PURPOSE To localize mRNA and protein of bradykinin (BK) receptors, BK precursor polypeptide (kininogen) mRNA, and to study functional biochemical pharmacology of the signal transduction processes mediated by B2-receptors in isolated human trabecular meshwork (h-TM) cells. Intraocular pressure (IOP) lowering effects of 2 kinins were also investigated. METHODS Previously documented procedures were utilized throughout these studies. RESULTS Kinninogen mRNA was most abundant in TM, ciliary body (CB), and optic nerve head and appeared elevated in glaucomatous h-TM tissue. High levels of B2-receptor mRNA were found in the sclera, iris, TM, and CB. B2-receptor subtype protein was localized in cells of the monkey and h-TM, and the treatment of isolated h-TM cells with transforming growth factor-β2 (5 ng/mL) caused significant (P<0.04) downregulation of B2-receptor mRNA. In isolated primary h-TM cells, BK (EC50=0.8±0.2 nM; n=19) and Met-Lys-BK (EC50=6.5±1.5 nM) mobilized intracellular Ca(2+) and induced the release of prostaglandins (PGs) that was blocked by 2 B2-receptor antagonists [HOE-140; (S)-WIN-64338]. The cyclooxygenase inhibitor, bromfenac, abolished BK-induced PGs production. BK concentration dependently increased cell impedance, and it significantly (P<0.05) decreased h-TM cell volume in vitro. Intravitreal (ivt) administration of BK (50 μg), but not a B1-agonist (Sar-[D-Phe(9)]-Des-Arg(9)-BK; also at 50 μg), efficaciously lowered IOP (22.9% to 37% from baseline) of Dutch-Belted rabbits that naturally have high IOPs (27-28 mmHg). CONCLUSIONS BK activates multiple signal transduction pathways in h-TM cells via B2-receptors that also mediate IOP reduction as observed in rabbits following ivt administration of BK. These ocular hypotensive effects of BK may be physiologically important and suggest a novel therapeutic potential of BK-related B2-agonists.

Purpose: To characterize the bradykinin (BK) receptor system in human non-pigmented ciliary epithelium (NPCE) using immunohistochemistry and functional cell-based techniques.Methods: B-2-receptor protein expression was studied in sections of human donor eyes and in Cynomolgus monkey eyes using immunohistochemical methods. The pharmacological characteristics of intracellular Ca2+ ([Ca2+](i)) mobilization in response to BK and related peptides, and blockade by two antagonists, was studied in primary human (p-h-NPCE) and in immortalized human NPCE ((im)h-NPCE) cells. Prostaglandins (PGs) release induced by BK was also studied in both cell-types using ELISA assays. Limited studies on primary human ciliary muscle (h-CM) cells and human trabecular meshwork (h-TM) cells and Chinese hamster ovary cells expressing human cloned B-2-receptors (CHO-B-2) were performed to compare with responses in both the NPCE cell-types.Results: B-2-receptor immunoreactivity was observed on human and Cynomolgus monkey NPCE cells on eye sections from both species. BK and related analog peptides differentially activated signaling mechanisms in NPCE cells by mobilizing [Ca2+](i), and the BK-evoked responses were blocked by B-2-receptor-selective antagonists, HOE-140 and (S)-WIN-64338. Relative agonist potencies (EC50, nM) in p-h-NPCE cells [and in (im)h-NPCE cells] were: BK = 3.4 +/- 0.4 [6.3 nM]; Hyp(3)-BK EC50 = 1.7 +/- 0.2 [6.0 nM], Lys-BK EC50 = 7.0 +/- 0.3 [19.8 nM]; Met-Lys-BK EC50 = 106 +/- 57.8 [125 nM]; Des-Arg(9)-BK EC50 = 410,000 [16 mu M]. The antagonist potencies for attenuating BK-induced mobilization of [Ca2+](i) in these cells were: HOE-140 (K-i = 7.9 +/- 1.8 nM, n = 4) and (S)-WIN-64338 (K-i = 451 +/- 44 nM, n = 4). These NPCE cell data correlated well with those obtained for h-CM and h-TM cells, and with B-2-receptor binding (r = 0.99, p<0.0001). However, BK failed to stimulate total PGs production in both NPCE cell-types even though 10% bovine serum increased PG release (by 4.9-fold above baseline), and even though BK stimulated PG release from h-CM, h-TM and in CHO-B-2 cells. BK (1 mu M) also failed to increase nitric oxide (NO) levels in NPCE cells even though sodium nitropruside increased NO production by 3-fold.Conclusions: Human and monkey NPCE express immunoreactive B-2-receptor proteins. These proteins were functionally active, since BK and related peptides potently stimulated mobilization of [Ca2+](i) in p-h-NPCE and imNPCE cells that was blocked by two B-2-selective antagonists. Down-stream signaling from B-2-receptor activation did not appear to involve PG synthesis/release (or NO production) in NPCE cell-types under the present conditions, even though h-CM, h-TM and CHO-B-2 cells exhibited robust PG synthesis and release in response to BK.

