Shravan K. Chintala

Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype.

The glaucomas are a group of optic neuropathies comprising the leading cause of irreversible blindness worldwide. Elevated intraocular pressure due to a reduction in normal aqueous outflow is a major causal risk factor. We found that endothelial leukocyte adhesion molecule-1 (ELAM-1), the earliest marker for the atherosclerotic plaque in the vasculature, was consistently present on trabecular meshwork (TM) cells in the outflow pathways of eyes with glaucomas of diverse etiology. We determined expression of ELAM-1 to be controlled by activation of an interleukin-1 (IL-1) autocrine feedback loop through transcription factor NF-κB, and activity of this signaling pathway was shown to protect TM cells against oxidative stress. These findings characterize a protective stress response specific to the eyes aqueous outflow pathways and provide the first known diagnostic indicator of glaucomatous TM cells. They further indicate that common mechanisms contribute to the pathophysiology of the glaucomas and vascular diseases.

Plasminogen activators promote excitotoxicity-induced retinal damage

Increased levels of extracellular L‐glutamate have been suggested to play a role in retinal damage in a number of blinding diseases such as glaucoma and diabetic retinopathy. Although glutamate can cause retinal damage in part by hyperstimulating its receptors (“excitotoxicity”), the downstream events that lead to retinal damage are poorly understood. In this study, we injected kainic acid (KA), a glutamate receptor agonist that specifically hyperstimulates non‐NMDA‐type receptors, into the vitreous humor of CD‐1 mice and have investigated the role of plasminogen activators (PAs) [tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA)] in excitotoxicity‐induced retinal damage. Injection of KA into the vitreous humor led to an up‐regulation in tPA and an induction in uPA activity in the retina and this was associated with activation of zymogen plasminogen to active plasmin. Immunocytochemical analysis indicated that retinal ganglion cells (RGCs), constitutively express tPA and release it into the extracellular space upon KA injection. Immunocytochemical analysis also indicated an increase in uPA in the nerve fiber layer after KA injection that was absent in the control retinas. These events were associated with apoptotic death of cells initially in the ganglion cell layer and subsequently in the inner and outer nuclear layer, associated with loss of RGCs and amacrine cells. These phenomena were inhibited when recombinant plasminogen activator inhibitor (rPAI‐1) or tPA‐STOP were injected into the vitreous humor with KA, whereas a plasmin inhibitor, α‐2‐antiplasmin, failed to attenuate KA‐induced retinal damage. Taken together, these results suggest that inhibition of plasminogen activators might attenuate retinal damage in blinding retinal diseases in which hyperstimulation of glutamate receptors is implicated as a causative factor to retinal damage. Mali, R. S., Cheng, M., Chintala, S. K. Plasminogen activators promote excitotoxicity‐induced retinal damage. FASEB J. 19, 1280–1289 (2005)

Optic nerve ligation leads to astrocyte-associated matrix metalloproteinase-9 induction in the mouse retina

Ischemic damage results in irreversible ganglion cell loss in the retina. While the mechanisms underlying ischemia-induced ganglion cell loss are not clearly understood, we have recently reported that ischemia, induced by optic nerve ligation, results in increased nerve fiber layer-associated matrix metalloproteinase-9 (MMP-9) induction and loss of ganglion cells from the retina. This study was conducted to determine the cellular source of MMP-9 using antibodies against MMP-9 and various cell types in the inner retina. The results presented in this study show that optic nerve ligation leads to induction of MMP-9 and activation of astrocytes. Double labeling studies using antibodies against MMP-9 and GFAP showed a greater overlap of MMP-9 with GFAP, compared to antibodies against glutamine synthetase and MMP-9 which showed no overlapping, suggesting that activated astrocytes contribute to MMP-9 expression in the retina. Further, double labeling studies using antibodies against von Willebrand factor and MMP-9 or Mac-1 and MMP-9 showed no overlapping of MMP-9 with any antibodies indicating that endothelial cells and microglial cells are not the principal source of MMP-9 in the retina following optic nerve ligation.

