Expression of genes with biomarker potential identified in skin from DSLD-affected horses increases with age.

Degenerative Suspensory Ligament Desmitis (DSLD) negatively impacts connective tissues in horses, which often leads to progressive chronic pain and lameness. DSLD has been shown to be a systemic disorder that affects multiple body systems, including tendons, sclerae, and the aorta. Currently, the diagnosis is confirmed by post mortem histological examination of a tendon or suspensory ligament. Histology reveals inappropriate accumulations of proteoglycans in the tendons and other tissues in DSLD-affected horses. Unfortunately, there is no reliable method to diagnose DSLD in living horses. Recently, bone morphogenetic protein 2 (BMP2) was identified in active DSLD lesions. In addition, recent data from RNA sequencing (RNA-seq) showed overexpression of numerous genes, among them BMP2, FOS and genes for keratins in DSLD skin biopsies-derived RNA. We hypothesized that some of these genes can be used as biomarkers for diagnosis of DSLD in a panel. Overexpression of some of them was verified in quantitative real time PCR. Immunohistochemistry and RNAscope in-situ hybridization (ISH) assays were used to determine the level of overexpression of specific genes in skin biopsies from control and DSLD-affected horses. The RNAscope ISH assay has shown to be more reliable and more specific that immunohistochemistry. ISH confirmed a significant increase in KRT83 and BMP-2 in hair follicles in DSLD cases, as well as abnormally high expression of FOS in the epidermis, especially in aging horses. Because statistically relevant specificity and sensitivity was documented only for FOS and BMP2, but not KRT83 we recommend the use of FOS and BMP2 panel to diagnose DSLD. We conclude that a panel of two markers from the studied group (BMP2 and FOS) can serve as an additional diagnostic tool for DSLD in living horses, especially in older animals. Further studies are necessary to confirm if this biomarker panel could be used as a prospective tool to identify DSLD in horses as they age.

Introduction.

Just like the first edition of this widely successful book the second edition provides latest updates of our understanding of pathophysiology, pathology, clinical presentation and treatment of heritable soft connective tissue diseases. In addition, new knowledge of not only structures but also of functions of basic components of connective tissues (e.g., collagen), and of organs such as tendons has been added as well. Moreover, readers will learn more about new syndromes and new subgroups of previously described syndromes and disorders as well. The authors are not only prominent investigators in their field, but they are also good writers and that should provide an additional incentive for interested readers.

Basic Components of Connective Tissues and Extracellular Matrix: Fibronectin, Fibrinogen, Laminin, Elastin, Fibrillins, Fibulins, Matrilins, Tenascins and Thrombospondins.

Collagens are the most abundant components of the extracellular matrix (ECM) and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. It is an insoluble polymer of the monomeric soluble precursor tropoelastin, and the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of transforming growth factors ß (TGFß) through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Many other molecules, though lower in quantity, function as essential, structural and/or functional components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its multidomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin it also binds to a variety of compounds, particularly to various growth factors, and as such, fibrinogen is a player in cardiovascular and extracellular matrix physiology. Laminins contribute to the structure of the ECM and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Fibrillins represent the predominant core of microfibrils in elastic as well as non-elastic extracellular matrixes, and interact closely with tropoelastin and integrins. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide basis for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Latent TGFß binding proteins (LTBPs) are included here as their structure is similar to fibrillins. Several categories of ECM components described after fibrillins are sub-classified as matricellular proteins, i.e., they are secreted into ECM, but do not provide structure. Rather they interact with cell membrane receptors, collagens, proteases, hormones and growth factors, communicating and directing cell-ECM traffic. Fibulins are tightly connected with basement membranes, elastic fibers and other components of extracellular matrix and participate in formation of elastic fibers. Matrilins have been emerging as a new group of supporting actors, and their role in connective tissue physiology and pathophysiology has not been fully characterized. Tenascins are ECM polymorphic glycoproteins found in many connective tissues in the body. Their expression is regulated by mechanical stress both during development and in adulthood. Tenascins mediate both inflammatory and fibrotic processes to enable effective tissue repair and play roles in pathogenesis of Ehlers-Danlos, heart disease, and regeneration and recovery of musculo-tendinous tissue. One of the roles of thrombospondin 1 is activation of TGFß. Increased expression of thrombospondin and TGFß activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially with physical activity, loading and post-injury. It plays a role in vascular wall remodeling and has been found in atherosclerotic plaques as well.

