Dimitrios Karamichos

In vitro model suggests oxidative stress involved in keratoconus disease

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

Novel in Vitro Model for Keratoconus Disease

Keratoconus is a disease where the cornea becomes cone-like due to structural thinning and ultimately leads to compromised corneal integrity and loss of vision. Currently, the therapeutic options are corrective lenses for early stages and surgery for advanced cases with no in vitro model available. In this study, we used human corneal fibroblasts (HCFs) and compared them to human Keratoconus fibroblasts (HKCs) cultured in a 3-dimensional (3D) model, in order to compare the expression and secretion of specific extracellular matrix (ECM) components. For four weeks, the cells were stimulated with a stable Vitamin C (VitC) derivative ± TGF-β1 or TGF-β3 (T1 and T3, respectively). After four weeks, HKCs stimulated with T1 and T3 were significantly thicker compared with Control (VitC only); however, HCF constructs were significantly thicker than HKCs under all conditions. Both cell types secreted copious amounts of type I and V collagens in their assembled, aligned collagen fibrils, which increased in the degree of alignment upon T3 stimulation. In contrast, only HKCs expressed high levels of corneal scarring markers, such as type III collagen, which was dramatically reduced with T3. HKCs expressed α-smooth muscle actin (SMA) under all conditions in contrast to HCFs, where T3 minimized SMA expression. Fast Fourier transform (FFT) data indicated that HKCs were more aligned when compared to HCFs, independent of treatments; however, HKC’s ECM showed the least degree of rotation. HKCs also secreted the most aligned type I collagen under T3 treatment, when compared to any condition and cell type. Overall, our model for Keratoconus disease studies is the first 3D in vitro tissue engineered model that can mimic the Keratoconus disease in vivo and may be a breakthrough in efforts to understand the progression of this disease.

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.

Quercetin Attenuates Lactate Production and Extracellular Matrix Secretion in Keratoconus

Keratoconus(KC) is an ecstatic corneal disease leading to corneal-thinning and the formation of a cone-like cornea. Elevated lactate levels, increased oxidative stress, and myofibroblast formation have all been previously reported. In the current study, we assess the role of Quercetin on collagen secretion and myofibroblast formation in KC in vitro. Human corneal fibroblasts(HCFs) and human keratoconus cells(HKCs) were treated with a stable Vitamin C derivative and cultured for 4 weeks, stimulating formation of a self-assembled extracellular matrix. All samples were analyzed using Western blots and targeted tandem mass spectrometry. Our data showed that Quercetin significantly down regulates myofibroblast differentiation and fibrotic markers, such as α-smooth muscle actin (α-SMA) and Collagen III (Col III), in both HCFs and HKCs. Collagen III secretion was reduced 80% in both HCFs and HKCs following Quercetin treatment. Furthermore, Quercetin reduced lactate production by HKCs to normal HCF levels. Quercetin down regulated TGF-βR2 and TGF-β2 expression in HKCs suggesting a significant link to the TGF-β pathway. These results assert that Quercetin is a key regulator of fibrotic markers and ECM assembly by modulating cellular metabolism and TGF-β signaling. Our study suggests that Quercetin is a potential therapeutic for treatment of corneal dystrophies, such as KC.

A Systematic Review of the Role of Dysfunctional Wound Healing in the Pathogenesis and Treatment of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disorder showcasing an interaction between genetic predisposition and environmental risks. This usually involves the coaction of a mixture of cell types associated with abnormal wound healing, leading to structural distortion and loss of gas exchange function. IPF bears fatal prognosis due to respiratory failure, revealing a median survival of approximately 2 to 3 years. This review showcases the ongoing progress in understanding the complex pathophysiology of IPF and it highlights the latest potential clinical treatments. In IPF, various components of the immune system, particularly clotting cascade and shortened telomeres, are highly involved in disease pathobiology and progression. This review also illustrates two US Food and Drug Administration (FDA)-approved drugs, nintedanib (OFEV, Boehringer Ingelheim, Ingelheim am Rhein, Germany) and pirfenidone (Esbriet, Roche, Basel, Switzerland), that slow IPF progression, but unfortunately neither drug can reverse the course of the disease. Although the mechanisms underlying IPF remain poorly understood, this review unveils the past and current advances that encourage the detection of new IPF pathogenic pathways and the development of effective treatment methods for the near future.

