Inhibition of FGF-Induced ␣A-Crystallin Promoter Activity in Lens Epithelial Explants by TGF␤

PURPOSE. Fibroblast growth factor (FGF) plays a key role in normal lens biology, and recent studies suggest that transforming growth factor (TGF)-␤ is involved in the origin of certain forms of cataract. In the current study, the effects of FGF and TGF␤ on ␣A-crystallin promoter activity were investigated. METHODS. Rat lens epithelial explants were cultured with or without growth factors after transfecting with the firefly luciferase reporter gene driven by either the mouse ␣A-crystallin promoter region or a control simian virus (SV)40 promoter. RESULTS. FGF-2, at a concentration that induced lens fiber differentiation, strongly stimulated ␣A-crystallin promoter activity in explants at 3 to 4 days of culture, whereas SV40 promoter control specimens showed no comparable increase. At lower concentrations of FGF, sufficient to induce cell proliferation but not differentiation, there was only a slight increase in ␣A-crystallin promoter activity. Stimulation of ␣A-crystallin promoter activity induced by the fiber-differentiating concentration of FGF was virtually abolished by as little as 25 pg/ml TGF␤2, but the onset of fiber-specific ␤-crystallin accumulation was not prevented at this concentration. Phase-contrast microscopy revealed overt cataractous changes only at concentrations of TGF␤ more than 25 pg/ml. CONCLUSIONS. The stimulation of ␣A-crystallin promoter activity by FGF is consistent with its role in inducing accumulation of crystallins in explants. The blocking effect of TGF␤ on this process, even at a concentration too low to induce obvious pathologic changes, indicates the potential for TGF␤ to disturb ␣A-crystallin gene expression during early fiber differentiation. (Invest Ophthalmol Vis Sci

Secreted frizzled-related protein disrupts PCP in eye lens fiber cells that have polarised primary cilia

AbstractPlanar cell polarity (PCP) signaling polarises cells along tissue axes. Although pathways involved are becoming better understood, outstanding issues include; (i) existence/identity of cues that orchestrate global polarisation in tissues, and (ii) the generality of the link between polarisation of primary cilia and asymmetric localisation of PCP proteins. Mammalian lenses are mainly comprised of epithelial-derived fiber cells. Concentrically arranged fibers are precisely aligned as they elongate along the anterior–posterior axis and orientate towards lens poles where they meet fibers from other segments to form characteristic sutures. We show that lens exhibits PCP, with each fiber cell having an apically situated cilium and in most cases this is polarised towards the anterior pole. Frizzled and other PCP proteins are also asymmetrically localised along the equatorial–anterior axis. Mutations in core PCP genes Van Gogh-like 2 and Celsr1 perturb oriented fiber alignment and suture formation. Suppression of the PCP pathway by overexpressing Sfrp2 shows that whilst local groups of fibers are often similarly oriented, they lack global orientation; consequently when local groups of fibers with different orientations meet they form multiple, small, ectopic suture-like configurations. This indicates that this extracellular inhibitor disrupts a global polarising signal that utilises a PCP-mediated mechanism to coordinate the global alignment and orientation of fibers to lens poles

Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial

<div><p>Background</p><p>Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension.</p><p>Methods and findings</p><p>To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow.</p><p>Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; <i>p <</i> 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population.</p><p>Conclusions</p><p>Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required.</p><p>Trial registration</p><p>ClinicalTrials.gov <a href="https://clinicaltrials.gov/ct2/show/NCT01640964" target="_blank">NCT01640964</a></p></div

In vitro generation of functional lens-like structures with relevance to age-related nuclear cataract

PURPOSE. To investigate the capacity of lens epithelial cells, maintained in a modified explant culture system, to mimic normal patterns of lens cell differentiation and to regenerate lens structure and function. METHODS. Lens epithelial explants were set up in pairs with their apical surfaces facing each other. These explant pairs (EPs) were then cultured in vitreous for up to 43 days to promote their growth and differentiation. Immunohistochemistry and conventional light and electron microscopy were used to assess structural and functional properties of the lenslike structures that developed from EPs. RESULTS. EPs that were asymmetrically exposed to vitreous routinely produced biconvex, lens-like structures composed of ordered epithelial and fiber cells that were transparent and had some focusing and magnifying ability. In addition, characteristic of the lens in vivo, fiberlike cells that were peripherally situated in EPs contained markers of the relatively early stages of fiber differentiation, whereas centrally situated cells contained markers of terminally differentiated fibers. During longterm culture of the EPs, a central opacity appeared that had structural features similar to those reported for the early stages of human, age-related nuclear cataract. CONCLUSIONS. This study shows that, given appropriate culture conditions, lens epithelial cells can regenerate ordered lenslike structures with functional properties. This system represents a valuable new tool for the investigation of factors involved in the generation of normal lens structure and function and lens opacification

