Synthesis of extracellular matrix components by human ciliary muscle cells in culture

The production and spatial organization of connective tissue components in ciliary muscle cell cultures was studied with immunohistochemical and ultrastructural methods. Antibodies against collagen types IV and VI, fibronectin and laminin were used. Laminin stains as pericellular network surrounding individual muscle cells. Type IV collagen shows positive cytoplasmic staining and only small foci of extracellular immunofluorescence. Staining for type VI collagen and fibronectin is seen near the ends of the bipolar cells, while the lateral sides of the cells remain unstained. Electronmicroscopy shows that cultured ciliary muscle cells are surrounded by an incomplete basal lamina. In addition, bundles of 5-20 nm thick extracellular microfibrils are seen. The bundles are oriented parallel to the axis of the cells and are in close contact with the cell membrane in areas where membrane-bound dense bands are formed. Immunoelectronmicroscopy indicates that the bundles contain fibronectin and type VI collagen fibrils. While the fibronectin fibrils approach the cell membrane directly, type VI collagen fibrils are usually separated from the cell membrane by fine fibrillous material of different nature. Quality and spatial organization of the extracellular material in ciliary muscle cell cultures shows marked similarities with the extracellular matrix of ciliary muscle in situ

Elektronenmikroskopische und inununhistochemische Untersuchungenzur augendrucksenkenden Wirkung von Prostaglandin F2alpha [Electron microscopy and immunohistochemical studies of the intraocular pressure lowering effect of prostaglandin F2 alpha]

Topically applied prostaglandin F2 alpha (PGF2 alpha) has been shown to lower intraocular pressure in cynomolgus monkeys. In this study the morphological changes following topical treatment of seven cynomolgus monkeys with 4 micrograms PGF2 alpha isopropylester for 5-8 days were investigated and compared with the eyes of five normal animals. Quantitative light microscopical, ultrastructural and immunhistochemical methods were used. No cellular signs of inflammation were seen in any of the eyes. Slight edema in the most anterior part of the ciliary processes occurred in most eyes, but only in parts of the circumference. The most pronounced change was dilation of the intramuscular spaces within the ciliary muscle. No changes were observed in the extracellular matrix within the muscle bundles, which consist partly of type IV and VI collagen, laminin and fibronectin. In the connective tissue between the muscle bundles, loss of collagen type I and III fibrils was observed. Additionally, macrophages were found with phagolysosomes containing phagocytized collagen fibrils. We suggest that loss of extracellular material leads to a widening of the uveoscleral outflow pathways of ciliary muscle and thereby to a reduction in intraocular pressure

The β4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary β-subunits to KCa1.1 activity in the outflow pathway is unknown. Methods: Using quantitative polymerase chain reaction, we measured expression of β-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of β4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks β4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack β4. Results: Kcnmb4 was the most highly expressed β-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. β4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed β-subunit in human TM cells, and the sole β-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%). Conclusions: The β4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting β4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma

Matrix metalloproteinase-3 (MMP-3)-mediated gene therapy for glaucoma.

Approximately 80 million people globally are affected by glaucoma, with a projected increase to over 110 million by 2040. Substantial issues surrounding patient compliance remain with topical eye drops, and up to 10% of patients become treatment resistant, putting them at risk of permanent vision loss. The major risk factor for glaucoma is elevated intraocular pressure, which is regulated by the balance between the secretion of aqueous humor and the resistance to its flow across the conventional outflow pathway. Here, we show that adeno-associated virus 9 (AAV9)-mediated expression of matrix metalloproteinase-3 (MMP-3) can increase outflow in two murine models of glaucoma and in nonhuman primates. We show that long-term AAV9 transduction of the corneal endothelium in the nonhuman primate is safe and well tolerated. Last, MMP-3 increases outflow in donor human eyes. Collectively, our data suggest that glaucoma can be readily treated with gene therapy-based methods, paving the way for deployment in clinical trials

Enhancement of outflow facility in the murine eye by targeting selected tight-junctions of Schlemm's canal endothelia

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm’s canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system

Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F2 alpha

Topically applied prostaglandin F2 alpha has been shown to lower intraocular pressure in cynomolgus monkeys. In this study the morphological changes, following topical treatment with 4-50 micrograms of prostaglandin F2 alpha for 4-8 days, were investigated. Semiquantitation of (1) accumulation of white blood cells as one sign of inflammation, (2) edema and (3) enlarged spaces between ciliary muscle cells were performed. Eighty sections per eye encompassing the whole circumference were investigated. No accumulation of white blood cells was seen in any of the eyes. Slight edema in the most anterior part of the ciliary processes occurred in most eyes, but only in part of the circumference. These changes could be either directly induced by the prostaglandin treatment or secondary to the decrease in intraocular pressure. The most pronounced change was the dilatation of the intramuscular spaces. These enlarged spaces could explain the physiologically shown increase in uveoscleral outflow