Proteoglycans and Diseases of Soft Tissues.

Proteoglycans consist of protein cores to which at least one glycosaminoglycan chain is attached. They play important roles in the physiology and biomechanical function of tendons, ligaments, cardiovascular system, and other systems through their involvement in regulation of assembly and maintenance of extracellular matrix, and through their participation in cell proliferation together with growth factors. They can be divided into two main groups, small and large proteoglycans. The small proteoglycans are also known as small leucine-rich proteoglycans (SLRPs) which are encoded by 18 genes and are further subclassified into Classes I-V. Several members of Class I and II, such as decorin and biglycan from Class I, and Class II fibromodulin and lumican, are known to regulate collagen fibrillogenesis. Decorin limits the diameter of collagen fibrils during fibrillogenesis. The function of biglycan in fibrillogenesis is similar to that of decorin. Though biomechanical function of tendon is compromised in decorin-deficient mice, decorin can substitute for lack of biglycan in biglycan-deficient mice. New data also indicate an important role for biglycan in disorders of the cardiovascular system, including aortic valve stenosis and aortic dissection. Two members of the Class II of SLRPs, fibromodulin and lumican bind to the same site within the collagen molecule and can substitute for each other in fibromodulin- or lumican-deficient mice.Aggrecan and versican are the major representatives of the large proteoglycans. Though they are mainly found in the cartilage where they provide resilience and toughness, they are present also in tensile portions of tendons and, in slightly different biochemical form in fibrocartilage. Degradation by aggrecanase is responsible for the appearance of different forms of aggrecan and versican in different parts of the tendon where these cleaved forms play different roles. In addition, they are important components of the ventricularis of cardiac valves. Mutations in the gene for versican or in the gene for elastin (which binds to versican ) lead to severe disruptions of normal developmental of the heart at least in mice.

Growth Factor Roles in Soft Tissue Physiology and Pathophysiology.

Repair and healing of injured and diseased tendons has been traditionally fraught with apprehension and difficulties, and often led to rather unsatisfactory results. The burgeoning research field of growth factors has opened new venues for treatment of tendon disorders and injuries, and possibly for treatment of disorders of the aorta and major arteries as well. Several chapters in this volume elucidate the role of transforming growth factor ß (TGFß) in pathogenesis of several heritable disorders affecting soft tissues, such as aorta, cardiac valves, and tendons and ligaments. Several members of the bone morphogenetic group either have been approved by the FDA for treatment of non-healing fractures or have been undergoing intensive clinical and experimental testing for use of healing bone fractures and tendon injuries. Because fibroblast growth factors (FGFs) are involved in embryonic development of tendons and muscles among other tissues and organs, the hope is that applied research on FGF biological effects will lead to the development of some new treatment strategies providing that we can control angiogenicity of these growth factors. The problem, or rather question, regarding practical use of imsulin-like growth factor I (IGF-I) in tendon repair is whether IGF-I acts independently or under the guidance of growth hormone. FGF2 or platelet-derived growth factor (PDGF) alone or in combination with IGF-I stimulates regeneration of periodontal ligament: a matter of importance in Marfan patients with periodontitis. In contrast, vascular endothelial growth factor (VEGF) appears to have rather deleterious effects on experimental tendon healing, perhaps because of its angiogenic activity and stimulation of matrix metalloproteinases-proteases whose increased expression has been documented in a variety of ruptured tendons. Other modalities, such as local administration of platelet-rich plasma (PRP) and/or of mesenchymal stem cells have been explored extensively in tendon healing. Though treatment with PRP and mesenchymal stem cells has met with some success in horses (who experience a lot of tendon injuries and other tendon problems), the use of PRP and mesenchymal stem cells in people has been more problematic and requires more studies before PRP and mesenchymal stem cells can become reliable tools in management of soft tissue injuries and disorders.

Connective Tissue Disorders in Domestic Animals.

