found to activate a membrane serine/threonine kinase signaling mechanism in a variety of cell types, but the downstream effectors of BMP signaling in chondrocyte differentiation remain unidentified. We have previously reported that BMP‐2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that type X collagen mRNA levels in chondrocytes cultured under serum‐free (SF) conditions are elevated 3‐ to 5‐fold within 24 h. To better define the molecular mechanisms of induction of chondrocyte hypertrophy by BMPs, we examined the effect of BMPs on type X collagen production by 15‐day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP‐2, BMP‐4, or BMP‐7. Two populations of chondrocytes were used: one representing resting cartilage isolated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna. BMP‐2, BMP‐4, and BMP‐7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline phosphatase, diminished levels of type II collagen, and induction of the hypertrophic chondrocyte‐specific marker, type X collagen. To test whether BMP control of type X collagen expression occurs at the transcriptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5′ flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP‐2, BMP‐4, or BMP‐7. A 533 bp region located 2.4–2.9 kb upstream from the type X collagen transcriptional start site was both necessary and sufficient for strong BMP responsiveness in cells destined for hypertrophy, but not in chondrocytes derived from the lower sterna.

Diminished Type III Collagen Promotes Myofibroblast Differentiation and Increases Scar Deposition in Cutaneous Wound Healing

The repair of cutaneous wounds in the postnatal animal is associated with the development of scar tissue. Directing cell activities to efficiently heal wounds while minimizing the development of scar tissue is a major goal of wound management and the focus of intensive research efforts. Type III collagen (Col3), expressed in early granulation tissue, has been proposed to play a prominent role in cutaneous wound repair, although little is known about its role in this process. To establish the role of Col3 in cutaneous wound repair, we examined the healing of excisional wounds in a previously described murine model of Col3 deficiency. Col3 deficiency (Col3+/–) in aged mice resulted in accelerated wound closure with increased wound contraction. In addition, Col3-deficient mice had increased myofibroblast density in the wound granulation tissue as evidenced by an increased expression of the myofibroblast marker, α-smooth muscle actin. In vitro, dermal fibroblasts obtained from Col3-deficient embryos (Col3+/– and –/–) were more efficient at collagen gel contraction and also displayed increased myofibroblast differentiation compared to those harvested from wild-type (Col3+/+) embryos. Finally, wounds from Col3-deficient mice also had significantly more scar tissue area on day 21 postwounding compared to wild-type mice. The effect of Col3 expression on myofibroblast differentiation and scar formation in this model suggests a previously undefined role for this ECM protein in tissue regeneration and repair.

Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection

While people infected with Leishmania can become refractory to reinfection, human vaccines have not yet been achieved. Glennie et al. identify a population of skin-resident Leishmania-specific memory T cells that produce IFN-γ and recruit circulating T cells to the skin in response to a subsequent parasitic infection. The findings indicate that a successful vaccine may be dependent on generating skin-resident memory T cells for an effective immune response.

IL-33-Dependent Group 2 Innate Lymphoid Cells Promote Cutaneous Wound Healing.

Breaches in the skin barrier initiate an inflammatory immune response that is critical for successful wound healing. Innate lymphoid cells (ILCs) are a recently identified population of immune cells that reside at epithelial barrier surfaces such as the skin, lung and gut and promote pro-inflammatory or epithelial repair functions following exposure to allergens, pathogens or chemical irritants. However, the potential role of ILCs in regulating cutaneous wound healing remains undefined. Here, we demonstrate that cutaneous injury promotes an IL-33-dependent group 2 ILC (ILC2) response and that abrogation of this response impairs re-epithelialization and efficient wound closure. Additionally, we provide evidence suggesting that an analogous ILC2 response is operational in acute wounds of human skin. Together, these results indicate that IL-33-responsive ILC2s are an important link between the cutaneous epithelium and the immune system, acting to promote the restoration of skin integrity following injury.

Regulating the regulators of chondrocyte hypertrophy.

