ItemSex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia(BioMed Central Ltd., 2023-11-12) Mabry, Steve; Wilson, E. Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Fadeyibi, Oluwadarasimi; Vera, Edward, Jr.; Osikoya, Oluwatobiloba; Cushen, Spencer C.; Karamichos, Dimitrios; Goulopoulou, Styliani; Cunningham, Rebecca L.BACKGROUND: Gestational sleep apnea is a hypoxic sleep disorder that affects 8-26% of pregnancies and increases the risk for central nervous system dysfunction in offspring. Specifically, there are sex differences in the sensitivity of the fetal hippocampus to hypoxic insults, and hippocampal impairments are associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. Yet, it is unclear whether gestational sleep apnea impacts these hippocampal-associated functions and if sex and age modify these effects. To examine the relationship between gestational sleep apnea and hippocampal-associated behaviors, we used chronic intermittent hypoxia (CIH) to model late gestational sleep apnea in pregnant rats. We hypothesized that late gestational CIH would produce sex- and age-specific social, anxiety-like, repetitive, and cognitive impairments in offspring. METHODS: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine gestational hypoxia-induced behavioral phenotypes, we quantified hippocampal-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal neuronal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, early growth response protein 1, and doublecortin), and circulating hormones in offspring. RESULTS: Late gestational CIH induced sex- and age-specific differences in social, repetitive, and memory functions in offspring. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and elevated circulating corticosterone levels but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH on social behaviors were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed in anxiety-like behaviors, hippocampal neuronal activity, or circulating testosterone and estradiol levels, regardless of sex or age of offspring. CONCLUSIONS: Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for behavioral and physiological outcomes in offspring, such as social dysfunction, repetitive behaviors, and cognitive impairment, that are dependent on sex and age. Sleep apnea during late pregnancy is a common pregnancy complication that can impact the brain development of children born to mothers with sleep apnea. Children with impaired brain development may present with decreased social skills, memory issues, anxiety, and compulsivity. It is unclear if there is a cause and effect relationship between sleep apnea during late pregnancy and behavioral changes in offspring. Additionally, it is unknown whether male or female sex or age of the offspring affects these relationships. In this study, we exposed pregnant rats to a model of sleep apnea called chronic intermittent hypoxia (CIH) within late gestation and examined the behavior of the offspring and brain activity during puberty and young adulthood. We found that CIH during late pregnancy had long-term effects in the offspring that were different in males and females. Notably, female offspring displayed social impairments in response to late gestation CIH, whereas male offspring displayed cognitive dysfunction. ItemQuercetin and Related Analogs as Therapeutics to Promote Tissue Repair(MDPI, 2023-10-28) McKay, Tina B.; Emmitte, Kyle A.; German, Carrie; Karamichos, DimitriosQuercetin is a polyphenol of the flavonoid class of secondary metabolites that is widely distributed in the plant kingdom. Quercetin has been found to exhibit potent bioactivity in the areas of wound healing, neuroprotection, and anti-aging research. Naturally found in highly glycosylated forms, aglycone quercetin has low solubility in aqueous environments, which has heavily limited its clinical applications. To improve the stability and bioavailability of quercetin, efforts have been made to chemically modify quercetin and related flavonoids so as to improve aqueous solubility while retaining bioactivity. In this review, we provide an updated overview of the biological properties of quercetin and proposed mechanisms of actions in the context of wound healing and aging. We also provide a description of recent developments in synthetic approaches to improve the solubility and stability of quercetin and related analogs for therapeutic applications. Further research in these areas is expected to enable translational applications to improve ocular wound healing and tissue repair. ItemProspective Observational Study Evaluating Systemic Hormones and Corneal Crosslinking Effects in Keratoconus(Elsevier B.V., 