Though soft tissue disorders have been recognized and described to some detail in several types of domestic animals and small mammals for some years, they remain uncommon. Because of their low prevalence, not much progress has been made not only in improved diagnosis but also in our understanding of the biochemical basis and pathogenesis of these diseases in animals. Ehlers-Danlos syndrome (EDS) described in dogs already in 1943 and later in cats has only minor impact on the well-being of the dog as its effects on skin of these animals are rather limited. The involved skin is thin and hyperextensible with easily inflicted injuries resulting in hemorrhagic wounds and atrophic scars. Joint laxity and dislocation common in people are less frequently found in dogs. No systemic complications, such as organ rupture or cardiovascular problems which have devastating consequences in people have been described in cats and dogs. The diagnosis is based on clinical presentation and on light or electron microscopic features of disorganized and fragmented collagen fibrils. Several case of bovine and ovine dermatosparaxis analogous to human Ehlers-Danlos syndrome type VIIC were found to be caused by mutations in the procollagen I N-proteinase (pnPI) or ADAMTS2 gene, though mutations in other sites are likely responsible for other types of dermatosparaxis. Cattle suffering from a form of Marfan syndrome (MFS) were described to have aortic dilatation and aneurysm together with ocular abnormalities and skeletal involvement. As in people, mutations at different sites of bovine FBN1 may be responsible for Marfan phenotype. Hereditary equine regional dermal asthenia (HERDA), or hyperelastosis cutis, has been recognized in several horse breeds as affecting primarily skin, and, occasionally, tendons. A mutation in cyclophilin B, a chaperon involved in proper folding of collagens, has been identified in some cases. Warmblood fragile foal syndrome (WFFS) is another Ehlers-Danlos-like disorder in horses, affecting primarily Warmbloods who present with skin fragility and joint hyperextensibility. Degenerative suspensory ligament desmitis (DSLD) affects primarily tendons and ligaments of certain horse breeds. Data from our laboratory showed excessive accumulation of proteoglycans in organs with high content of connective tissues. We have identified increased presence of bone morphogenetic protein 2 (BMP2) in active foci of DSLD and an abnormal form of decorin in proteoglycan deposits. Our most recent data obtained from next generation sequencing showed disturbances in expression of genes for numerous proteoglycans and collagens.

Differential gene expression in skin RNA of horses affected with degenerative suspensory ligament desmitis.

BACKGROUND: Equine degenerative suspensory ligament desmitis (DSLD) is a systemic connective tissue disorder first identified in Peruvian Paso horses but afflicting other horse breeds as well. Inappropriate accumulation of proteoglycans in connective tissues, most prominently in tendons and ligaments, leads to progressive and debilitating lameness and pain. It is largely unknown what drives the overproduction of proteoglycans, but our previous studies suggest involvement of bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-ß (TGFß) family, impacting synthesis of proteoglycans. To identify potential players in pathogenesis of DSLD a new approach utilizing next generation sequencing was undertaken. METHODS: Next generation sequencing was performed using RNA extracted from skin biopsies of six control Peruvian Pasos and six horses with DSLD (4 Peruvian Pasos and 2 warmbloods). The CuffDiff result sets were validated with algorithms used to run them. This was based on the determined false discovery rates derived from the P values adjusted for multiple testing for any given result. RESULTS: Bioinformatics analysis of transcriptomes revealed differential expression of over 1500 genes, including increased expression of genes for several growth factors (most prominently BMP2, FGF5, CTGF, many members of the EGF family), and mediators of signaling (Fos, Myc, MAPK system), and keratins. Two genes encoding for enzymes involved in synthesis of hyaluronan were also overexpressed. Gene expression was decreased for protein cores of many proteoglycans, several growth factors, most collagens, and many peptides with immune function. CONCLUSIONS: The overexpression of BMP2 correlates well with our previous data. However, the decrease in expression of numerous proteoglycans was unexpected. A mutation in a gene of a less characterized proteoglycan and/or glycosyltransferase with subsequent increased production of hyaluronan and/or a proteoglycan(s) undetected in our study could account for the systemic proteoglycan deposition. Decreased collagen gene expression indicates abnormal connective tissue metabolism. The increased expression of keratin genes and FGF5 supports reports of skin abnormalities in DSLD. Underexpression of immune function genes corresponds with lack of inflammation in DSLD tissues. Finally, though the proteoglycan and/or glycosaminoglycan abundant in DSLD has not been identified, we validated our previous data, including overexpression of BMP2, and systemic nature of DSLD due to disturbed metabolism of the extracellular matrix.

