Gross Cystic Disease Fluid Protein-15/Prolactin-Inducible Protein as a Biomarker for Keratoconus Disease

Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-β) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-β isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease

Catalytic N-oxidation of tertiary amines on RuO2NPs anchored graphene nanoplatelets

Ultrafine ruthenium oxide nanoparticles (RuO2NPs) with an average diameter of 1.3 nm were anchored on graphene nanoplatelets (GNPs) using a Ru(acac)3 precursor by a very simple dry synthesis method. The resultant material (GNPs–RuO2NPs) was used as a heterogeneous catalyst for the N-oxidation of tertiary amines for the first time. The transmission electron microscopy (TEM) images of the GNPs–RuO2NPs showed the excellent attachment of RuO2NPs on GNPs. The loading of Ru in GNPs–RuO2NPs was 2.68 wt%, as confirmed by scanning electron microscope-energy dispersive spectroscopy (SEM-EDS). The X-ray photoelectron spectrum (XPS) and the X-ray diffraction pattern (XRD) of GNPs–RuO2NPs revealed that the chemical state of Ru on GNPs was +4. After the optimization of reaction conditions for N-oxidation of triethylamine, the scope of the reaction was extended to various aliphatic, alicyclic and aromatic tertiary amines. The GNPs–RuO2NPs showed excellent catalytic activity in terms of yields even at a very low amount of Ru catalyst (0.13 mol%). The GNPs–RuO2NPs was heterogeneous in nature, chemically as well as physically, very stable and could be reused up to 5 times.ArticleCATALYSIS SCIENCE & TECHNOLOGY. 4(7):2099-2106 (2014)journal articl

Establishment of a 3D In Vitro Model to Accelerate the Development of Human Therapies against Corneal Diabetes

The authors thank Dr. John M Asara, Min Yuan, and Susanne Breitkopf for their technical help with metabolomics experiments, Dr. Ben Fowler for his technical help with TEM experiments and also Tina B McKay for many thoughtful discussions and scientific insights during the study.Purpose To establish an in vitro model that would mirror the in vivo corneal stromal environment in diabetes (DM) patients. Methods Human corneal fibroblasts from Healthy (HCFs), Type 1DM (T1DM) and Type 2DM (T2DM) donors were isolated and cultured for 4 weeks with Vitamin C stimulation in order to allow for extracellular matrix (ECM) secretion and assembly. Results Our data indicated altered cellular morphology, increased cellular migration, increased ECM assembly, and severe mitochondrial damage in both T1DM and T2DMs when compared to HCFs. Furthermore, we found significant downregulation of Collagen I and Collagen V expression in both T1DM and T2DMs. Furthermore, a significant up regulation of fibrotic markers was seen, including α-smooth muscle actin in T2DM and Collagen III in both T1DM and T2DMs. Metabolic analysis suggested impaired Glycolysis and Tricarboxylic acid cycle (TCA) pathway. Conclusion DM has significant effects on physiological and clinical aspects of the human cornea. The benefits in developing and fully characterizing our 3D in vitro model are enormous and might provide clues for the development of novel therapeutics.Yeshttp://www.plosone.org/static/editorial#pee

Sex Hormones, Growth Hormone, and the Cornea

The growth and maintenance of nearly every tissue in the body is influenced by systemic hormones during embryonic development through puberty and into adulthood. Of the ~130 different hormones expressed in the human body, steroid hormones and peptide hormones are highly abundant in circulation and are known to regulate anabolic processes and wound healing in a tissue-dependent manner. Of interest, differential levels of sex hormones have been associated with ocular pathologies, including dry eye disease and keratoconus. In this review, we discuss key studies that have revealed a role for androgens and estrogens in the cornea with focus on ocular surface homeostasis, wound healing, and stromal thickness. We also review studies of human growth hormone and insulin growth factor-1 in influencing ocular growth and epithelial regeneration. While it is unclear if endogenous hormones contribute to differential corneal wound healing in common animal models, the abundance of evidence suggests that systemic hormone levels, as a function of age, should be considered as an experimental variable in studies of corneal health and disease

Human Keratoconus Cell Contractility is Mediated by Transforming Growth Factor-Beta Isoforms

Keratoconus (KC) is a progressive disease linked to defects in the structural components of the corneal stroma. The extracellular matrix (ECM) is secreted and assembled by corneal keratocytes and regulated by transforming growth factor-β (TGF-β). We have previously identified alterations in the TGF-β pathway in human keratoconus cells (HKCs) compared to normal corneal fibroblasts (HCFs). In our current study, we seeded HKCs and HCFs in 3D-collagen gels to identify variations in contractility, and expression of matrix metalloproteases (MMPs) by HKCs in response the TGF-β isoforms. HKCs showed delayed contractility with decreased Collagen I:Collagen V ratios. TGF-β1 significantly increased ECM contraction, Collagen I, and Collagen V expression by HKCs. We also found that HKCs have significantly decreased Collagen I:Collagen III ratios suggesting a potential link to altered collagen isoform expression in KC. Our findings show that HKCs have significant variations in collagen secretion in a 3D collagen gel and have delayed contraction of the matrix compared to HCFs. For the first time, we utilize a collagen gel model to characterize the contractility and MMP expression by HKCs that may contribute to the pathobiology of KC

Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells <i>in vitro</i>

<div><p>Keratoconus (KC) is a progressive corneal ectasia linked to thinning of the central cornea. Hard contact lenses, rigid gas permeable lenses, and scleral lenses are the primary treatment modalities for early to mid- stages of KC to correct refractive error and astigmatism that develops as a result of an irregular corneal structure. These treatments are associated with significant drawbacks, including reduced availability of the tear film and oxygen to the corneal epithelium and stroma. However, it remains unknown whether hypoxia affects corneal integrity in the KC pathobiology. A number of studies have associated elevated oxidative stress with KC both <i>in vitro</i> and <i>ex vivo</i>. We hypothesized that KC-derived corneal fibroblasts are more susceptible to hypoxia-induced oxidative stress compared to healthy controls leading to exacerbation of corneal thinning in KC. This study investigated the effects of hypoxia on ECM secretion, assembly, and matrix metalloproteinase (MMP) expression in human corneal fibroblasts from healthy controls (HCFs) and KC patients (HKCs) <i>in vitro</i>. HCFs and HKCs were cultured in 3D constructs for 3 weeks and maintained or transferred to normoxic (21% O₂) or hypoxic (2% O₂) conditions, respectively, for 1 additional week. At the 4 week time-point, constructs were isolated and probed for Collagen I, III, and V, keratocan and MMP-1, -2, -3, -9, and -13, as well as hypoxia markers, hypoxia inducible factor-1α and lactoferrin. Conditioned media was also collected and probed for Collagen I, III, and V by Western blot. Thickness of the ECM assembled by HCFs and HKCs was measured using immunofluorescence microscopy. Results showed that hypoxia significantly reduced Collagen I secretion in HKCs, as well as upregulated the expression of MMP-1 and -2 with no significant effects on MMP-3, -9, or -13. ECM thickness was reduced in both cell types following 1 week in a low oxygen environment. Our study shows that hypoxia influences collagen and MMP expression by HKCs, which may have consequential effects on ECM structure in the context of KC.</p></div

Secretion of the major collagens, Col I, III, and V, detected in the media following exposure to normoxic or hypoxic (2% O₂) conditions.

<p>(A) Representative western blots and (B-D) quantification by densitometry showing a reduction in Col I in both HCFs and HKCs following hypoxia exposure. Error bars represent standard error of the mean. n = 3 for Col I and III, n = 2 for Col V. A two-way ANOVA was used to determine statistical significance with *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.</p

Regulation of hypoxia-inducible proteins and malonyl CoA levels by hypoxia.

<p>(A) Protein expression of hypoxia-inducible proteins, including HIF-1α, aryl hydrocarbon receptor nuclear transport (ARNT), and lactoferrin. (B) Schematic depicting regulation of malonyl CoA levels by acetyl CoA carboxylase (ACC) and malonyl CoA decarboxylase (MCD). Protein expression of ACC and MCD measured by Western blot with quantification determined using densitometry with background subtraction. The contrast and brightness were altered to 46% and -20%, respectively, uniformly throughout the ARNT blot in order to enable increased distinction of the band from the background. The unmodified, uncropped western blots are provided in the supplemental material. Malonyl CoA levels in HCFs and HKCs were measured by ELISA. All data was normalized to HCF-Normoxic control. n≥3, error bars represent SEM. An ANOVA was used to determine statistical significance with *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.</p

ECM thickness after 4 weeks at normoxic conditions or maintained for 3 weeks at normoxia and then transferred to a hypoxia environment (2% O₂) for 1 week.

<p>The distance from top to bottom cell layer was measured by confocal microscopy. n = 3, mean±S.E.M. Statistical significance determined by ANOVA, comparing all values to HCF-Normoxic controls.</p

Effects of hypoxia on MMP, collagen expression, and FAK signaling in HCFs and HKCs under normoxic or hypoxic (2% O₂) conditions.

<p>(A) Representative western blots and (B-F) quantification for MMP-9, MMP-2, MMP-1, MMP-13, and MMP-3 expression. (G) Representative western blots of cytosolic collagen and keratocan expression and quantification of (H-K) Collagens I, III, V, and keratocan expression. (L) Representative western blot of pFAK and FAK expression and (M) quantification of the ratio of pFAK/FAK. Quantification determined using densitometry. n = 3, statistical significance determined by an ANOVA with * = p≤0.05, ** = p≤0.01, *** = p≤0.001, and **** = p≤0.0001.</p