Endocrine and Metabolic Pathways Linked to Keratoconus: Implications for the Role of Hormones in the Stromal Microenvironment

Hormones play a critical role in regulating tissue function by promoting cell survival, proliferation, and differentiation. Our study explores the influence of endocrine function in regulating metabolism and inflammatory pathways in Keratoconus (KC), which is a corneal thinning disease associated with reduced stromal deposition. KC is known to be a multifactorial disease with an elusive pathogenesis. We utilized a cross-sectional study analyzing clinical features and saliva samples from sixty-four KC patients and fourteen healthy controls. In order to determine if endocrine function varied between healthy controls and KC, we measured hormone levels in saliva and found significantly increased dehydroepiandrosterone sulfate (DHEA-S) and reduced estrone levels in KC patients compared to healthy controls. We measured significant variations in metabolites associated with pro-inflammatory processes, including myoinositol and 1-methyl-histidine, by targeted mass spectrometry. We also measured significantly increased IL-16 and stem cell factor in KC saliva samples compared to healthy controls, with higher expression of these pro-inflammatory proteins correlating with increased KC clinical grade, corneal curvature, and stromal thinning. Our results identify a novel mechanism linking KC and pro-inflammatory markers and suggest that altered hormone levels modulate metabolism, cytokine, and growth factor expression leading to increased severity of the KC condition.

Complete metabolome and lipidome analysis reveals novel biomarkers in the human diabetic corneal stroma

Prolonged hyperglycemia during diabetes mellitus can cause severe ophthalmic complications affecting both the anterior and posterior ocular segments leading to impaired vision or blindness. Diabetes-induced corneal pathologies are associated with decreased wound healing capacity, corneal edema, and altered epithelial basement membrane. The mechanism by which diabetes modulates structure and function within the corneal stroma are unknown. In our study, we characterized the effects of diabetes on extracellular matrix, lipid transport, and cellular metabolism by defining the entire metabolome and lipidome of Type 1 and Type 2 human diabetic corneal stroma. Significant increases in Collagen I and III were found in diabetic corneas suggesting that diabetes promotes defects in matrix structure leading to scarring. Furthermore, increased lipid content, including sphingosine-1-phosphate and dihydrosphingosine, in diabetic corneas compared to healthy controls were measured suggesting altered lipid retention. Metabolomics analysis identified elevated tryptophan metabolites, independent of glucose metabolism, which correlated with upregulation of the Kynurenine pathway in diabetic corneas. We also found significant upregulation of novel biomarkers aminoadipic acid, D,L-pipecolic acid, and dihydroorotate. Our study links aberrant tryptophan metabolism to end-stage pathologies associated with diabetes indicating the potential of the Kynurenine pathway as a therapeutic target for inhibiting diabetes-associated defects in the eye.

Description of the Sphingolipid Content and Subspecies in the Diabetic Cornea

Abstract Purpose: Diabetes mellitus (DM) is characterized by high blood sugar levels over a prolonged period. Long term complications include but not limited heart disease, stroke, kidney failure, and ocular damage. An estimated 382 million people are diagnosed with Type 2 DM accounting for 90% of the cases. Common corneal dysfunctions associated with DM result in impaired vision due to decreased wound healing, corneal edema, and altered epithelial basement membrane. Lipids play a fundamental role in tissue metabolism and disease states. We attempt to determine the role of sphingolipids (SPL) in human Type I and Type II diabetic corneas. Materials and Methods: Cadaver corneas from healthy (non-diabetic/no ocular trauma), Type I (T1DM), and Type II diabetic (T2DM) donors were obtained and processed for lipidomics using LC-MS/MS. Results: Our data show significant differences in the SPL composition between control, T1DM and T2DM corneas. Both T1DM and T2DM showed a 10-folddownregulation of sphingomyelin(SM), 5-fold up regulation of Ceramides (Cer) and 2-fold upregulation of monohexosylceramides (MHC). Differences were also seen in total amounts of SPL where Cer was increased by approximately 3 fold in both T1DM and T2DM where SM decreased by 50% in both T1DM and T2DM when compared to healthy controls. No differences were seen in MHC amounts. Conclusions: Overall, our data indicate major differences in SPL distribution in human diabetic corneas. Information on the sphingolipids role in cornea, corneal cell physiology, and diseases are very limitedwhich highlights the importance of these findings.

Keratoconus: Tissue Engineering and Biomaterials

Keratoconus (KC) is a bilateral, asymmetric, corneal disorder that is characterized by progressive thinning, steepening, and potential scarring. The prevalence of KC is stated to be 1 in 2000 persons worldwide; however, numbers vary depending on size of the study and regions. KC appears more often in South Asian, Eastern Mediterranean, and North African populations. The cause remains unknown, although a variety of factors have been considered. Genetics, cellular, and mechanical changes have all been reported; however, most of these studies have proven inconclusive. Clearly, the major problem here, like with any other ocular disease, is quality of life and the threat of vision loss. While most KC cases progress until the third or fourth decade, it varies between individuals. Patients may experience periods of several months with significant changes followed by months or years of no change, followed by another period of rapid changes. Despite the major advancements, it is still uncertain how to treat KC at early stages and prevent vision impairment. There are currently limited tissue engineering techniques and/or “smart” biomaterials that can help arrest the progression of KC. This review will focus on current treatments and how biomaterials may hold promise for the future.

Acute hypoxia influences collagen and matrix metalloproteinase expression by human keratoconus cells in vitro.

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 in vitro and ex vivo. 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) in vitro. 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.