Connective Tissue and Age-Related Diseases.

We begin this chapter by describing normal characteristics of several pertinent connective tissue components, and some of the basic changes they undergo with ageing. These alterations are not necessarily tied to any specific disease or disorders, but rather an essential part of the normal ageing process. The general features of age-induced changes, such as skin wrinkles, in selected organs with high content of connective or soft tissues are discussed in the next part of the chapter. This is followed by a section dealing with age-related changes in specific diseases that fall into at least two categories. The first category encompasses common diseases with high prevalence among mostly ageing populations where both genetic and environmental factors play roles. They include but may not be limited to atherosclerosis and coronary heart disease, type II diabetes, osteopenia and osteoporosis, osteoarthritis, tendon dysfunction and injury, age-related disorders of spine and joints. Disorders where genetics plays the primary role in pathogenesis and progression include certain types of progeria, such as Werner syndrome and Hutchinson-Gilford progeria belong to the second category discussed in this chapter. These disorders are characterized by accelerated signs and symptoms of ageing. Other hereditary diseases or syndromes that arise from mutations of genes encoding for components of connective tissue and are less common than diseases included in the first group will be discussed briefly as well, though they may not be directly associated with ageing, but their connective tissue undergoes some changes compatible with ageing. Marfan and Ehlers-Danlos syndromes are primary examples of such disorders. We will probe the role of specific components of connective tissue and extracellular matrix if not in each of the diseases, then at least in the main representatives of these disorders.

Does BMP2 play a role in the pathogenesis of equine degenerative suspensory ligament desmitis?

OBJECTIVE: Horses afflicted with degenerative suspensory ligament desmitis (DSLD) suffer from progressive leg pain and lameness without history of trauma. DSLD is a systemic disorder caused by abnormal accumulation of proteoglycans in many connective tissues. One proteoglycan found in higher quantities in DSLD is decorin. The accumulated decorin has an abnormally glycosylated glycosaminoglycan chain in DSLD. In addition to acellular accumulations of proteoglycans foci of active fibroblasts/tenoblasts were observed in some tendons and suspensory ligaments (SLs) from DSLD cases We have hypothesized that this represents an early event in DSLD and that production of chondrogenic growth factors, such as BMP2, and/or enzyme participating in glycosylation of glycosaminoglycans is a major factor in initiation and progression of DSLD. RESULTS: Using immunohistochemistry we have identified BMP2 in these cellular foci, indicating association with proteoglycan production, but not in other cells in the tendon and SLs. In contrast, very little staining for TGFß and dermatan sulfate epimerase, an enzyme involved in glycosylation of glycosaminoglycan chains, was observed in these foci and other cells in both control and DSLD-affected tendons and SLs. Our data support our hypothesis that chondrogenic growth factors may be responsible, at least in part for progression of DSLD in horses.

Basic Components of Vascular Connective Tissue and Extracellular Matrix.

Though the composition of the three layers constituting the blood vessel wall varies among the different types of blood vessels, and some layers may even be missing in capillaries, certain basic components, and properties are shared by all blood vessels, though each histologically distinct layer contains a unique complement of extracellular components, growth factors and cytokines, and cell types as well. The structure and composition of vessel layers informs and is informed by the function of the particular blood vessel. The adaptation of the composition and the resulting function of the extracellular matrix (ECM) to changes in circulation/blood flow and a variety of other extravascular stimuli can be characterized as remodeling spearheaded by vascular cells. There is a surprising amount of cell traffic among the three layers. It starts with endothelial cell mediated transmigration of inflammatory cells from the bloodstream into the subendothelium, and then into tissue adjoining the blood vessel. Smooth muscle cells and a variety of adventitial cells reside in tunica media and tunica externa, respectively. The latter cells are a mixture of progenitor/stem cells, fibroblasts, myofibroblasts, pericytes, macrophages, and dendritic cells and respond to endothelial injury by transdifferentiation as they travel into the two inner layers, intima and media for corrective mission in the ECM composition. This chapter addresses the role of various vascular cell types and ECM components synthesized by them in maintenance of normal structure and in their contribution to major pathological processes, such as atherosclerosis, organ fibrosis, and diabetic retinopathy.

