Topical ocular sodium 4-phenylbutyrate rescues glaucoma in a myocilin mouse model of primary open-angle glaucoma

PURPOSE. Mutations in the myocilin gene (MYOC) are the most common known genetic cause of primary open-angle glaucoma (POAG). The purpose of this study was to determine whether topical ocular sodium 4-phenylbutyrate (PBA) treatment rescues glaucoma phenotypes in a mouse model of myocilin-associated glaucoma (Tg-MYOC Y437H mice). METHODS. Tg-MYOC Y437H mice were treated with PBA eye drops (n ϭ 10) or sterile PBS (n ϭ 8) twice daily for 5 months. Long-term safety and effectiveness of topical PBA (0.2%) on glaucoma phenotypes were examined by measuring intraocular pressure (IOP) and pattern ERG (PERG), performing slit lamp evaluation of the anterior chamber, analyzing histologic sections of the anterior segment, and comparing myocilin levels in the aqueous humor and trabecular meshwork of Tg-MYOC Y437H mice. Sci. 2012;53: 1557-1565 RESULTS. Tg-MYO

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings

Bone Morphogenetic Protein 4 inhibits TGF-beta2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation

Zode, Gulab Shalikram, Bone Morphogenetic Protein 4 Inhibits TGF-β2 Stimulation of Extracellular Matrix Proteins in Optic Nerve Head Cells: Role of Gremlin in ECM Modulation . Doctor of Philosophy (Cell Biology and Genetics), May 2008; 177pp; 34 figures; bibliography, 192 titles. The glaucomatous neuropathy is caused by irreversible loss of retinal ganglion axons in the optic nerve head (ONH). The extensive remodeling of the extracellular matrix (ECM) in the glaucomatous ONH including increased synthesis and deposition of ECM (increased collagens, basement proteins, and elastin) is associated with loss of axons. Transforming growth factor-beta2 (TGF-β2) is increased in glaucomatous ONH and is thought to be responsible for increased synthesis and deposition of ECM proteins of the ONH. Bone morphogenetic proteins (BMPs) normally maintain the balance of ECM proteins via opposing TGF-β2 stimulated ECM proteins in various cell types. BMP antagonist gremlin inhibits BMPs function, thus may plan an important role in ECM modulation. We previously demonstrated that human ONH expresses BMP-4, BMP receptor and BMP antagonist gremlin. Therefore, we hypothesize that elevated TGF-β2 in the glaucomatous ONH induces gremlin expression that blocks BMP-4 inhibition of TGF-β2 signaling, leading to increased ECM synthesis and deposition. First, we examined whether human ONH tissues and ONH cells express the canonical BMP signaling pathway. This study demonstrated that ONH tissues and ONH cells express BMP-4 and Smad signaling pathway. Treatment of ONH cells with BMP-4 increased phosphorylation of R-Smad/1/58/ phosphorylation and interaction with Co-Smad4 indicating activation of the Smad signaling pathway. Therefore, cells within the human ONH can respond to locally released BMP via activation of Smad signaling. Second, we examined the signaling pathways utilized by TGF-β2 to stimulate ECM in ONH cells. This study demonstrated that TGF-β2 is increased in glaucomatous ONH. Recombinant TGF-β2 increased ECM deposition in ONH cells. TGF-β2 activated phosphorylation of R-smad2/3 but did not alter phosphorylation of ERK1/2, p38, and JNK1/2 in ONH cells. Inhibition of either TGF-β I receptor activity or phosphorylation of R-Smad3 or knockdown of R-Smad2/3 via siRNA reduced TGF-β2 stimulated ECM in ONH cells. Thus, TGF-β2 requires R-Smad2/3 to stimulate ECM proteins in ONH cells. Lastly, we investigated the potential effects of BMP-4 and gremlin on TGF-β2 stimulated ECM in ONH cells. BMP-4 significantly reduced TGF-β2 stimulation of ECM proteins. Addition of gremlin blocked the BMP-4 effect, increasing ECM proteins in ONH cells. Gremlin levels were significantly increased in the human glaucomatous ONH tissues. Interestingly, recombinant gremlin also increased ECM proteins in ONH cells. Gremlin stimulation of ECM proteins required activation of the TGF-β receptor and R-Smad3. TGF-β2 increased gremlin mRNA and protein in ONH cells. Thus, TGF-β2 induced gremlin expression intensifies TGF-β2 effects on ECM metabolism by inhibiting BMP-4 antagonism of TGF-β2 signaling. In conclusion, elevated TGF-β2 and gremlin in the glaucomatous ONH are involved in the pathogenesis of glaucomatous ONH. Elevated TGF-β2 directly increases ECM and also induces gremlin expression, which further aids TGF-β2 to stimulate ECM via inhibiting BMPs antagonism of TGF-β2 signaling, leading to unopposed TGF-β2 stimulated ECM proteins. Interestingly, R-smad3 is required for TGF-β2 or gremlin induced ECM remodeling in ONH cells. Therefore, modulation of R-smad3 provides a novel therapeutic target for preventing ECM remodeling in glaucoma

TGFβ2 induces chronic endoplasmic reticulum stress in trabecular meshwork cells.

Purpose: TGFβ2-induced extracellular matrix (ECM) accumulation in trabecular meshwork (TM) is associated with aqueous humor outflow resistance and IOP elevation. Recently, we have demonstrated that abnormal ECM accumulation leads to endoplasmic reticulum (ER) stress in TM. Here, we examined whether TGFβ2 induces ER stress in human TM cells. Methods: GTM3 or primary human TM cells (n=2) were treated with vehicle or recombinant TGFβ2 (5 ng/ml) in 0.5% FBS containing DMEM medium for 3 days & 7 days respectively. ER stress markers (Grp78, Grp94, ATF4 and CHOP) and ECM proteins (Fibronectin & Collagen IV) were examined by Western blot and immunostaining. GTM3 cells were transfected with plasmids expressing CRISPR-Cas9 targeting ATF4 or CHOP and subsequently treated with TGFβ2 for 48 hours. Cellular lysates were examined for ER stress and ECM proteins. Results: Western blot analysis demonstrated that TGFβ2 treatment led to ER stress as evident from increased levels of Grp78, Grp94, ATF4 and CHOP proteins compared to the vehicle treatment. TGFβ2-induced ER stress markers were also associated with ECM protein (fibronectin and Collagen IV) levels. Moreover, TGFβ2 treatment increased fibronectin staining and its colocalization with ER stress markers, suggesting TGFβ2- induced ECM proteins are associated with ER stress. Knockdown of key chronic ER stress transcriptional factors, ATF4 or CHOP prevented TGFβ2-induced ECM deposition and also reduced ER stress in GTM3 cells. Conclusions: Our preliminary findings clearly indicate that TGFβ2 can directly induce chronic ER stress in cultured human TM cells

Correction: Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork.

[This corrects the article DOI: 10.1371/journal.pone.0232111.]