Planar cell polarity in the mammalian eye lens

The major role of the eye lens is to transmit and focus images onto the retina. For this function, the lens needs to develop and maintain the correct shape, notably, the precise curvature and high-level order and organization of its elements. The lens is mainly comprised of highly elongated fiber cells with hexagonal cross-sectional profiles that facilitate regular packing. Collectively, they form concentrically arranged layers around the anterior-posterior polar axis, and their convex curvature contributes to the spheroidal shape of the lens. Although the lens has been a popular system for developmental studies, little is known about the mechanism(s) that underlies the development of its exquisite three-dimensional cellular architecture. In this review, we will describe our recent work, which shows how planar cell polarity (PCP) operates in lens and contributes to its morphogenesis. We believe that the lens will be a useful model system to study PCP in general and gain insights into mechanisms that generate high-level cellular order during development

Save Sight Institute

PURPOSE. Cataract is the most common cause of blindness in the world today, and yet there is no generally accepted treatment other than surgical intervention. Studies in rodent models designed to increase understanding of the molecular basis of cataract have shown that transforming growth factor (TGF)-␤ induces morphologic and molecular changes similar to those associated with some forms of human cataract. Because aging is the most widely recognized risk factor for cataract, it is important that any animal model be examined in this context. This was a study of the effects of aging on susceptibility to TGF␤-induced cataract. METHODS. Lenses from weanling, adult, and senile rats were cultured in defined serum-free medium with a range of concentrations of TGF␤2. The lenses were cultured for up to 7 days, photographed daily, fixed, and prepared for histology and immunolocalization. Opacification was quantified by image analysis. RESULTS. Lenses from weanling, adult, and senile rats all underwent similar cataractous changes when exposed to TGF␤. This included opacification, the formation of anterior subcapsular plaques, and accumulation of type I collagen and ␣-smooth muscle actin. Lenses from adult and senile animals, however, were generally more adversely affected by TGF␤ than lenses from weanlings. This study also showed that a low dose of TGF␤ administered over a prolonged period had an effect similar to that of a higher dose administered over a shorter period. CONCLUSIONS. An elevation of TGF␤ activity, either acute or chronic, and/or an age-related increase in lens cell susceptibility to TGF␤ may be triggering factors in the etiology of certain forms of cataract. (Invest Ophthalmol Vis Sci. 2000;41:3544 -3551) C ataract is the leading cause of blindness worldwide. 2 Despite the extent of the problem and considerable research effort over the years, there is no generally accepted pharmacologic agent that can be applied to prevent or slow the onset or progression of this disease. Development of such a therapeutic agent depends on increased understanding of the molecular basis of cataractogenesis. In this laboratory we have been investigating the effects of various growth factors on lens biology and disease. We have shown that in vitro all three mammalian isoforms of transforming growth factor (TGF)-␤ induce responses in lens cells that mimic events in cataractogenesis. Lens epithelial cells in explants 3,4 and cultured whole lenses 5 are induced to undergo molecular and morphologic changes that are typically associated with subcapsular cataracts and with aftercataract, the subcapsular opacification that often arises from lens cells remaining after cataract surgery. 6 TGF␤ induces distinct anterior subcapsular opacities in cultured lenses from weanling rats. 5 These opacities correspond histologically with subcapsular plaques, which contain aberrant cells and are virtually indistinguishable from early-stage anterior subcapsular cataracts in humans. Lenses and lens explants cultured with TGF␤ express molecular markers for subcapsular cataract, type I collagen, and ␣-smooth muscle actin. 7 Taken together, these findings point to a role for TGF␤ in the etiology of major forms of cataract. A variety of risk factors predispose toward cataract, but the most widely recognized of these is aging

Growth Factor Signaling in Vitreous Humor–Induced Lens Fiber Differentiation

Based on the characterization of the ERK1/2- and Akt-signaling pathways involved in vitreous-induced lens fiber cell differentiation, it is clear that FGF is the key growth factor involved in this process, and that other ocular growth factors, such as IGF, PDGF, and EGF, may be involved in fine tuning the signaling required for the initiation and/or maintenance of lens fiber differentiation in situ