Basic components of connective tissues and extracellular matrix: elastin, fibrillin, fibulins, fibrinogen, fibronectin, laminin, tenascins and thrombospondins.

Collagens are the most abundant components of the extracellular matrix and many types of soft tissues. Elastin is another major component of certain soft tissues, such as arterial walls and ligaments. Many other molecules, though lower in quantity, function as essential components of the extracellular matrix in soft tissues. Some of these are reviewed in this chapter. Besides their basic structure, biochemistry and physiology, their roles in disorders of soft tissues are discussed only briefly as most chapters in this volume deal with relevant individual compounds. Fibronectin with its muldomain structure plays a role of "master organizer" in matrix assembly as it forms a bridge between cell surface receptors, e.g., integrins, and compounds such collagen, proteoglycans and other focal adhesion molecules. It also plays an essential role in the assembly of fibrillin-1 into a structured network. Laminins contribute to the structure of the extracellular matrix (ECM) and modulate cellular functions such as adhesion, differentiation, migration, stability of phenotype, and resistance towards apoptosis. Though the primary role of fibrinogen is in clot formation, after conversion to fibrin by thrombin, it also binds to a variety of compounds, particularly to various growth factors, and as such fibrinogen is a player in cardiovascular and extracellular matrix physiology. Elastin, an insoluble polymer of the monomeric soluble precursor tropoelastin, is the main component of elastic fibers in matrix tissue where it provides elastic recoil and resilience to a variety of connective tissues, e.g., aorta and ligaments. Elastic fibers regulate activity of TGFßs through their association with fibrillin microfibrils. Elastin also plays a role in cell adhesion, cell migration, and has the ability to participate in cell signaling. Mutations in the elastin gene lead to cutis laxa. Fibrillins represent the predominant core of the microfibrils in elastic as well as non-elastic extracellular matrixes, and interact closely with tropoelastin and integrins. Not only do microfibrils provide structural integrity of specific organ systems, but they also provide a scaffold for elastogenesis in elastic tissues. Fibrillin is important for the assembly of elastin into elastic fibers. Mutations in the fibrillin-1 gene are closely associated with Marfan syndrome. Fibulins are tightly connected with basement membranes, elastic fibers and other components of extracellular matrix and participate in formation of elastic fibers. Tenascins are ECM polymorphic glycoproteins found in many connective tissues in the body. Their expression is regulated by mechanical stress both during development and in adulthood. Tenascins mediate both inflammatory and fibrotic processes to enable effective tissue repair and play roles in pathogenesis of Ehlers-Danlos, heart disease, and regeneration and recovery of musculo-tendinous tissue. One of the roles of thrombospondin 1 is activation of TGFß. Increased expression of thrombospondin and TGFß activity was observed in fibrotic skin disorders such as keloids and scleroderma. Cartilage oligomeric matrix protein (COMP) or thrombospondin-5 is primarily present in the cartilage. High levels of COMP are present in fibrotic scars and systemic sclerosis of the skin, and in tendon, especially with physical activity, loading and post-injury. It plays a role in vascular wall remodeling and has been found in atherosclerotic plaques as well.

Proteoglycans and diseases of soft tissues.

Proteoglycans consist of a protein core to which at least one glycosaminoglycan chain is attached. They play important roles in the physiology and biomechanical function of tendons, ligaments and cardiovascular system through their involvement in regulation of assembly and maintenance of extracellular matrix, and as they participate in cell proliferation through their interactions with growth factors. They can be divided into two main groups of small and large proteoglycans. The small proteoglycans are also known as small leucine-rich proteoglycans (or SLRPs) which are encoded by 17 genes and are further subclassified into Classes I-V. Several members of Class I and II, such as decorin and biglycan from Class I, and Class II fibromodulin and lumican, are known to regulate collagen fibrillogenesis. Decorin limits the diameter of collagen fibrils during fibrillogenesis. The function of biglycan in fibrillogenesis is similar to that of decorin. Though biomechanical function of tendon is compromised in decorin-deficient mice, decorin can substitute for lack of biglycan in biglycan-deficient mice. New data also indicate an important role for biglycan in disorders of the cardiovascular system, including aortic valve stenosis and aortic dissection. Two members of the Class II of SLRPs, fibromodulin and lumican bind to the same site within the collagen molecule and can substitute for each other in fibromodulin- or lumican-deficient mice.Aggrecan and versican are the major representatives of the large proteoglycans. Though they are mainly found in the cartilage where they provide resilience and toughness, they are also present in tensile portions of tendons and, in slightly different biochemical form in fibrocartilage. Degradation with aggrecanase is responsible for the appearance of different forms of aggrecan and versican in different parts of the tendon where these cleaved forms play different roles. In addition, they are important components of the ventricularis of cardiac valves. Mutations in the gene for versican or in the gene for elastin (which binds to versican) lead to severe disruptions of normal developmental of the heart at least in mice.