Different cell populations in bovine trabecular meshwork: an ultrastructural and immunocytochemical study

Bovine outflow tissue differs markedly from that of humans. Tissue culture studies on the cells of this region are often compared with those of primate trabecular meshwork cells. A thorough cytological and immunocytochemical characterization of the cells of the bovine chamber angle is lacking. We have therefore investigated the cells of the pectinate ligament, the reticular meshwork, the region adjacent to the aqueous plexus, the connective tissue region between reticular meshwork and ciliary muscle and the ciliary muscle itself, ultrastructurally and immunocytochemically with staining for the cytoskeletal proteins vimentin and desmin, for alpha-smooth muscle-actin and rough endoplasmic reticulum (rER). In the pectinate ligament and in the region adjacent to the aqueous plexus, the cells were found to have especially abundant rER and glycogen in their cytoplasm. Vimentin was abundant in the reticular meshwork as positive staining was seen both in frozen and paraffin sections. Alpha-smooth muscle-actin could be found in the region connecting ciliary muscle and reticular meshwork as well as in a small area adjacent to the posterior capillary loops of the aqueous plexus. Ultrastructurally, these cells resembled myofibroblasts. The ciliary muscle cells stained both for vimentin and for alpha-smooth muscle actin

Age changes in rhesus monkey ciliary muscle: light and electron microscopy

The ciliary muscle from 44 rhesus monkeys ranging in age from 137-day fetuses and a 3-week neonate to 35-year-old adults was studied by light and electron microscopy. In the fetuses and the neonate, the muscle cells appeared immature, and the muscle consisted mainly of longitudinally oriented fibers. By age 1 year, the muscle fibers were aggregated into bundles, and within each bundle the individual fibers were packed tightly together. In sagittal section, the muscle now exhibited longitudinal, reticular and circular portions. The individual muscle cells exhibited ultrastructural characteristics which set them apart from other smooth muscle cells. Age-related structural changes occurred in the ciliary muscle and intramuscular nerves, beginning at 6 years. These included increasing numbers of lysosomes, 'fingerprints', and the appearance of myelin figures within some nerve endings and nerve fibers. These alterations became more frequent and pronounced with age, and in elderly animals were seen in all regions of the muscle and around its entire circumference. Additionally, the elderly animals exhibited overt degeneration of some muscle cells and some myelinated nerve fibers. The time-course of these age-related structural neuromuscular alterations parallels the decline of functional accommodative amplitude (i.e. presbyopia) and of the ciliary muscle's configurational response to peripheral pharmacologic and central electrical stimulation. This suggests a pathophysiologic, albeit (given the relatively moderate overall abnormalities) not necessarily causal, relationship. An age-related increase in the number of pigmented cells between the ciliary muscle bundles also occurred, with the anterior longitudinal region being affected last. This might reflect an age-related decline in uveoscleral drainage of aqueous humor

Age-related changes of the ciliary muscle in comparison with changes induced by treatment with prostaglandin F2 alpha. An ultrastructural study in rhesus and cynomolgus monkeys

The relationship between individual ciliary muscle cells and the surrounding connective tissue was studied in the eyes of three normal, young (3-4 years) cynomolgus monkeys (Macaca fascicularis), three aged (34-36 years) rhesus monkeys (Macaca mulatta) and seven young (3-7 years) cynomolgus monkeys topically treated with prostaglandin F2 alpha (PGF2 alpha) for 4-8 days. In normal eyes, collagen fibrils and microfibrils are in places in continuity with the muscle cells' basal lamina, which is connected to the cell membrane by fine fibrillous material. In old eyes, the basal lamina is markedly thickened, masking the connection of fibrils with the muscle cells' membrane. A distinctive finding in several muscle cells of old eyes are electronlucent clefts, 60-80 nm wide, between basal lamina and cell membrane, which are not transversed by fibrils or fibrillous material. The cell membrane of these muscle cells shows large folds filled with disarranged myofilaments. Additionally, these cells contain inclusion bodies consisting of concentrically arranged double membranes. Following treatment with PGF2 alpha, similar changes are seen in young animals, too. Here, the muscle cells have lost their connection to the extracellular fibrils due to a PGF2 alpha-induced lysis of extracellular material. Lack of attachment between basal lamina and altered muscle cells in aged eyes might indicate an involvement of the extracellular matrix in age-related changes of the individual ciliary muscle cells