Activation of the NFAT-Calcium Signaling Pathway in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2+]i and ECM gene expression in normal and glaucoma LC cells. Methods: [Ca2+]i was measured with dual-wavelength Ca2+ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-ß1 [TGFß1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. Results: Basal and hypotonic cell membrane stretch-induced [Ca2+]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2+]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2-induced elevation in profibrotic ECM genes (TGFß1, Col1A1, and periostin), both in normal and in glaucoma LC cells. Conclusions: Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

Homoeopathy and homeostasis in the vascular system - Part I

This two-part paper offers a novel, experimentally derived explanation of how homoeopathic remedies may work. It expands, at the kind suggestion of the Editor of the British Homoeopathic Journal, on a short article which I submitted earlier on the same subject to the Newsletter of the Homoeopathic Association of Ireland. The first part of the paper presents evidence of a homeostatic mechanism in a dog's vein which provides a physiological basis for the Law of Similars. The second part offers evidence that the same mechanism operates throughout the body and that homoeopathic physicians already make empirical use of it. Part 1 of the paper has three sections. The first explains what the Law of Similars is and what it implies. It suggests the reasons why, I believe, main-line pharmacologists and allopathic physicians deny its plausibility. It describes the first evidence I obtained of a physiological process which would allow the Law of Similars to operate in the lateral saphenous vein of the dog. The second section describes experiments in which noradrenaline, isoprenaline and acetylcholine were used to both constrict and dilate the saphenous vein by direct action on its smooth muscle. Section three deals with the vasa vasorum which are a crucial element in vascular homeostasis and in the operation of the Law of Similars in the dog's vein. It describes some of the special features which characterize the action of drugs released through the vasa vasorum. Section four in part 2 shows that the homeostatic mechanism which is present in the vein operates also in the circulatory system in general and the coronary vessels in particular. It offers evidence which suggests that the mechanism operates also in the kidney and the brain and in the whole body. Section five examines the nature of homoeopathy in the light of the new knowledge. It suggests how homoeopathy works and how the homoeopathic physician can maximize the effectiveness of his prescribed drugs. It shows how the Law of Similars is implicated in the aetiology of many common degenerative diseases and in ageing. It closes with a look at environmental pollution and shows why standard testing techniques fail to detect the true extent of this problem