phenylmercuric-acetate and Inflammation

Local and chronic inflammation induced by amyloid-β (Aβ) plays a central role in the development of age-related macular degeneration. The retina is an immune-privileged site due to local tissue barrier. Yet, the manner by which immune cells pass through this barrier and accumulate in the retina remains unclear. Matrix metalloproteinases (MMPs) induce barrier disruption via proteolysis of epithelial tight junction (TJ) proteins. We hypothesized that Aβ-induced MMP secretion causes disruption of epithelial barrier integrity. To test this hypothesis, human adult retinal pigment epithelial (haRPE) cells were exposed to Aβ, and the expression of MMP-2 and MMP-9 was detected using gelatin zymography. To demonstrate the key role of MMPs in modulating epithelial barrier structure, the MMP agonist 4-aminophenylmercuric acetate (APMA), an MMP inhibitor (GM6001) and siRNA against MMP-9 were employed for comparison. We found that MMP-9, secreted by Aβ- or APMA-stimulated cells, mediated low transepithelial electrical resistance (TER) and high transepithelial permeability by disrupting TJ proteins. However, these alterations were reduced by the MMP inhibitor GM6001 or by silencing of the MMP-9 gene. Our findings suggest that the degradation of TJ proteins such as zonula occludens-1, occludin and F-actin by MMP-9 secreted by Aβ-stimulated cells constitutes an important mechanism in the breakdown of the barrier which contributes to chronic inflammation in the retina of age-related macular degeneration.

Topics: Adult; Amyloid beta-Peptides; Cell Death; Cell Shape; Dipeptides; Epithelial Cells; Humans; Inflammation; Inflammation Mediators; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Phenylmercuric Acetate; Protein Structure, Quaternary; Retinal Pigment Epithelium; RNA, Small Interfering; Tight Junction Proteins

Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis.. To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation.. Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subjected to tension and processed for transmission electron microscopy (TEM).. Without glucose, or with APMA, explants under tension separated at the dermo-epidermal junction. This in vitro separation occurred via 2 different ultrastructural processes. Lack of glucose reduced hemidesmosomes (HDs) numbers until they disappeared and the basal cell cytoskeleton collapsed. Anchoring filaments (AFs), connecting the basal cell plasmalemma to the BM, were unaffected although they failed under tension. APMA activation of constituent lamellar MMPs did not affect HDs but caused AFs to disappear, also leading to dermo-epidermal separation under tension.. Natural laminitis may occur in situations where glucose uptake by lamellar basal cells is compromised (e.g. equine Cushing's disease, obesity, hyperlipaemia, ischaemia and septicaemia) or when lamellar MMPs are activated (alimentary carbohydrate overload).. Therapies designed to facilitate peripheral glucose uptake and inhibit lamellar MMP activation may prevent or ameliorate laminitis.

Topics: Acute Disease; Animals; Basement Membrane; Culture Techniques; Enzyme Activation; Epidermis; Foot Diseases; Glucose; Hoof and Claw; Horse Diseases; Horses; Inflammation; Matrix Metalloproteinases; Microscopy, Electron, Transmission; Phenylmercuric Acetate

Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1beta, but not IL-1alpha, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1beta was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1beta by MMP-2 was identified at the Glu25-Leu26 bond. These results suggest that IL-1beta stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1beta.

Topics: Amino Acid Sequence; Cell Line; Collagenases; Dinoprostone; Enzyme Activation; Fibroblasts; Fibrosarcoma; Gelatinases; Glycoproteins; Humans; Inflammation; Interleukin-1; Kinetics; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Metalloendopeptidases; Molecular Sequence Data; Phenylmercuric Acetate; Substrate Specificity; Sulfhydryl Reagents; Tissue Inhibitor of Metalloproteinases