2023-10-23) Van, Lyly; Bennett, Sashia; Nicholas, Sarah E.; Hjortdal, Jesper; McKay, Tina B.; Karamichos, DimitriosPURPOSE: To evaluate associations between hormone levels and corneal parameters in patients with keratoconus (KC), before and after photooxidative corneal collagen crosslinking (CXL). DESIGN: Prospective, observational cohort study. PARTICIPANTS: Twenty-eight patients with KC who were scheduled for CXL at Aarhus University Hospital in Denmark. METHODS: Androgen (dehydroepiandrosterone sulfate [DHEA-S]) and estrogen (estrone and estriol) plasma levels were measured and clinical assessments were performed before CXL and 2 to 3 months post-CXL, comparing the CXL eye with the control eye from the same participant. MAIN OUTCOME MEASURES: Associations between hormone levels and maximum corneal curvature (K(max)) and minimum central corneal thickness (CCt(min)) before and after CXL. RESULTS: Corneal collagen crosslinking was associated with a 2% reduction in K(max) values in the CXL eye, post-CXL, from baseline (median, 56.8 diopters [D]; 95% confidence interval [CI], 50.4-60.3) to the second visit (55.7 D; 95% CI, 50.4-58.8; P < 0.001). Systemic DHEA-S levels were 5 to 6 orders of magnitude higher than estriol or estrone concentrations in plasma. Importantly, estriol levels, rather than DHEA-S or estrone levels, were more closely correlated with K(max) before CXL (Spearman's r = 0.55, P = 0.01). Post-CXL K(max) and CCt(min) were not associated with DHEA-S, estrone, or estriol plasma levels at the same timepoint. CONCLUSIONS: This study provides supporting evidence based on a KC clinical population that systemic estrogen levels may influence corneal parameters (curvature and thickness) pre-CXL. Further studies evaluating the interplay between the therapeutic benefits of CXL and systemic hormone distributions are needed to determine if perturbation of the local corneal microenvironment influences endocrine function. FINANCIAL DISCLOSURES: The authors have no proprietary or commercial interest in any materials discussed in this article. ItemA Method for Real-Time Assessment of Mitochondrial Respiration Using Murine Corneal Biopsy(Association for Research in Vision and Ophthalmology, 2023-08-29) Liang, Wentao; Huang, Li; Yuan, Tian; Cheng, Rui; Takahashi, Yusuke; Moiseyev, Gennadiy P.; Karamichos, Dimitrios; Ma, Jian-XingPURPOSE: To develop and optimize a method to monitor real-time mitochondrial function by measuring the oxygen consumption rate (OCR) in murine corneal biopsy punches with a Seahorse extracellular flux analyzer. METHODS: Murine corneal biopsies were obtained using a biopsy punch immediately after euthanasia. The corneal metabolic profile was assessed using a Seahorse XFe96 pro analyzer, and mitochondrial respiration was analyzed with specific settings. RESULTS: Real-time adenosine triphosphate rate assay showed that mitochondrial oxidative phosphorylation is a major source of adenosine triphosphate production in ex vivo live murine corneal biopsies. Euthanasia methods (carbon dioxide asphyxiation vs. overdosing on anesthetic drugs) did not affect corneal OCR values. Mouse corneal biopsy punches in 1.5-mm diameter generated higher and more reproducible OCR values than those in 1.0-mm diameter. The biopsy punches from the central and off-central cornea did not show significant differences in OCR values. There was no difference in OCR reading by the tissue orientations (the epithelium side up vs. the endothelium side up). No significant differences were found in corneal OCR levels between sexes, strains (C57BL/6J vs. BALB/cJ), or ages (4, 8, and 32 weeks). Using this method, we showed that the wound healing process in the mouse cornea affected mitochondrial activity. CONCLUSIONS: The present study validated a new strategy to measure real-time mitochondrial function in fresh mouse corneal tissues. This procedure should be helpful for studies of the ex vivo live corneal metabolism in response to genetic manipulations, disease conditions, or pharmacological treatments in mouse models. ItemNovel Correlation between TGF-beta1/-beta3 and Hormone Receptors in the Human Corneal Stroma(MDPI, 2023-09-09) Choi, Alexander J.; Hefley, Brenna S.; Nicholas, Sarah E.; Cunningham, Rebecca L.; Karamichos, DimitriosThis study investigated the interplay between transforming growth factor beta (TGF-beta1/T1 and TGF-beta3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 10(6) cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERalpha) and beta (ERbeta), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERalpha, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERalpha, ERbeta, and GnRHR. T3 caused significant upregulation