PurposeNetarsudil is a Rho kinase/norepinephrine transporter inhibitor currently in phase 3 clinical development for glaucoma treatment. We investigated the effects of its active metabolite, netarsudil-M1, on outflow facility (C), outflow hydrodynamics, and morphology of the conventional outflow pathway in enucleated human eyes.MethodsPaired human eyes (n = 5) were perfused with either 0.3 μM netarsudil-M1 or vehicle solution at constant pressure (15 mm Hg). After 3 hours, fluorescent microspheres were added to perfusion media to trace the outflow patterns before perfusion-fixation. The percentage effective filtration length (PEFL) was calculated from the measured lengths of tracer distribution in the trabecular meshwork (TM), episcleral veins (ESVs), and along the inner wall (IW) of Schlemm's canal after global and confocal imaging. Morphologic changes along the trabecular outflow pathway were investigated by confocal, light, and electron microscopy.ResultsPerfusion with netarsudil-M1 significantly increased C when compared to baseline (51%, P < 0.01) and to paired controls (102%, P < 0.01), as well as significantly increased PEFL in both IW (P < 0.05) and ESVs (P < 0.01). In treated eyes, PEFL was significantly higher in ESVs than in the IW (P < 0.01) and was associated with increased cross-sectional area of ESVs (P < 0.01). Percentage effective filtration length in ESVs positively correlated with the percentage change in C (R2 = 0.58, P = 0.01). A significant increase in juxtacanalicular connective tissue (JCT) thickness (P < 0.05) was found in treated eyes compared to controls.ConclusionsNetarsudil acutely increased C by expansion of the JCT and dilating the ESVs, which led to redistribution of aqueous outflow through a larger area of the IW and ESVs.

Aqueous outflow in mice is segmental. SPARC-null mice demonstrated a more uniform outflow pattern and decreased collagen fibril diameter. Areas of high flow had less compact juxtacanalicular connective tissue ECM, and IOP was inversely correlated with PEFL. Our data show a correlation between morphology, aqueous outflow, and IOP, indicating a modulatory role of SPARC in IOP regulation.

Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic 6 LC using the cytomegalovirus ( IntroductionThe systemic amyloidoses are a diverse group of protein misfolding diseases in which proteins aggregate and form fibrillar deposits in tissues. Amyloidosis can be genetic in origin (familial amyloidosis, AF) or can occur in the setting of chronic inflammation or infection (amyloidosis because of deposition of the acute phase serum amyloid A protein, AA). However the most commonly diagnosed form, amyloid light chain (AL) amyloidosis, is because of deposition of an immunoglobulin light chain (LC) usually produced by clonal plasma cells in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as LC deposits may rapidly accumulate in organs such as the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. 1 Patients with AL amyloidosis are treated with chemotherapy to eradicate the plasma cell clone in the bone marrow that is the source of the amyloidogenic protein. Unfortunately, chemotherapeutics and even newer anti-plasma cell drugs with novel mechanisms of action can cause significant toxicity in AL amyloidosis patients. Although the pathophysiology of AL amyloidosis is still not completely understood, it is hoped that patient outcomes will be improved with the development of therapies that specifically target the process of protein aggregation, fibril formation, amyloid deposition, and organ damage.Although it is clear that the overexpression of a clonal amyloidogenic LC causes AL amyloidosis, it is not clear what structural features of amyloidogenic LC are responsible for misfolding and aggregation. Furthermore, although it is well-established that glycoaminoglycans 2 and serum amyloid P component 3 can interact with LC proteins, and are found in association with amyloid fibrils, the role of these accessory molecules in fibril formation in vivo is not well understood. The role of prefibrillar LCs in organ dysfunction remains a major question in the disease pathogenesis. Data from our group have demonstrated that amyloidogenic LC can be acutely toxic to target organs, inducing oxidative stress in cells and organ culture model systems. 4,5 Amyloidogenic LCs can be internalized into cells, regulating the expression of proteoglycans and possibly mediating interactions leading to the activation of stress and other signaling pathways. 6,7 Moreover, other investigators have demonstrat...

Y27632 increases outflow facility in human eyes. This increase correlates positively with an increase in EFL, which is associated with an increased expansion in the JCT. Our data suggest that EFL could serve as a novel parameter to correlate with outflow facility.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase α1–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the α1 and β1 subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

Our goal was to develop a 3-D multi-cellular construct using primary human corneal fibroblasts cultured on a disorganized collagen substrate in a scaffold-free environment and to use it to determine the regulation of proteoglycans over an extended period of time (11 weeks). Electron micrographs revealed multi-layered constructs with cells present in between alternating parallel and perpendicular arrays of fibrils. Type I collagen increased 2–4-fold. Stromal proteoglycans including lumican, syndecan4, decorin, biglycan, mimecan, and perlecan were expressed. The presence of glycosaminoglycan chains was demonstrated for a subset of the core proteins (lumican, biglycan, and decorin) using lyase digestion. Cuprolinic blue–stained cultures showed that sulfated proteoglycans were present throughout the construct and most prominent in its mid-region. The size of the Cuprolinic-positive filaments resembled those previously reported in a human corneal stroma. Under the current culture conditions, the cells mimic a development or nonfibrotic repair phenotype.

Amyloidosis is a disease of protein misfolding that ultimately impairs organ function. Previously, we demonstrated that amyloidogenic light chains (1, 6, and 3 subtypes), internalized by cardiac fibroblasts, enhanced sulfation of secreted glycosaminoglycans. In this study, we investigated the internalization and cellular trafficking of urinary immunoglobulin light chains into cardiac fibroblasts. We demonstrate that these light chains have the ability to form annular rings in solution. Internalization was assessed by incubating cells in the presence of light chain conjugated to Oregon Green 488 followed by monitoring with live cell confocal imaging. The rate of light chain internalization was reduced by treatment with methyl-␤-cyclodextrin but not filipin. Amyloid light chain did co-localize with dextranTexas Red. Once internalized, the light chains were detected in lysosomes and then secreted into the extracellular medium. The light chain detected in the cell lysate and medium possessed a lower hydrophobic species. Nocodazole, a microtubule inhibitor, did not disperse aggregates. In addition, internalization and retention of the light chain proteins was altered in the presence of the proteasomal inhibitor MG132. These results indicate that the cell internalizes light chain by a fluid phase endocytosis, which is then modified and ultimately compromises the cell.

In the absence of the P2X(7) receptor, the expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of receptor resulted in morphologic changes in the stroma, including changes in alignment of collagen fibrils, decreased expression of collagen, and smaller fibrils with fewer PGs per fibril.