Browsing by Author "McNew, James A"

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    A novel paradigm for non-fibrotic regeneration of the cornea: The role of TGF-beta superfamily during embryonic cornea regeneration
    (2015-12-16) Spurlin, James W; Farach-Carson, Mary C; Grande-Allen, Jane; Lwigale, Peter Y; McNew, James A; Stern, Micheal
    Damage to the cornea results in fibrotic scarring, leading to the loss of tissue transparency and reduced visual acuity. In fact, corneal opacity is the world’s third leading cause of blindness. Other than transplantation of the affected tissue, there is no treatment to prevent corneal scarring. For these reasons, there is a need to develop anti-fibrotic therapies to promote corneal regeneration after injury. Embryonic tissue has a remarkable regenerative capacity. However, prior to this study, it was not known if the embryonic cornea possessed the ability to regenerate. I hypothesized wounded embryonic corneas wound exhibit non-fibrotic regeneration, and could be used to elucidate novel mechanisms of cornea regeneration. I developed a multistep microdissection method that allows access to the embryonic cornea and several other tissues undergoing organogenesis. Utilizing this methodology, I found embryonic corneal wounds induce a transient population of scar-forming myofibroblast, and ultimately regenerate scar-free. Immunohistological analysis of wounded embryonic corneas revealed transient change in expression of ECM components, which is restored to normal levels in the healed corneas. Furthermore, I showed that Sema3A mRNA is elevated and innervation of wounded embryonic corneas is inhibited during healing, but regenerated corneas are fully innervated. These findings contribute to the understanding of the events that orchestrate scar-free regeneration of wounded corneas. Since embryonic corneas possess an intrinsic regenerative capacity, the embryonic wound healing model serves as a great tool to study regulatory mechanisms that facilitate non-fibrotic healing. Because scar associated myofibroblasts are inherently transient in the embryonic cornea wound, I sought to determine mechanistic regulation of this cell population during cornea regeneration. I hypothesized the embryonic cornea wound would exhibit unique regulation of myofibroblast inductive growth factor, TGF-beta, during regeneration. Through studying gene expression profiles in the embryonic cornea wound healing model, I determined the spatiotemporal distribution of TGF-beta transcripts and the subsequent activation of the myofibroblast population. Moreover, I identified the expression of candidate TGF-beta antagonists when myofibroblasts are found to exit the regenerating cornea. My data shows BMP3 as a novel antagonist to TGF-beta mediated myofibroblast differentiation in isolated embryonic corneal cells. Interestingly, TGF-beta mediated accumulation of focal adhesion appears to be attenuated by BMP3, implicating the role of cellular adhesion in promoting the myofibroblast phenotype. Collectively, this work demonstrates the utility of the embryonic cornea wound healing model to identify novel mechanisms of scar-free cornea regeneration. Additionally, this novel mechanism of BMP3 antagonism on TGF-beta mediated fibrotic response suggests targeting aspects of cellular adhesion signaling may provide viable therapeutics to mitigate corneal fibrosis.
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    Biochemical Analysis of Atlastin’s Membrane Anchor: Morphology, Dynamics, and Function
    (2018-12-18) Betancourt, Miguel A; McNew, James A
    This project sheds new light into the endoplasmic reticulum (ER) fusion protein, atlastin. We studied its membrane anchor and its interaction with the lipid bilayer. The endoplasmic reticulum is composed flattened sheets and interconnected tubules that extend throughout the cytosol and contact other organelles. These discrete ER morphologies require specialized proteins that drive membrane curvature, dynamics, and mediate their maintenance. The GTPase atlastin is required for homotypic fusion of ER tubules. All atlastin homologs possess a conserved domain architecture consisting of a GTPase domain, a three-helix bundle middle domain, a hydrophobic membrane anchor, and a C-terminal tail. We analyzed atlastin’s hydrophobic anchor with recombinant atlastin and different mutants reconstituted into preformed liposomes, as model membranes. While traditionally atlastin’s membrane anchor was assumed to be two transmembrane segments that fully span the lipid bilayer; we have found it consists of two intramembranous hairpin loops. The topology of these hairpins remains static during membrane fusion and do not appear to play an active role in lipid mixing. We also analyzed the membrane domain topology of the mitochondrial fusion protein mitofusin-1 and ER resident protein Sac1 and found that they also have a dual intramembranous hairpin membrane anchor. This points to a conserved topology that may be expanded to ER resident protein and atlastin homologs. We were also able to recapitulate an ER-like network with atlastin proteoliposomes in polylysine coated coverslips; thus, showing that atlastin’s can form and maintain tubular structures, this result is consistent with the dual hairpin intramembrane loop topology. We also determined that co-reconstitution of atlastin with reticulon, an ER tube-forming protein, did not influence GTPase activity or membrane fusion, however both have the propensity to inhabit high curvature membranes. We also analyzed atlastin’s GTP binding pocket and found that inter- and intra-molecular salt bridging is important in GTP hydrolysis.
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    Regulation of the neurovascular patterning by growth factors and cytokines during anterior ocular development
    (2015-12-16) Ojeda Cardenas, Ana; Bennett, George N; Jacot, Jeffrey G; McNew, James A; Tao, Yizhi J
    The cornea is a transparent, avascular, and one of the most innervated tissue of the body. Corneal diseases including injuries, neovascularization, congenital eye defects and degenerations, represent a major public health burden. Although, studies have been focused on understanding the basis of transparency, innervation, and neovascularization of the adult cornea, little is known about the molecular mechanisms that lead to this specialized structure results in a highly innervated but avascular tissue during embryogenesis. The purpose of this work was to identify molecular regulators of the neurovascular patterning during cornea development. First, Sema3A, a well-known chemorepulser of axons, was identified as a key modulator in the establishment of cornea avascularity in both, avian and murine models. Moreover, I demonstrated that chemokines, initially described for their function in controlling immune cell migration, also play an important role in axon guidance and vasculogenesis during ocular development. Examination of the expression of the chemokine CXCL14 by in situ hybridization and immunohistochemistry revealed novel patterns of localization in the corneal stroma, iris, lens epithelium, retina and trigeminal ganglion. Comparison in the expression of CXCL14 and CXCL12 shows that they are expressed in complementary patterns in most tissues during ocular development, suggesting an interactive regulation of these chemokines. Visual examination of Retrovirus-mediated Knockdown of CXCL14 embryos revealed relatively smaller eyes compared to controls, and immunohistochemical analysis of ocular nerves indicated exacerbated projection of sensory nerves into the corneal stroma, corneal epithelium and iris, which subsequently elevated nerve density in these tissues. In vitro analyses revealed that CXCL14 has an inhibitory effect on CXCL12-induced axon growth of trigeminal ganglion sensory neurons. Furthermore, Knockdown of CXCL14 in Tg(tie1: H2B:eYFP) transgenic Japanese quail embryos resulted in ectopic migration of YFP fluorescently labeled angioblasts into the cornea and exogenous CXCL14 inhibits VEGF- and CXCL12-induced angioblast migration into the cornea. This is the first time that CXCL14 has been shown to have a critical function during embryogenesis that may be mediated through inhibition of CXCL12 signaling. Collectively, these results demonstrate that neurovascular patterning of the anterior eye during development depends on an intricate process and fine balance of growth factors and cytokines. These findings will contribute to a better understanding of the molecular mechanisms involved in pathological conditions such as cornea neovascularization, anterior segment ocular dysgeneses and wound healing, where angiogenesis and nerve regeneration are critically compromised.