in expressions PR, ERalpha, ERbeta, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERalpha, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-beta isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms. ItemCollagen Crosslinking for Keratoconus: Cellular Signaling Mechanisms(MDPI, 2023-05-16) Karamichos, Dimitrios; Nicholas, Sarah E.; Khan, Asher; Riaz, Kamran M.Collagen crosslinking (CXL) is a widely used treatment to halt the progression of keratoconus (KC). Unfortunately, a significant number of patients with progressive KC will not qualify for CXL, including those with corneas thinner than 400 microm. The present study aimed to investigate the molecular effects of CXL using in vitro models, mirroring the normal, as well as thinner corneal stroma seen in KCs. Primary human corneal stromal cells were isolated from healthy (HCFs) and keratoconus (HKCs) donors. Cells were cultured and stimulated with stable Vitamin C resulting in 3D self-assembled extracellular matrix (ECM), cell-embedded, constructs. CXL was performed on (a) thin ECM with CXL performed at week 2 and (b) normal ECM with CXL performed at week 4. Constructs without CXL served as controls. All constructs were processed for protein analysis. The results showed modulation of Wnt signaling, following CXL treatment, as measured by the protein levels of Wnt7b and Wnt10a, correlated to the expression of alpha-smooth muscle actin (SMA). Further, the expression of a recently identified KC biomarker candidate, prolactin-induced protein (PIP), was positively impacted by CXL in HKCs. CXL-driven upregulation of PGC-1 and the downregulation of SRC and Cyclin D1 in HKCs were also noted. Although the cellular/molecular impacts of CXL are largely understudied, our studies provide an approximation to the complex mechanisms of KC and CXL. Further studies are warranted to determine factors influencing CXL outcomes. ItemUnravelling the Impact of Cyclic Mechanical Stretch in Keratoconus-A Transcriptomic Profiling Study(MDPI, 2023-04-28) Akoto, Theresa; Cai, Jingwen; Nicholas, Sarah; McCord, Hayden; Estes, Amy J.; Xu, Hongyan; Karamichos, Dimitrios; Liu, YutaoBiomechanical and molecular stresses may contribute to the pathogenesis of keratoconus (KC). We aimed to profile the transcriptomic changes in healthy primary human corneal (HCF) and KC-derived cells (HKC) combined with TGFbeta1 treatment and cyclic mechanical stretch (CMS), mimicking the pathophysiological condition in KC. HCFs (n = 4) and HKCs (n = 4) were cultured in flexible-bottom collagen-coated 6-well plates treated with 0, 5, and 10 ng/mL of TGFbeta1 with or without 15% CMS (1 cycle/s, 24 h) using a computer-controlled Flexcell FX-6000T Tension system. We used stranded total RNA-Seq to profile expression changes in 48 HCF/HKC samples (100 bp PE, 70-90 million reads per sample), followed by bioinformatics analysis using an established pipeline with Partek Flow software. A multi-factor ANOVA model, including KC, TGFbeta1 treatment, and CMS, was used to identify differentially expressed genes (DEGs, |fold change| >/= 1.5, FDR /= 10 in >/=1 sample) in HKCs (n = 24) vs. HCFs (n = 24) and those responsive to TGFbeta1 and/or CMS. PANTHER classification system and the DAVID bioinformatics resources were used to identify significantly enriched pathways (FDR ItemSalivary Exosomes in Health and Disease: Future Prospects in the Eye(MDPI, 2023-04-14) Liu, Angela; Hefley, Brenna; Escandon, Paulina; Nicholas, Sarah E.; Karamichos, DimitriosExosomes are a group of vesicles that package and transport DNA, RNA, proteins, and lipids to recipient cells. They can be derived from blood, saliva, urine, and/or other biological tissues. Their impact on several diseases, such as neurodegenerative, autoimmune, and ocular diseases, have been reported, but not fully unraveled. The exosomes that are derived from saliva are less studied, but offer significant advantages over exosomes from other sources, due to their accessibility and ease of collection. Thus, their role in the pathophysiology of diseases is largely unknown. In the context of ocular diseases, salivary exosomes have been under-utilized, thus creating an enormous gap in the literature. The current review discusses the state of exosomes research on systemic and ocular diseases and highlights the role and potential of salivary exosomes as future ocular therapeutic vehicles. ItemPeroxisome proliferator-activated receptor-alpha (PPARalpha) regulates wound healing and mitochondrial metabolism in the cornea(National Academy of Science, 2023-03-22) Liang, Wentao; Huang, Li; Whelchel, Amy E.; Yuan, Tian; Ma, Xiang; Cheng, Rui; Takahashi, Yusuke; Karamichos, Dimitrios; Ma, Jian-XingDiabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-alpha (PPARalpha) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARalpha knockout mice and corneal epithelium-specific PPARalpha conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARalpha agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARalpha transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARalpha agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARalpha, suggesting a PPARalpha-dependent mechanism. These findings suggest that downregulation of PPARalpha plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes. ItemThe Underlying Relationship between Keratoconus and Down Syndrome(MDPI, 2022-09-24) Akoto, Theresa; Li, Jiemin J.; Estes, Amy J.; Karamichos, Dimitrios; Liu, YutaoKeratoconus (KC) is one of the most significant corneal disorders worldwide, characterized by the progressive thinning and cone-shaped protrusion of the cornea, which can lead to severe visual impairment. The prevalence of KC varies greatly by ethnic groups and geographic regions and has been observed to be higher in recent years. Although studies reveal a possible link between KC and genetics, hormonal disturbances, environmental factors, and specific comorbidities such as Down Syndrome (DS), the exact cause of KC remains unknown. The incidence of KC ranges from 0% to 71% in DS patients, implying that as the worldwide population of DS patients grows, the number of KC patients may continue to rise significantly. As a result, this review aims to shed more light on the underlying relationship between KC and DS by examining the genetics relating to the cornea, central corneal thickness (CCT), and mechanical forces on the cornea, such as vigorous eye rubbing. Furthermore, this review discusses KC diagnostic and treatment strategies that may help detect KC in DS patients, as well as the available DS mouse models that could be used in modeling KC in DS patients. In summary, this review will provide improved clinical knowledge of KC in DS patients and promote additional KC-related research in these patients to enhance their eyesight and provide suitable treatment targets. ItemAblation of Sphingosine Kinase 1 Protects Cornea from Neovascularization in a Mouse Corneal Injury Model(MDPI, 2022-09-24) Wilkerson, Joseph L.; Basu, Sandip K.; Stiles, Megan A.; Prislovsky, Amanda; Grambergs, Richard C.; Nicholas, Sarah E.; Karamichos, Dimitrios; Allegood, Jeremy C.; Proia, Richard L.; Mandal, NawajesThe purpose of this study was to investigate the role of sphingosine kinase 1 (SphK1), which generates sphingosine-1-phosphate (S1P), in corneal neovascularization (NV). Wild-type (WT) and Sphk1 knockout (Sphk1(-/-)) mice received corneal alkali-burn treatment to induce corneal NV by placing a 2 mm round piece of Whatman No. 1 filter paper soaked in 1N NaOH on the center of the cornea for 20 s. Corneal sphingolipid species were extracted and identified using liquid chromatography/mass spectrometry (LC/MS). The total number of tip cells and those positive for ethynyl deoxy uridine (EdU) were quantified. Immunocytochemistry was done to examine whether pericytes were present on newly forming blood vessels. Cytokine signaling and angiogenic markers were compared between the two groups using multiplex assays. Data were analyzed using appropriate statistical tests. Here, we show that ablation of SphK1 can significantly reduce NV invasion in the cornea following injury. Corneal sphingolipid analysis showed that total levels of ceramides, monohexosyl ceramides (HexCer), and sphingomyelin were significantly elevated in Sphk(-/-) corneas compared to WT corneas, with a comparable level of sphingosine among the two genotypes. The numbers of total and proliferating endothelial tip cells were also lower in the Sphk1(-/-) corneas following injury. This study underscores the role of S1P in post-injury corneal NV and raises further questions about the roles played by ceramide, HexCer, and sphingomyelin in regulating corneal NV. Further studies are needed to unravel the role played by bioactive sphingolipids in maintenance of corneal transparency and clear vision. ItemLatest in Cellular Pathology Research(MDPI, 2022-12-24) Karamichos, DimitriosThe year 2021 marked the 10th anniversary of the publication of Cells. To celebrate this milestone, a Special Issue entitled "10th Anniversary of Cells—Advances in Cellular Pathology" was launched. The goal of this Special Issue was the collection of impactful research/review articles in the Cellular Pathology field. The final roster of published articles for the Special Issue is an incredible collection of research articles and reviews, covering