Activation of TLR3 Promotes the Degeneration of Retinal Ganglion Cells by Upregulating the Protein Levels of JNK3

PURPOSEnTo investigate whether activation of Toll-like receptor 3 (TLR3) promotes the degeneration of retinal ganglion cells (RGCs) by upregulating the protein levels of c-jun N-terminal kinase 3 (JNK3).nnnMETHODSnToll-like receptor 3-specific activator, Poly(I:C) (polyinosinic-polycytidylic acid), or PBS was injected into the vitreous humor of Thy1-YFP mice. At 24, 48, and 72 hours after treatments, degeneration of RGCs was assessed by using antibodies against brain-specific homeobox/POU domain protein 3a (Brn3a). A TLR3-specific inhibitor was injected into the vitreous humor with or without Poly(I:C). Western blot assays were performed to determine relative levels of TLR3, JNK3, pJNK3, and sterile alpha and HEAT/Armadillo motif-containing 1 (SARM1) proteins in retinal protein extracts, and immunohistochemistry assays were performed to determine their cellular localization in the retina. Mouse eyes were treated with Poly(I:C) or PBS along with MitoTracker Red, and colocalization of MitoTracker Red and JNK3 in the retinas was determined by using antibodies against JNK3.nnnRESULTSnPoly(I:C) activated TLR3 and upregulated its downstream target protein JNK3 but not SARM1 in the retina. Poly(I:C) activated TLR3 and upregulated JNK3 specifically in RGCs and promoted a significant degeneration of RGCs over a 72-hour time period. Toll-like receptor 3 upregulated the levels of JNK3 protein in the cytoplasm of RGCs, but not in the mitochondria. Toll-like receptor 3-specific inhibitor downregulated Poly(I:C)-mediated upregulation of JNK3 protein, and, in turn, significantly attenuated TLR3-induced degeneration of RGCs.nnnCONCLUSIONSnResults presented in this study show that the activation of TLR3 alone promotes the degeneration of RGCs by upregulating the protein levels of JNK3.

Mechanisms regulating plasminogen activators in transformed retinal ganglion cells.

Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs death. In this study, by employing RGC-5 cells, we report that tPA and uPA elicit their direct effect through the low-density lipoprotein-related receptor-1 (LRP-1). We also show that blockade of protease-LRP-1 interaction leads to a complete reduction in autocrine synthesis of tPA and uPA, and prevents protease-mediated death of RGC-5 cells. RGC-5 cells were cultured in serum-free medium and treated with 2.0 microM Staurosporine to induce their differentiation. Neurite outgrowth was observed by a phase contrast microscope and quantified by NeuroJ imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by MTT assays. Compared to untreated RGC-5 cells, cells treated with Staurosporine differentiated, synthesized and secreted elevated levels of tPA and uPA, and underwent cell death. In contrast, when RGC-5 cells were treated with Staurosporine along with the receptor associated protein (RAP), proteolytic activities of both tPA and uPA were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived and showed increased neurite outgrowth. These results indicate that LRP-1 regulates autocrine synthesis of tPA and uPA in RGC-5 cells and suggest that the use of RAP to antagonize the effect of proteases may be a way to prevent RGC death in glaucoma.

Tissue and urokinase plasminogen activators instigate the degeneration of retinal ganglion cells in a mouse model of glaucoma

Elevated intraocular pressure (IOP) promotes the degeneration of retinal ganglion cells (RGCs) during the progression of Primary Open-Angle Glaucoma (POAG). However, the molecular mechanisms underpinning IOP-mediated degeneration of RGCs remain unclear. Therefore, by employing a mouse model of POAG, this study examined whether elevated IOP promotes the degeneration of RGCs by up-regulating tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) in the retina. IOP was elevated in mouse eyes by injecting fluorescent-microbeads into the anterior chamber. Once a week, for eight weeks, IOP in mouse eyes was measured by using Tono-Pen XL. At various time periods after injecting microbeads, proteolytic activity of tPA and uPA in retinal protein extracts was determined by fibrinogen/plasminogen zymography assays. Localization of tPA and uPA, and their receptor LRP-1 (low-density receptor-related protein-1) in the retina was determined by immunohistochemistry. RGCs degeneration was assessed by immunostaining with antibodies against Brn3a. Injection of microbeads into the anterior chamber led to a progressive elevation in IOP, increased the proteolytic activity of tPA and uPA in the retina, activated plasminogen into plasmin, and promoted a significant degeneration of RGCs. Elevated IOP up-regulated tPA and LRP-1 in RGCs, and uPA in astrocytes. At four weeks after injecting microbeads, RAP (receptor associated protein; 0.5 and 1.0xa0μM) or tPA-Stop (1.0 and 4.0xa0μM) was injected into the vitreous humor. Treatment of IOP-elevated eyes with RAP led to a significant decrease in proteolytic activity of both tPA and uPA, and a significant decrease in IOP-mediated degeneration of RGCs. Also, treatment of IOP-elevated eyes with tPA-Stop decreased the proteolytic activity of both tPA and uPA, and, in turn, significantly attenuated IOP-mediated degeneration of RGCs. Results presented in this study provide evidence that elevated IOP promotes the degeneration of RGCs by up-regulating the levels of proteolytically active tPA and uPA.

Dynasore protects the ocular surface against damaging stress

“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated in this case, and two of the inhibitors did not block dye uptake. The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.