Ischemic preconditioning, retinal neuroprotection and histone deacetylase activities.

Increased histone deacetylase (HDAC) activity and the resulting dysregulation of protein acetylation is an integral event in retinal degenerations associated with ischemia and ocular hypertension. This study investigates the role of preconditioning on the process of acetylation in ischemic retinal injury. Rat eyes were unilaterally subjected to retinal injury by 45 min of acute ischemia, and retinal neuroprotection induced by 5 min of an ischemic preconditioning (IPC) event. HDAC activity was evaluated by a fluorometric enzymatic assay with selective isoform inhibitors. Retinal localization of acetylated histone-H3 was determined by immunohistochemistry on retina cross sections. Cleaved caspase-3 level was evaluated by Western blots. Electroretinogram (ERG) analyses were used to assess differences in retinal function seven days following ischemic injury. In control eyes, analysis of HDAC isoforms demonstrated that HDAC1/2 accounted for 28.4 ± 1.6%, HDAC3 for 42.4 ± 1.5% and HDAC6 activity 27.3 ± 3.5% of total activity. Following ischemia, total Class-I HDAC activity increased by 21.2 ± 6.2%, and this increase resulted solely from a rise in HDAC1/2 activity. No change in HDAC3 activity was measured. Activity of Class-II HDACs and HDAC8 was negligible. IPC stimulus prior to ischemic injury also suppressed the rise in Class-I HDAC activity, cleaved caspase-3 levels, and increased acetylated histone-H3 in the retina. In control animals 7 days post ischemia, ERG a- and b-wave amplitudes were significantly reduced by 34.9 ± 3.1% and 42.4 ± 6.3%, respectively. In rats receiving an IPC stimulus, the ischemia-induced decline in ERG a- and b-wave amplitudes was blocked. Although multiple HDACs were detected in the retina, these studies provide evidence that hypoacetylation associated with ischemic injury results from the selective rise in HDAC1/2 activity and that neuroprotection induced by IPC is mediated in part by suppressing HDAC activity.

Acetylation preserves retinal ganglion cell structure and function in a chronic model of ocular hypertension.

PURPOSE: The current studies investigate if the histone deacetylase (HDAC) inhibitor, valproic acid (VPA), can limit retinal ganglion cell (RGC) degeneration in an ocular-hypertensive rat model. METHODS: Intraocular pressure (IOP) was elevated unilaterally in Brown Norway rats by hypertonic saline injection. Rats received either vehicle or VPA (100 mg/kg) treatment for 28 days. Retinal ganglion cell function and number were assessed by pattern electroretinogram (pERG) and retrograde FluoroGold labeling. Western blotting and a fluorescence assay were used for determination of histone H3 acetylation and HDAC activity, respectively, at 3-day, 1-week, and 2-week time points. RESULTS: Hypertonic saline injections increased IOPs by 7 to 14 mm Hg. In vehicle-treated animals, ocular hypertension resulted in a 29.1% and 39.4% decrease in pERG amplitudes at 2 and 4 weeks, respectively, and a 42.9% decrease in mean RGC density at 4 weeks. In comparison, VPA treatment yielded significant amplitude preservation at 2 and 4 weeks and showed significant RGC density preservation at 4 weeks. No significant difference in RGC densities or IOPs was measured between control eyes of vehicle- and VPA-treated rats. In ocular-hypertensive eyes, class I HDAC activity was significantly elevated within 1 week (13.3 ± 2.2%) and histone H3 acetylation was significantly reduced within 2 weeks following the induction of ocular hypertension. CONCLUSIONS: Increase in HDAC activity is a relatively early retinal event induced by elevated IOP, and suppressing HDAC activity can protect RGCs from ocular-hypertensive stress. Together these data provide a basis for developing HDAC inhibitors for the treatment of optic neuropathies.

Latanoprost-induced changes in rat intraocular pressure: direct or indirect?

INTRODUCTION: The topical application of prostaglandin F(2 ) (FP)-receptor agonists has been shown to significantly lower intraocular pressure (IOP) in humans and is now considered the first-line treatment for ocular hypertension. Despite the prominent role FP-receptor agonists play in the treatment of glaucoma, our understanding of how these agents lower IOP remains incomplete. The present study was designed to evaluate the role of matrix metalloproteinase (MMP) activation and the cytokine, tumor necrosis factor alpha (TNF-alpha), in latanoprost-induced changes in IOP. METHODS: Changes in IOP following an acute topical administration of latanoprost (60 ng) in normotensive Brown Norway rats were evaluated by means of a commercially available rebound tonometer. To examine the role of MMPs and TNF-alpha in this response, the rats were pretreated with a broad-spectrum MMP inhibitor, GM-6001 (100 microg), or the TNF-alpha inhibitor, thalidomide (25 microg). RESULTS: The topical administration of latanoprost (60 ng) alone produced a biphasic change in ipsilateral IOP: an initial hypertension (4.21 +/- 0.52), followed by a prolonged hypotension (-4.79 +/- 0.65). In rats, pretreatment with GM-6001 blocked the latanoprost-induced reduction in IOP but did not prevent the initial rise in IOP. Pretreatment with thalidomide also blocked the ocular hypotension induced by latanoprost; however, thalidomide pretreatment enhanced the duration of the initial hypertension. CONCLUSIONS: These results provide evidence that the secretion and activation of MMPs and the release of TNF-alpha play a central role in the ocular hypotension induced by FP-agonists. The administration of FP-agonists appears to lower IOP directly by inducing the activation of MMPs within the ciliary body, leading to improved uveoscleral outflow and indirectly through the release of TNF within the ciliary body. Secreted TNF-alpha may then activate TNF-receptors in the uvea and trabecular meshwork, increasing both uveoscleral and conventional outflow.