topics from cancer, diabetes, and ocular manifestations, all of which are truly consequential to the quality of life. ItemTreatment of Non-Infectious Corneal Injury: Review of Diagnostic Agents, Therapeutic Medications, and Future Targets(Springer Nature, 2022-01-13) Dang, Deanna H.; Riaz, Kamran M.; Karamichos, DimitriosCorneal injuries can occur secondary to traumatic, chemical, inflammatory, metabolic, autoimmune, and iatrogenic causes. Ocular infection may frequently occur concurrent to corneal injury; however, antimicrobial agents are excluded from this present review. While practitioners may primarily rely on clinical examination techniques to assess these injuries, several pharmacological agents, such as fluorescein, lissamine green, and rose bengal, can be used to formulate a diagnosis and develop effective treatment strategies. Practitioners may choose from several analgesic medications to help with patient comfort without risking further injury or delaying ocular healing. Atropine, cyclopentolate, scopolamine, and homatropine are among the most frequently used medications for this purpose. Additional topical analgesic agents may be used judiciously to augment patient comfort to facilitate diagnosis. Steroidal anti-inflammatory agents are frequently used as part of the therapeutic regimen. A variety of commonly used agents, including prednisolone acetate, loteprednol, difluprednate, dexamethasone, fluorometholone, and methylprednisolone are discussed. While these medications are effective for controlling ocular inflammation, side effects, such as elevated intraocular pressure and cataract formation, must be monitored by clinicians. Non-steroidal medications, such as ketorolac, bromfenac, nepafenac, and diclofenac, are additionally used for their efficacy in controlling ocular inflammation without incurring side effects seen with steroids. However, these agents have their own respective side effects, warranting close monitoring by clinicians. Additionally, ophthalmologists routinely employ several agents in an off-label manner for supplementary control of inflammation and treatment of corneal injuries. Patients with corneal injuries not infrequently have significant ocular surface disease, either as a concurrent pathology or as an exacerbation of previously existing disease. Several agents used in the management of ocular surface disease have also been found to be useful as part of the therapeutic armamentarium for treatment of corneal injuries. For example, several antibiotics, such as doxycycline and macrolides, have been used for their anti-inflammatory effects on specific cytokines that are upregulated during acute injuries. There has been a recent wave of interest in amniotic membrane therapies (AMTs), including topical, cryopreserved and dehydrated variants. AMT is particularly effective in ocular injuries with violation of corneal surface integrity due to its ability to promote re-epithelialization of the corneal epithelium. Blood-based therapies, including autologous serum tears, plasma-enriched growth factor eyedrops and autologous blood drops, have additionally been explored in small case series for effectiveness in challenging and recalcitrant cases. Protection of the ocular surface is also a vital component in the treatment of corneal injuries. Temporary protective methods, such as bandage contact lenses and mechanical closure of the eyelids (tarsorrhaphy) can be particularly helpful in selective cases. Glue therapies, including biologic and non-biologic variants, can also be used in cases of severe injury and risk of corneal perforation. Finally, there are a variety of recently introduced and in-development agents that may be used as adjuvant therapies in challenging patient populations. Neurotrophic corneal disease may occur as a result of severe or chronic injury. In such cases, recombinant human nerve growth factor (cenegermin), topical insulin, and several other novel agents may be an alternate and effective option for clinicians to consider. ItemUnravelling Novel Roles of Salivary Exosomes in the Regulation of Human Corneal Stromal Cell Migration and Wound Healing(MDPI, 2022-04-14) Escandon, Paulina; Liu, Angela; Nicholas, Sarah E.; Khan, Asher; Riaz, Kamran M.; Karamichos, DimitriosSalivary exosomes have demonstrated vast therapeutic and diagnostic potential in numerous diseases. This study pioneers previously unexplored roles of SE in the context of corneal wound healing by utilizing primary corneal stromal cells from healthy (HCFs), type I diabetes mellitus (T1DMs), type II DM (T2DMs), and keratoconus (HKCs) subjects. Purified, healthy human SEs carrying tetraspanins CD9+, CD63+, and