Purpose: To examine the bradykinin (BK) B-2-receptor system in human and monkey ciliary muscle (CM) using immunohistochemical techniques, and to pharmacologically characterize the associated biochemical signal transduction systems in human CM (h-CM) cells. BK-induced modulation of intraocular pressure (IOP) in pigmented Dutch-Belt rabbits and cynomolgus monkeys was also studied.Methods: Previously published procedures were used throughout these studies.Results: The human and monkey ciliary bodies expressed high levels of B-2-receptor protein immunoreactivity. Various kinins differentially stimulated [Ca2+](i) mobilization in primary h-CM cells (BK EC50=2.4 +/- 0.2 nM > Hyp(3), beta-(2-thienyl)-Ala(5),Tyr(Me)(8)-((R))-Arg(9))-BK (RMP-7) > Des-Arg(9)-BK EC50=4.2 mu M [n=3-6]), and this was blocked by B-2-selective antagonists, HOE-140 (IC50=1.4 +/- 0.1 nM) and WIN-63448 (IC50=174 nM). A phospholipase C inhibitor (U73122; 10-30 mu M) and ethylene glycol tetraacetic acid (1-2 mM) abolished the BK-induced [Ca2+](i) mobilization. Total prostaglandin (primarily PGE(2)) secretion stimulated by BK and other kinins in h-CM cells was attenuated by the cyclooxygenase inhibitors bromfenac and flurbiprofen, and by the B-2-antagonists. BK and RMP-7 (100 nM) induced a twofold increase in extracellular signal-regulated kinase-1/2 phosphorylation, and BK (0.1-1 mu M; at 24 h) caused a 1.4-3.1-fold increase in promatrix metalloproteinases-1-3 release. Topical ocular BK (100 mu g) failed to alter IOP in cynomolgus monkeys. However, intravitreal injection of 50 mu g of BK, but not Des-Arg(9)-BK, lowered IOP in rabbit eyes (22.9 +/- 7.3% and 37.0 +/- 5.6% at 5 h and 8 h post-injection; n=7-10).Conclusions: These studies have provided evidence of a functional endogenously expressed B-2-receptor system in the CM that appears to be involved in modulating IOP.

PURPOSE:Increasing evidence supports a role for complement in the pathogenesis of age-related macular degeneration (AMD). This study evaluated retinal microglia, T-lymphocytes, and complement deposition in a light-induced retinopathy model. The effect of a serotonin (5-hydroxytryptamine, 5-HT(1A)) agonist on these processes was investigated.METHODS:Rats were dark adapted for 24 hours before a 6-hour blue light exposure. Some animals were predosed subcutaneously with AL-8309A. Retinas were evaluated at different times after light exposure. Paraffin sections were stained with antibody for a microglial marker (Iba1), a T-lymphocyte marker (CD3), and complement components C1q, C3, factor B, factor H, and membrane attack complex (MAC).RESULTS:Light exposure resulted in substantial photoreceptor and RPE loss. Robust microglia activation and migration to the outer retina occurred rapidly. Substantial T-lymphocyte recruitment did not occur. Complement alternative pathway was strongly activated, resulting in the deposition of C3, factor B, factor H, and MAC in the area of photic lesions. Dosing with AL-8309A prevented retinal lesions and decreased microglia activation/recruitment and complement deposition in the outer retina.CONCLUSIONS:In blue light exposed retinas, microglia were activated and migrated toward the outer retina, whereas a T-lymphocyte response was minimal. The innate immune system was markedly activated, with substantial complement deposition in the outer retina after light exposure. This complement deposition was prevented by AL-8309A. This model may be useful in the evaluation of complement inhibitors and other neuroprotectants intended for ocular use. AL-8309 is under evaluation in the clinic and may be useful in the treatment of AMD.

Purpose: Gene transduction using adenoviral vectors is an important research tool. To assess and optimize this technique for glaucoma research, we characterized green fluorescent protein (GFP) expression in the mouse eye after intraocular injection of adenoviral vector encoding GFP (Ad5.CMV-GFP) and evaluated the effect of anti-CD40L antibody administration on GFP expression.Methods: Mice were injected with Ad5.CMV-GFP intracamerally (IC) or intravitreally (IVT) with or without anti-CD40L antibody treatment. GFP expression was assessed by in vivo fluorescence intensity with a standardized grading scale. Location of expression was analyzed histologically by fluorescence microscopy.Results: Intraocular injection of Ad5.CMV-GFP induced titer-dependent expression of GFP in the anterior segment. In vivo fluorescence was detectable but low after IC injection. After IVT injection, fluorescence in the eye peaked at days 47 with a fluorescence grade of 3.0 +/- 0.0 (mean +/- SEM, n=6; injection with 1x10(8) pfu vector). After day 7, GFP expression declined significantly. Treatment with anti-CD40L antibody increased fluorescence intensity after IC injection, and prolonged GFP expression in the IVT group. At day 43, fluorescence grades of the IVT group were 2.8 +/- 0.7 (with anti-CD40L) and 1.2 +/- 0.6 (without antibody). Three-Way ANOVA confirmed that GFP expression was significantly higher in the anti-CD40L than the no antibody group (p=0.013), significantly higher in the IVT than the IC group (p=0.003), and significantly higher in the high viral titer (1x10(8) pfu) than the low titer (1x10(7) pfu) group (p=0.010). Fluorescence microscopic examination of cross-sections of eyes indicated GFP expression in the trabecular meshwork (TM), corneal endothelium, and sporadically iris, ciliary body and lens epithelium.Conclusions: Intraocular injection of Ad5.CMV-GFP induced GFP expression in the mouse anterior segment, including the TM. Expression was more prominent after IVT injection than IC injection. Anti-CD40L antibody treatment increased both intensity and duration of GFP expression. These findings provide important and practical means to improve duration and efficiency of adenovirus-mediated transgene expression in the eye.