Advances in the use of growth factors for treatment of disorders of soft tissues.

Repair and healing of injured and diseased tendons have been traditionally fraught with apprehension and difficulties, and often lead to rather unsatisfactory results. The burgeoning research field of growth factors has opened new venues for treatment of tendon disorders and injuries, and possibly for treatment of disorders of the aorta and major arteries as well. Several chapters in this volume elucidate the role of transforming growth factor ß in pathogenesis of several heritable disorders affecting soft tissues, such as aorta, cardiac valves, and tendons and ligaments. Several members of the bone morphogenetic group either have been approved by the FDA for treatment of non-healing fractures or have been undergoing intensive clinical and experimental testing for use in healing of bone fractures and tendon injuries. Because FGFs are involved in embryonic development of tendons and muscles among other tissues and organs the hope is that their testing would lead to the development of some new treatment strategies providing that we can control angiogenicity of these growth factors. The problem, or rather question regarding practical use of IGF-I in tendon repair is whether IGF-I acts independently or under the guidance of growth hormone. FGF2 or PDGF alone or in combination with IGF-I stimulated regeneration of periodontal ligament, a matter of importance in Marfan patients with periodontitis. In contrast, VEGF appears to have rather deleterious effect on experimental tendon healing, perhaps because of its angiogenic activity and stimulation of matrix metalloproteinases, proteases whose increased expression has been documented in a variety of ruptured tendons. Other modalities, such as local administration of platelet-rich plasma (PRP) and/or of mesenchymal stem cells have been explored extensively in tendon healing. Though treatment with PRP and mesenchymal stem cells has met with some success in horses (who experience a lot of tendon injuries and other tendon problems), the use of PRP and mesenchymal stem cells in people has been more problematic and requires more studies before PRP and mesenchymal stem cells can become reliable tools in management of soft tissue injuries and disorders.

Connective tissue disorders in domestic animals.

Though soft tissue disorders have been recognized and described to some detail in several types of domestic animals and small mammals for some years, not much progress has been made in our understanding of the biochemical basis and pathogenesis of these diseases in animals. Ehlers-Danlos syndrome described in dogs already in 1943 and later in cats affects mainly skin in these animals. The involved skin is thin and hyperextensible with easily inflicted injuries resulting in hemorrhagic wounds and atrophic scars. Joint laxity and dislocation common in people are less frequently found in dogs. No systemic complications, such as organ rupture or cardiovascular problems which have devastating consequences in people have been described in cats and dogs. The diagnosis is based on clinical presentation and on light or electron microscopic features of disorganized and fragmented collagen fibrils. Several cases of bovine and ovine dermatosparaxis analogous to human Ehlers-Danlos syndrome type VIIC were found to be caused by mutations in the procollagen I N-proteinase (pnPI) or ADAMTS2 gene, though mutations in other sites are likely responsible for other types of dermatosparaxis. Cattle suffering from a form of Marfan syndrome were described to have aortic dilatation and aneurysm together with ocular abnormalities and skeletal involvement. As in people mutations at different sites of bovine FBN1 may be responsible for Marfan phenotype. Hereditary equine regional dermal asthenia (HERDA), or hyperelastosis cutis, has been recognized in several horse breeds as affecting primarily skin, and, occasionally, tendons. A mutation in cyclophilin B, a chaperon involved in proper folding of collagens, has been identified in some cases. Degenerative suspensory ligament desmitis (DSLD) affects primarily tendons and ligaments of certain horse breeds. New data from our laboratory showed excessive accumulation of proteoglycans in organs with high content of connective tissues. We have identified an abnormal form of decorin with altered biological activity in these proteoglycan deposits, and more recently changes in processing of aggrecan were found by us and other investigators.The naturally occurring diseases of soft tissues in domestic animals described here have a potential to serve as good models for analogous human diseases. This is the case particularly relevant to dogs as a half out of the more than 400 naturally occurring hereditary canine diseases has the potential to serve as a model for human disease.