CD81+ were utilized. Scratch and cell migration assays were performed after 0, 6, 12, 24, and 48 h following SE stimulation (5 and 25 microg/mL). Significantly slower wound closure was observed at 6 and 12 h in HCFs with 5 mug/mL SE and T1DMs with 5 and 25 mug/mL SE. All wounds were closed by 24-hour, post-wounding. HKCs, T1DMs, and T2DMs with 25microg/mL SE exhibited a significant upregulation of cleaved vimentin compared to controls. Thrombospondin 1 was significantly upregulated in HCFs, HKCs, and T2DMs with 25 microg/mL SE. Lastly, HKCs, T1DMs, and T2DMs exhibited a significant downregulation of fibronectin with 25 mug/mL SE. Whether SEs can be utilized to clinical settings in restoring corneal defects is unknown. This is the first-ever study exploring the role of SEs in corneal wound healing. While the sample size was small, results are highly novel and provide a strong foundation for future studies. ItemFAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro(MDPI, 2021-11-12) Yeung, Vincent; Sriram, Sriniwas; Tran, Jennifer A.; Guo, Xiaoqing; Hutcheon, Audrey E. K.; Zieske, James D.; Karamichos, Dimitrios; Ciolino, Joseph B.Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-beta1 and -beta3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of alpha-smooth muscle actin (alphaSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-beta1, TGF-beta3, or TGF-beta1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-beta1 or TGF-beta3 impart distinct effects on genes involved in wound healing and fibrosis-ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-beta1 enhanced alphaSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-beta3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-beta1 + FAKi attenuated TGF-beta1-mediated myofibroblast differentiation, as shown by abrogated alphaSMA expression. This study concludes that FAK signaling is important for the onset of TGF-beta1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring. ItemThe Role of Estriol and Estrone in Keratoconic Stromal Sex Hormone Receptors(MDPI, 2022-01-14) Escandon, Paulina; Nicholas, Sarah E.; Cunningham, Rebecca L.; Murphy, David A.; Riaz, Kamran M.; Karamichos, DimitriosKeratoconus (KC) is a progressive corneal thinning disease that manifests in puberty and worsens during pregnancy. KC onset and progression are attributed to diverse factors that include: environmental, genetics, and hormonal imbalances; however, the pathobiology remains elusive. This study aims to determine the role of corneal stroma sex hormone receptors in KC and their interplay with estrone (E1) and estriol (E3) using our established 3D in vitro model. Healthy cornea stromal cells (HCFs) and KC cornea stromal cells (HKCs), both male and female, were stimulated with various concentrations of E1 and E3. Significant changes were observed between cell types, as well as between males and females in the sex hormone receptors tested; androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERalpha), and estrogen receptor beta (ERbeta) using Western blot analysis. E1 and E3 stimulations in HCF females showed AR, PR, and ERbeta were significantly upregulated compared to HCF males. In contrast, ERalpha and ERbeta had significantly higher expression in HKC's females than HKC's males. Our data suggest that the human cornea is a sex-dependent, hormone-responsive tissue that is significantly influenced by E1 and E3. Therefore, it is plausible that E1, E3, and sex hormone receptors are involved in the KC pathobiology, warranting further investigation. ItemQuercetin Decreases Corneal Haze In Vivo and Influences Gene Expression of TGF-Beta Mediators In Vitro(MDPI, 2022-07-07) McKay, Tina B.; Kivanany, Pourisika B.; Nicholas, Sarah E.; Nag, Okhil K.; Elliott, Michael H.; Petroll, W. Matthew; Karamichos, DimitriosWe have previously reported the flavonoid, quercetin, as a metabolic regulator and inhibitor of myofibroblast differentiation in vitro. Our current study evaluated the effects of topical application of quercetin on corneal scar development using two different animal models followed by RNA analysis in vitro. Wild-type C57BL/6J mice were anesthetized and the corneal epithelium and stroma were manually debrided, followed by quercetin (0.5, 1, 5, or 50 mM) or vehicle application. Corneal scarring was assessed for 3 weeks by slit lamp imaging and clinically scored. In a separate animal study, six New Zealand White rabbits underwent lamellar keratectomy surgery, followed by treatment with 5 mM quercetin or vehicle twice daily for three days. Stromal backscattering was assessed at week 3 by in vivo confocal microscopy. In mice, a single dose of 5 mM quercetin reduced corneal