Acid-sensing ion channel 3 expressed in type B synoviocytes and chondrocytes modulates hyaluronan expression and release.

BACKGROUND: Rheumatoid arthritis is an inflammatory disease marked by intra-articular decreases in pH, aberrant hyaluronan regulation and destruction of bone and cartilage. Acid-sensing ion channels (ASICs) are the primary acid sensors in the nervous system, particularly in sensory neurons and are important in nociception. ASIC3 was recently discovered in synoviocytes, non-neuronal joint cells critical to the inflammatory process. OBJECTIVES: To investigate the role of ASIC3 in joint tissue, specifically the relationship between ASIC3 and hyaluronan and the response to decreased pH. METHODS: Histochemical methods were used to compare morphology, hyaluronan expression and ASIC3 expression in ASIC3+/+ and ASIC3-/- mouse knee joints. Isolated fibroblast-like synoviocytes (FLS) were used to examine hyaluronan release and intracellular calcium in response to decreases in pH. RESULTS: In tissue sections from ASIC3+/+ mice, ASIC3 localised to articular cartilage, growth plate, meniscus and type B synoviocytes. In cultured FLS, ASIC3 mRNA and protein was also expressed. In FLS cultures, pH 5.5 increased hyaluronan release in ASIC3+/+ FLS, but not ASIC3-/- FLS. In FLS from ASIC3+/+ mice, approximately 50% of cells (25/53) increased intracellular calcium while only 24% (14/59) showed an increase in ASIC3-/- FLS. Of the cells that responded to pH 5.5, there was significantly less intracellular calcium increases in ASIC3-/- FLS compared to ASIC3+/+ FLS. CONCLUSION: ASIC3 may serve as a pH sensor in synoviocytes and be important for modulation of expression of hyaluronan within joint tissue.

Role of ASIC3 in the primary and secondary hyperalgesia produced by joint inflammation in mice.

The acid sensing ion channel 3 (ASIC3) is critical for the development of secondary hyperalgesia as measured by mechanical stimulation of the paw following muscle insult. We designed experiments to test whether ASIC3 was necessary for the development of both primary and secondary mechanical hyperalgesia that develops after joint inflammation. We used ASIC3 -/- mice and examined the primary (response to tweezers) and secondary hyperalgesia (von-Frey filaments) that develops after joint inflammation comparing to ASIC3 +/+ mice. We also examined the localization of ASIC3 to the knee joint afferents innervating the synovium using immunohistochemical techniques before and after joint inflammation. We show that secondary mechanical hyperalgesia does not develop in ASIC3 -/- mice. However, the primary mechanical hyperalgesia of the inflamed knee joint still develops in ASIC3 -/- mice and is similar to ASIC3 +/+ mice. In knee joint synovium from ASIC3 +/+ mice without joint inflammation, ASIC3 was not localized to joint afferents that were stained with an antibody to protein gene product (PGP) 9.5 or calcitonin gene-related peptide (CGRP). ASIC3 was found, however, in synoviocytes of the knee joint of uninflamed mice. In ASIC3 +/+ mice with joint inflammation, ASIC3 co-localized with PGP 9.5 or CGRP in joint afferents innervating the synovium. We conclude that the decreased pH that occurs after inflammation would activate ASIC3 on primary afferent fibers innervating the knee joint, increasing the input to the spinal cord resulting in central sensitization manifested behaviorally as secondary hyperalgesia of the paw.

Confocal imaging of early heart development in Xenopus laevis.

Xenopus laevis provides a number of advantages to studies on cardiovascular development. The embryos are fairly large, are easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole-mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin, which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24-46 are presented.

Expression of type VI collagen in the developing mouse heart.