Type I collagen-based fibrous capsule enhances integration of tissue-engineered cartilage with native articular cartilage.

Successful integration of engineered constructs with host tissues is crucial for cartilage repair, yet achieving it remains challenging. A collagen I-based fibrous capsule characterized by increased cell density and decreased glycosaminoglycan deposition usually forms at the periphery of tissue-engineered cartilage. The current study aimed to evaluate the effects of a solid fibrous capsule on construct integration with native articular cartilage. To this end, capsule-containing (CC) and capsule-free (CF) constructs were grown by culturing chondrocyte-seeded scaffolds with insulin-like growth factor-1 and transforming growth factor-ß1, respectively, in a wavy-walled bioreactor that imparts hydrodynamic forces for 4 weeks. The ability of harvested constructs to integrate with native cartilage was determined using a cartilage explant model. Our results revealed that adhesive stress between native cartilage and the CC constructs was 57% higher than that in the CF group, potentially due to the absence of glycosaminoglycans and increased cell density in the capsule region and deposition of denser and thicker collagen fibrils at the integration site. The present work demonstrates that the fibrous capsule can effectively enhance early integration of engineered and native cartilage tissues and thus suggests the need to include the capsule as a variable in the development of cartilage tissue engineering strategies.

Glycan profiling of a defect in decorin glycosylation in equine systemic proteoglycan accumulation, a potential model of progeroid form of Ehlers-Danlos syndrome.

Defects in glycosylation of decorin can result in systemic hereditary disease. A mutation in the galactosyl transferase I gene is the underlying defect of a progeroid form of Ehlers-Danlos syndrome. We have previously described pathological changes in equine systemic proteoglycan accumulation (ESPA, formerly degenerative suspensory ligament desmitis) as consisting of excessive presence of decorin and other proteoglycans in organs and structures with a high content of connective tissue. Using liquid chromatography/mass spectrometry, and one- and two-dimensional immunoblotting we have determined that decorin from ESPA-tendons had a higher molecular weight than decorin from non-affected control tendons. Glycosaminoglycan structure and monosaccharide composition were determined with HPLC analysis of chondroitinase ABC-digested glycosaminoglycans and gas chromatography/mass spectrometry. This analysis revealed an increase in the total content of sulfated disaccharides, particularly due to enhanced sulfation at 6-position of N-acetyl galactosamine (GalNAc) with a subsequent decrease in the ratio of 4-sulfation to 6-sulfation disaccharides in the ESPA decorin. The ESPA-affected decorin also exhibited altered biological activity resulting in (1) diminished binding of TGFbeta1 (and of anti-decorin antibody) to ESPA decorin, and (2) increased expression of TGFbeta1 in ESPA tissues.

Effect of Lactobacillus acidophilus supernatants on body weight and leptin expression in rats.

BACKGROUND: Lactobacillus extracts and supernatants have been used as probiotics in human and veterinary medicine for their ability to enhance wound healing and immunity. Previous data from our laboratory demonstrated that Lactobacillus supernatant (LS) stimulated wound healing, angiogenesis and proliferation of embryonic cells after topical application. This current study shows that LS after its administration into the cerebral ventricles of male rats exerts systemic effects. METHODS: The right lateral cerebral ventricle of young male rats was accessed through intracerebroventricular cannulation (ICV) under anesthesia and aseptic conditions. One group of control rats received saline solution, a second control group received 0.8 M lactic acid solution (to control for acidity of LS), and a third group received LS. The animals were sacrificed 12, 24, 48, 96 and 120 hours after the injection. Selected tissues were collected, fixed in 10% buffered formalin and used for immunohistochemistry and in situ hybridization. Other tissues were frozen and extracted for immunoblotting RESULTS: LS-injected animals had a slight decrease in body weight when compared to their initial weight and to both control groups. Using immunohistochemistry and in situ hybridization leptin expression was studied in multiple brain sections and peripheral adipose tissue of control and LS-injected rats. Strong cytoplasmic stain was observed by both techniques in neurons of the cerebral cortex, thalamus, hypothalamus, hippocampus and, to lesser degree, in the cells of the choroid plexus in the LS-injected rats. Control animals demonstrated much less intense staining in neurons located in the same regions using immunohistochemistry and almost no staining with in situ hybridization technique. Adipose tissue exhibited slight presence of leptin in LS-treated animals. In contrast no immunohistochemical staining for GM-CSF and TNFalpha was observed in brains from control and treated rats. Western blotting showed mild increase in leptin and leptin receptors in intestines and retroperitoneal adipose tissues of LS-injected rats. CONCLUSION: This study demonstrates that direct administration of LS into rat CNS leads to a decrease in body weight of rats and an increase in the expression of leptin in specific areas of the brain and retroperitoneal adipose tissue.

