Dimitrios Karamichos, Ph.D.
Permanent URI for this communityhttps://hdl.handle.net/20.500.12503/31785
Executive Director and Endowed Chair, North Texas Eye Research Institute
Professor, Pharmaceutical Sciences
Professor, Pharmacology & Neuroscience
Email: Dimitrios.Karamichos@unthsc.edu
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Browsing Dimitrios Karamichos, Ph.D. by Subject "Animals"
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Item A 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.Item A 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.Item Ablation 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.Item Peroxisome 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.Item Sex 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.Item The 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.