During development, the embryonic atrioventricular (AV) endocardial cushions undergo a morphogenic process to form mature valve leaflets and the membranous septa in the heart. Several extracellular matrix (ECM) proteins are expressed in the developing AV endocardial cushions, but it remains to be established if any specific ECM proteins are necessary for normal cushion morphogenesis. Abnormal development of the cardiac AV valves is a frequent cause of congenital heart defects, particularly in infants with trisomy 21 (Down syndrome). The genes encoding the alpha1 and alpha2 chains of type VI collagen are located on human chromosome 21 within the region thought to be critical for congenital heart defects in trisomy 21 infants. This suggests that the type VI collagen alpha1(VI) and alpha2(VI) chains may be important in normal AV valve morphogenesis. As a first step in understanding the role of type VI collagen in valve development, the authors examined the normal spatial and temporal expression patterns of mRNA and protein for type VI collagen in the embryonic mouse heart. Ribonuclease protection assay analysis demonstrates cardiac expression of the type VI collagen for alpha1(VI), alpha2(VI), and alpha3(VI) transcripts beginning at embryonic days 11-11.5 of mouse development. In situ hybridization studies demonstrate a coordinated pattern of cardiac expression within the AV valves for each type VI collagen chain from embryonic day 11.5 through the neonatal period. Immunohistochemical studies confirm a concentrated type VI collagen localization pattern in the endocardial cushions from the earliest stages of valve development through the neonatal period. These data indicate that type VI collagen is expressed in the developing AV canal in a pattern consistent with cushion tissue mesenchymal cell migration and proliferation, and suggest that type VI collagen plays a role in the morphogenesis of the developing cardiac AV endocardial cushions into the valve leaflets and membranous septa of the heart.

Type VI collagen in the cardiac valves and connective tissue septa during heart development.

A variety of extracellular matrix (ECM) proteins have been shown to be present in the embryonic heart during the morphogenesis of the valves and membranous septa. It is not known if any specific ECM protein is required for the normal morphogenesis of these tissues, but this is of great interest since there is a high incidence of congenital malformations which affect valvular and septal tissues. Interestingly, the alpha 1 and alpha 2 genes of type VI collagen are located within the region of human chromosome 21 thought to be involved in the congenital heart defect phenotype associated with trisomy 21 (Down's syndrome). In this study we examined the distribution and investigated the function of type VI collagen in the cardiac valves and septa of chicken and mouse embryos during various stages of development. Immunohistochemical and in situ hybridization studies revealed a pattern of cardiac expression of type VI collagen which is present from the earliest stages of valve and septum development through the neonatal period. Results from an in vitro bioassay suggest that type VI collagen may play a role in the formation and migration of specific cells in the forming valves and septa. These data support molecular genetic studies which have indicated that type VI collagen is involved in the heart defect phenotype seen in trisomy 21.

Modulation of cell migration and vessel formation by vascular endothelial growth factor and basic fibroblast growth factor in cultured embryonic heart.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate endothelial cell proliferation, migration, and vascular tube formation. We tested the hypotheses that these growth factors stimulate (1) cell migration and (2) assembly into cord-like structures in embryonic rat heart explants cultured on collagen gels. Atrial and ventricular explants from rat embryos at 12 (E12, avascular) and 14 (E14, early vascularization stage) days of gestation were cultured on a collagen substrate. Western blot analysis of the explants indicated that endogenous VEGF was present in both atria and ventricles during incubation. Addition of bFGF to E12 explants markedly increased cell migration, whereas VEGF had no significant effect. In E14 explants neither growth factor influenced cell migration. Cotreatment with VEGF and bFGF did not have a synergistic effect on the migration distance of cells from either E12 or E14 embryonic hearts. However, VEGF stimulated the appearance of cord-like structures in E14, but not E12, explants. Transmission electron microscopy analysis showed that these cord-like structures consist of elongated cells, some of which aggregate into clusters, or form tube-like structures, similar to capillaries. Serial sections of monolayers revealed that tube formation occurs beneath the surface of collagen gel. We conclude that in this model system VEGF and bFGF play distinct roles, at specific time points, in coronary vascular tube formation in the developing heart.