THE DEVELOPMENT, growth, and maintenance of a functional skeleton relies on the existence of several different types of chondrocytes with markedly different fates. The articular chondrocytes on the surface of joints are expected to persist throughout life, producing extracellular matrix components which provide a cushion to distribute load on bone at joints. In contrast, it is the changing dynamics of the underlying cartilage at the epiphyseal growth plate that permits a very different function: growth and development. Chondrocytes of this region undergo a series of changes including hypertrophy, release of extracellular matrix vesicles, and alterations in production of extracellular matrix proteins, prior to mineralization and replacement by bone. As chondrocytes progress toward terminal differentiation, morphological, and biosynthetic changes occur. Proliferating chondrocytes exit the cell cycle and become flattened before fully maturing into round, hypertrophic chondrocytes which secrete and organize a different extracellular matrix, characterized by high levels of alkaline phosphatase (ALP), diminished levels of collagens type II and IX, and production of a new hypertrophic chondrocytespecific product, type X collagen. In addition to normal bone formation during development of the skeleton, this process of endochondral ossification has also been implicated in fracture repair and such pathologic processes as osteoarthritis and ectopic bone formation. The fundamental importance of chondrocyte maturation in the growth, development, and repair of the skeleton has led to intense investigation of the growth factors that regulate terminal differentiation of chondrocytes. Although several growth factors have been indentified which regulate chondrocyte maturation, it remains unclear how these factors interact to coordinate the process leading to bone elongation and endochondral ossification. In this issue, Grismud and colleagues propose a role for bone morphogenetic protein-6 (BMP-6) as a positive regulator of chondrocyte maturation. Their report also provides insight into how parathyroid hormone-related protein (PTHrP) and indian hedgehog, other growth factors known to modulate chondrocyte hypertrophy, may interact with BMP-6 in the growth plate. BMPs belong to the transforming growth factor-b (TGFb) superfamily of secreted growth and differentiation polypeptides, which also includes activins, growth and differentiation factors, and Mullerian inhibiting substance. For over 30 years, it has been known that BMPs are capable of inducing the formation of new cartilage and bone when implanted extraskeletally. However, it was not until 1988 that three of the proteins involved (BMP2–4) were cloned. Although their name stresses their critical impact on the skeletal system, more than 20 members of the BMP family have been identified and determined to have a broader biological impact than just skeletogenesis. This impact includes establishment of basic embryonic patterning and growth and differentiation of nearly all body systems including the cardiovascular, gastrointestinal, respiratory, nervous, and urogenital systems and the integument (see review by Hogan). As their name implies, most BMPs can initiate new cartilage and bone formation in a cascade of events reminiscent of endochondral bone formation. BMP-2, BMP-4, BMP-5, and BMP-7 have all been shown to possess the ability to promote bone formation in vivo. Additional evidence with cultured cells also point to considerable overlap in BMP function; recombinant BMP-2, BMP-4, BMP-6, and BMP-7 have all been shown to induce both hypertrophy in cultured chondrocytes and osteogenesis from mesenchymal stem cells. However, information derived from BMP localization studies and null mutations in mice suggests more specific roles for individual BMPs in normal development. The temporal and spatial patterns of expression of BMPs in embryonic mouse forelimbs support roles for BMP-2, BMP-4, BMP-6, and BMP-7 in skeletal formation, and multiple BMPs, including BMP-2, BMP-4, BMP-6, and BMP-7, are produced by chondrocytes undergoing endochondral ossification. Recent detailed analysis of expression patterns for BMPs in the embryonic mouse humerus by Solloway et al. show that BMP-7 is expressed in both proliferating chondrocytes and the perichondrum, BMP-4 is found in chondrocytes of the transition zone immediately adjacent to the mature hypertrophic chondro-

Regenerative healing, scar-free healing and scar formation across the species: current concepts and future perspectives

All species have evolved mechanisms of repair to restore tissue function following injury. Skin scarring is an inevitable and permanent endpoint for many postnatal organisms except for non‐amniote vertebrates such as amphibians, which are capable of tissue regeneration. Furthermore, mammalian foetuses through mid‐gestation are capable of rapid wound repair in the absence of scar formation. Notably, excessive cutaneous scar formation, such as hypertrophic and keloid scars, is a species limited clinical entity as it occurs only in humans, although wounds on the distal limbs of horses are also prone to heal with fibroproliferative pathology known as equine exuberant granulation tissue. Currently, there are no reliable treatment options to eradicate or prevent scarring in humans and vertebrates. The limited number of vertebrate models for either hypertrophic or keloid scarring has been an impediment to mechanistic studies of these diseases and the development of therapies. In this viewpoint essay, we highlight the current concepts of regenerative, scar‐free and scar‐forming healing compared across a number of species and speculate on areas for future research. Furthermore, in‐depth investigative research into the mechanisms of scarless repair may allow for the development of improved animal models and novel targets for scar prevention. As the ability to heal in both a scarless manner and propensity for healing with excessive scar formation is highly species dependent, understanding similarities and differences in healing across species as it relates to the regenerative process may hold the key to improve scarring and guide translational wound‐healing studies.

Translating stem cell therapies: the role of companion animals in regenerative medicine.

Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell-based therapies involving hematopoietic stem cells and mesenchymal stem cells, as well as other adult regenerative cells and recently embryonic stem cells and induced pluripotent stem cells. In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the “One Health Initiative,” the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored.Veterinarians and veterinary medicine have been integral to the development of stem cell therapies. The contributions of large animal experimental models to the development and refinement of modern hematopoietic stem cell transplantation were noted nearly five decades ago. More recent advances in adult stem cell/regenerative cell therapies continue to expand knowledge of the basic biology and clinical applications of stem cells. A relatively liberal legal and ethical regulation of stem cell research in veterinary medicine has facilitated the development and in some instances clinical translation of a variety of cell‐based therapies involving hematopoietic stem cells and mesenchymal stem cells, as well as other adult regenerative cells and recently embryonic stem cells and induced pluripotent stem cells. In fact, many of the pioneering developments in these fields of stem cell research have been achieved through collaborations of veterinary and human scientists. This review aims to provide an overview of the contribution of large animal veterinary models in advancing stem cell therapies for both human and clinical veterinary applications. Moreover, in the context of the “One Health Initiative,” the role veterinary patients may play in the future evolution of stem cell therapies for both human and animal patients will be explored.

Acellular Hydrogels for Regenerative Burn Wound Healing: Translation from a Porcine Model.

Currently available skin grafts and skin substitute for healing following third-degree burn injuries is fraught with complications, often resulting in long-term physical and psychological sequelae. Synthetic treatment that can promote wound healing in a regenerative fashion would provide an off-the-shelf, non-immunogenic strategy to improve clinical care of severe burn wounds. Here, we demonstrate vulnerary efficacy and accelerated healing mechanism of dextran-based hydrogel in third-degree porcine burn model. The model was optimized to allow examination of the hydrogel treatment for clinical translation and its regenerative response mechanisms. Hydrogel treatment accelerated third-degree burn wound healing by rapid wound closure, improved reepithelialization, enhanced extracellular matrix remodeling, and greater nerve reinnervation, compared to the dressing treated group. These effects appear to be mediated through the ability of the hydrogel to facilitate a rapid but brief initial inflammatory response that coherently stimulates neovascularization within the granulation tissue during the first week of treatment, followed by an efficient vascular regression to promote a regenerative healing process. Our results suggest that the dextran-based hydrogels may substantially improve healing quality and reduce skin grafting incidents and thus pave the way for clinical studies to improve the care of severe burn injury patients.