scar formation. In rabbits, stromal backscattering was substantially lower in two out of three animals in the quercetin-treated group. In vitro studies of human corneal fibroblasts showed that quercetin modulated select factors of the transforming growth factor-beta (TGF-beta) signaling pathway. These results provide evidence that quercetin may inhibit corneal scarring. Further studies in a larger cohort are required to validate the efficacy and safety of quercetin for clinical applications. ItemSex Hormones, Growth Hormone, and the Cornea(MDPI, 2022-01-11) McKay, Tina B.; Priyadarsini, Shrestha; Karamichos, DimitriosThe 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. ItemA novel 3D culture model of fungal keratitis to explore host-pathogen interactions within the stromal environment(Elsevier Ltd., 2021-04-15) Brown, Marina E.; Montgomery, Micaela L.; Kamath, Manali M.; Nicholas, Sarah; Liu, Yutao; Karamichos, Dimitrios; Fuller, Kevin K.Fungal keratitis (FK) pathology is driven by both fungal growth and inflammation within the corneal stroma. Standard in vitro infection models involving co-culture of the pathogen and the corneal cells in tissue culture medium are sufficient to probe host responses to the fungus; however, they lack the physiological structure and nutrient composition of the stroma to accurately study fungal invasiveness and metabolic processes. We therefore sought to develop a culture model of FK that would allow for both host and fungal cell biology to be evaluated in parallel. Towards this end, we employed a previously described system in which primary human cornea fibroblasts (HCFs) are cultured on transwell membranes, whereupon they secrete a three-dimensional (3D) collagen matrix that resembles the human stroma. We demonstrated that two common mold agents of FK, Fusarium petroliphilum and Aspergillus fumigatus, penetrated into these constructs and caused a disruption of the collagen matrix that is characteristic of infection. HCF morphology appeared altered in the presence of fungus and electron microscopy revealed a clear internalization of fungal spores into these cells. Consistent with this apparent phagocyte-like activity of the HCFs, mRNA and protein levels for several pro-inflammatory cytokines/chemokines (including TNFalpha, IL-1beta, IL-6, and IL-8) were significantly upregulated compared to uninfected samples. We similarly found an upregulation of several HCF metalloproteases (MMPs), which are enzymes that breakdown collagen during wound healing and may further activate pro-inflammatory signaling molecules. Finally, several fungal collagenase genes were upregulated during growth in the constructs relative to growth in tissue culture media alone, suggesting a fungal metabolic shift towards protein catabolism. Taken together, our results indicate that this 3D-stromal model provides a physiologically relevant system to study host and fungal cell pathobiology during FK. ItemNerve influence on the metabolism of type I and type II diabetic corneal stroma: an in vitro study(Springer Nature, 2021-07-01) Whelchel, Amy E.; Nicholas, Sarah E.; Ma, Jian-Xing; Karamichos, DimitriosCorneal innervation plays a major role in the pathobiology of diabetic corneal disease. However, innervation impact has mainly been investigated in the context of diabetic epitheliopathy and wound healing. Further studies are warranted in the corneal stroma-nerve interactions. This study unravels the nerve influence on corneal stroma metabolism. Corneal stromal cells were isolated from healthy (HCFs) and diabetes mellitus (Type1DM and Type2 DM) donors. Cells were cultured on polycarbonate membranes, stimulated by stable Vitamin C, and stroma-only and stroma-nerve co-cultures were investigated for metabolic alterations. Innervated compared to stroma-only constructs exhibited significant alterations in pyrimidine, glycerol phosphate shuttle, electron transport chain and glycolysis. The most highly altered metabolites between healthy and T1DMs innervated were phosphatidylethanolamine biosynthesis, and pyrimidine, methionine, aspartate metabolism. Healthy and T2DMs main pathways included aspartate, glycerol phosphate shuttle, electron transport chain, and gluconeogenesis. The metabolic impact on T1DMs and T2DMs was pyrimidine, purine, aspartate, and methionine. Interestingly, the glucose-6-phosphate and oxaloacetate was higher in T2DMs compared to T1DMs. Our in vitro co-culture model allows the examination of key metabolic pathways corresponding to corneal innervation in the diabetic stroma. These novel findings can pave the way for future studies to fully understand the metabolic distinctions in the diabetic cornea.