Degenerative suspensory ligament desmitis as a systemic disorder characterized by proteoglycan accumulation.

BACKGROUND: Degenerative suspensory ligament desmitis (DSLD) is a debilitating disorder thought to be limited to suspensory ligaments of Peruvian Pasos, Peruvian Paso crosses, Arabians, American Saddlebreds, American Quarter Horses, Thoroughbreds, and some European breeds. It frequently leads to persistent, incurable lameness and need to euthanize affected horses. The pathogenesis remains unclear, though the disease appears to run in families. Treatment and prevention are empirical and supportive, and not effective in halting the progression of the disease. Presently, the presumptive diagnosis of DSLD is obtained from patient signalment and history, clinical examination, and ultrasonographic examination of clinically affected horses, and is confirmed at post mortem examination. Presently, there are no reliable methods of diagnosing DSLD in asymptomatic horses. The goal of this study was to characterize and define the disorder in terms of tissue involvement at the macroscopic and microscopic levels. RESULTS: We examined tissues and organs from 28 affected horses (22 Peruvian Pasos, 6 horses of other breeds) and from 8 control horses. Histopathological examination revealed the presence of excessive amounts of proteoglycans in the following tissues removed from DSLD-affected horses: suspensory ligaments, superficial and deep digital flexor tendons, patellar and nuchal ligaments, cardiovascular system, and sclerae. Electron microscopy demonstrated changes in diameters of collagen fibrils in the tendon, and in smooth muscle cells of the media of the aorta compatible with increased cell permeability in DSLD-affected cells. Separation of tendon extracts by gel chromatography revealed the presence of additional proteoglycan(s) in extracts from affected, but not control extracts. CONCLUSION: This study demonstrates for the first time that DSLD, a disease process previously thought to be limited to the suspensory ligaments of the distal limbs of affected horses, is in fact a systemic disorder involving tissues and organs with significant connective tissue component. Abnormal accumulation of proteoglycans between collagen and elastic fibers rather than specific collagen fibril abnormalities is the most prominent histological feature of DSLD. Because of this observation and because of the involvement of many other tendons and ligaments beside the suspensory ligament, and of non-ligamentous tissue we, therefore, propose that equine systemic proteoglycan accumulation or ESPA rather than DSLD is a more appropriate name for this condition.

Culture supernatant of Lactobacillus acidophilus stimulates proliferation of embryonic cells.

Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angiogenic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-alpha (TNF-alpha), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized that LS stimulates the growth and development of other rapidly dividing cells, including embryonic cells. The stimulatory effects of LS on a neuroblastoma cell line (Neuro-2a), chicken embryos, and bovine embryos were examined. The addition of LS to Neuro-2a cultures caused a proliferation of cells in a concentration-dependent manner. Pretreatment of LS at 56 degrees C for 30 mins did not affect its stimulatory activity. The administration of LS to the chorioallantoic membrane (CAM) of chicken-embryonated eggs for 1-2 days resulted in extensive thickening of the membrane. The thickening was due to the influx and proliferation of fibroblasts and inflammatory cells, the accumulation of loose connective tissue composed primarily of mucopolysaccharides, and/or the formation of blood vessels. Stimulatory effects of LS on bovine embryos were also observed. The treatment with LS significantly promoted the development of zygotes to the four-cell stage and from the four-cell stage to blastocysts. These results have confirmed our hypothesis that LS exerts a stimulatory effect on the cells of embryonic stages including neuroblastoma cells, the CAM of chicken embryos, and bovine embryos from zygotes to blastocysts.