Kinin modulation of conventional outflow facility in the bovine eye.

The aim of this study was to investigate the effects of bradykinin on conventional outflow facility in relation to kinin effects on matrix metalloproteinase (MMP) secretion. Conventional outflow facility was measured in isolated bovine segments perfused at a constant pressure of 10 mmHg. Experiments were also performed in primary cultures of bovine trabecular meshwork cells to assess bradykinin effects on the secretion of MMP-9 assessed by western blot. Administration of bradykinin (10(-7) M) to perfused anterior segments produced a 50% increase in outflow facility above basal levels. The effect was slow to develop, requiring 100 min for a significant increase in facility and 4 h for the peak response to be observed. Pretreatment of anterior segments with the B(2) kinin receptor antagonist, HOE-140 (10(-6) M), or with the nonselective MMP inhibitor, GM6001 (10(-5) M) blocked the response to bradykinin (10(-7) M). Treatment of cultured trabecular meshwork cells with bradykinin (10(-7) M) for 120 min stimulated secretion of MMP-9 into the extracellular media, and this response was inhibited by HOE-140 (10(-6) M). These results demonstrate that bradykinin activates B(2) kinin receptors to increase conventional outflow in the perfused bovine eye and provide evidence that secretion and activation of MMPs within the conventional pathway may mediate the effect.

Evidence for multiple P2Y receptors in trabecular meshwork cells.

The purpose of this study was to determine whether functional purinergic P2 receptors are present in trabecular meshwork cells. The human trabecular cell line HTM-3 and cultured bovine trabecular cells were used to assess the effects of P2 agonists on intracellular Ca(2+) levels, extracellular signal-regulated kinase (ERK1/2) activation, and P2Y receptor expression. ATP, UTP, ADP, and 2-methyl-thio-adenosine triphosphate (2-MeS-ATP) each produced a concentration-dependent increase in intracellular Ca(2+) in bovine trabecular cells and the HTM-3 cell line. The addition of UDP did not produce any detectable rise in intracellular Ca(2+). Pretreatment with the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine-3',5'-diphosphate (MRS-2179) blocked the ADP- and 2-MeS-ATP-induced rise in intracellular Ca(2+). However, the ATP- or UTP-induced rise in intracellular Ca(2+) was not inhibited by MRS-2179 pretreatment. The addition of ADP, 2-MeS-ATP, ATP, or UTP were also found to activate the ERK1/2 signaling pathway. This activation of ERK1/2 was blocked by pretreatment with the mitogen-activated protein kinase kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U-0126) or the protein kinase C inhibitor chelerythrine chloride, but not by MRS-2179. Analysis of mRNA from HTM-3 cells by reverse transcription-polymerase chain reaction revealed the expression of P2Y(1), P2Y(4), and P2Y(11) receptor subtypes. These data demonstrate that multiple P2Y receptors are present in trabecular cells. Our results are consistent with the idea that the mobilization of intracellular Ca(2+)results from the activation of P2Y(1) and P2Y(4) receptors, whereas the activation of the ERK1/2 pathway results from the activation of P2Y(4) receptors alone. However, a role for the P2Y(11) receptors in mobilization of Ca(2+), or activation of the ERK1/2 pathway, cannot be discounted.

Bradykinin enhancement of PGE2 signalling in bovine trabecular meshwork cells.

Kinins and prostaglandins activate signalling pathways in cells of the trabecular meshwork and have opposing effects on outflow resistance to aqueous humor. Consequently, interactions between these pathways may be important in the regulation of intraocular pressure. In the present study, the influence of bradykinin on PGE(2) signalling was examined in primary cultures of bovine trabecular meshwork cells. Incubation of cells with bradykinin produced a concentration-dependent (EC(50)=3.6+/-0.7 nM) elevation of intracellular free Ca(2+). At a maximal concentration of 100 nM, the increase in Ca(2+) was rapid, peaking in 30 sec, and then slowly returned to baseline. This effect was completely inhibited in cells pretreated with the selective B(2) kinin receptor antagonist, Hoe-140. Treatment of trabecular meshwork cells with PGE(2), in comparison, had no effect on cellular Ca(2+) but produced a concentration-dependent increase in adenosine 3', 5'-cyclic monophosphate (cAMP) formation. Bradykinin had no effect on basal cAMP. However, incubation of cells with PGE(2) in combination with bradykinin resulted in a 3- to 5-fold enhancement of PGE(2)-stimulated cAMP production. Bradykinin enhancement of cAMP stimulation was concentration-dependent with an EC(50) of 3.6+/-1.8 nM. Treatment of cells with bradykinin increased the response maximum for PGE(2) signalling, while the EC(50) for PGE(2) was not changed. This action of bradykinin was again blocked in cells pretreated with Hoe-140. Bradykinin also produced a 2- to 3-fold increase in isoproterenol and cholera toxin-stimulated cAMP accumulation. However, when adenylyl cyclase was stimulated directly with forskolin, bradykinin failed to alter cAMP production. These results indicate that bradykinin activates B(2) kinin receptors in trabecular meshwork cells to amplify PGE(2)-stimulated cAMP formation by facilitating the interaction between activated G(s) and the catalytic unit of adenylyl cyclase.