Browsing by Author "Lwigale, Peter Y"

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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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    Complex 3D Culture Models for the Study of Bone Metastatic Prostate Cancer
    (2020-04-24) Sablatura, Lindsey Kay; Farach-Carson, Mary C; Lwigale, Peter Y
    Prostate cancer (PCa) is the most prevalent form of cancer in men. Despite the enormous resource expenditures on research and development, few advances have been made that improve therapeutic outcomes for patients with relapsed or bone metastatic disease. The high attrition rate of potential anti-cancer drugs entering clinical trials indicates the need for a shift in our approach to modeling metastatic tumors in preclinical studies. As evidence mounts of the importance of microenvironmental cues in tumorigenesis, metastasis, treatment response, and recurrence, research has sought to bridge the gap between in vivo biological relevance and in vitro accessibility and throughput. Three-dimensional (3D) culture models allow experimental control over variables not easily manipulatable in vivo using mouse models, including the cell types and extracellular matrix cues present in the tumor microenvironment (TME). These high content models also support real time imaging of cancer cell behavior as they contact with other cells in the TME. Perfusable models can be used further, not only to add and remove nutrients and waste from cultured cells, but also to introduce perturbagens including anti-cancer drugs for testing personalized oncology. With this goal in mind, these studies tested the hypothesis that the use of complex 3D models containing cancer, stroma, and endothelial cells to reconstitute cellular interactions can replicate events that occur in the bone metastatic niche, allowing investigation into new methods of disrupting growth and progression of bone metastatic disease. Work described in chapters 2 and 3 developed a new high-content, perfusable, hydrogel-based system that permitted the tri-culture of diverse PCa patient-derived xenograft cells with microvascular endothelial cells and tumor stromal cells. The applications of this new pre-clinical model for drug screening and the study of cancer biology were demonstrated. Work in chapter 4 constructed a model of the bone marrow TME to examine endothelial cell promotion of PCa death/dormancy in the bone metastatic niche. Together, the work in these chapters establishes a new stepping stone for in vitro models of bone metastatic PCa, providing new tools for drug discovery and cancer research.
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    Role of Chemokine Signaling and Heparin Binding Growth Factors During Ocular Neurovascular Patterning
    (2019-08-07) Cui, Ruda; Lwigale, Peter Y; Carson, Daniel
    The embryonic eye provides a unique context in which we can study the mechanisms of neurovascular patterning and wound healing. The anterior eye requires highly regulated signaling for the proper formation of an avascular, highly innervated, and transparent cornea. By studying ocular development in avian and murine models, we can begin to understand how signals in the embryo regulate interactions of the neural crest cells and ocular ectoderm for the proper formation of the anterior eye structures. The purpose of this work is to elucidate the functions of a chemokine (CXCL12), and two heparin-binding growth factors (midkine and pleiotrophin) during anterior eye development and neurovascular patterning. CXCL12 and its receptor CXCR4 are required for the proper vascular patterning in other organ systems, but their functions in the eye have yet to be discovered. Here I demonstrate that CXCL12-CXCR4 signaling has two major functions during ocular development: first, CXCL12 expression in the trigeminal placode is required for the proper formation of the trigeminal ganglion; second, CXCL12 expression in the periocular neural crest is required for proper vascular remodeling during later ocular development. Disruption of CXCL12 signaling in either the placode or neural crest result in significant neurovascular defects of the anterior eye. Midkine (MDK) and pleiotrophin (PTN) are two secreted proteins which make up their own family of heparin-binding growth factors. MDK expression can be induced by retinoic acid, which is an important signal for general ocular development. Here I report MDK and PTN expression during corneal and retinal development. Both MDK and PTN were expressed in the migrating neural crest, but otherwise had generally complimentary expression patterns. Although MDK and PTN were conserved in the anterior eye between chick and mouse, they had differential expression during retinal development. I also demonstrate that MDK and PTN in the anterior eye may signal to trigeminal nerves through ITGB1 and RPTPZ. Lastly, I present a novel method to study embryonic wound healing in culture. The embryonic cornea possesses regenerative properties not found in the adult, and can maintain its transparency by healing nonfibrotically. However, current methods of studying embryonic wound healing are limited in its efficiency and adaptability. Here I provide a protocol to study mechanisms of wound healing in an ex vivo model that allows for higher throughput, reproducibility, and adaptability. The results presented in this document give a glimpse of the intricate and highly regulated signaling required during ocular development. Better understanding of the molecular mechanisms are required for the creation of better therapeutics for ocular neurovascular diseases and wound healing.