Shabnam Khashabi

ABERRANT ANGIOGENIC CHARACTERISTICS OF HUMAN BRAIN ARTERIOVENOUS MALFORMATION ENDOTHELIAL CELLS

OBJECTIVETo identify and characterize the phenotypic and functional differences of endothelial cells derived from cerebral arteriovenous malformations (AVM), as compared with endothelial cells derived from a normal brain. METHODSIsolated AVM brain endothelial cells and control brain endothelial cells were evaluated immunohistochemically for expression of the endothelial cell markers von Willebrand factor and CD31, as well as angiogenic factors including vascular endothelial growth factor A, interleukin-8, and endothelin-1. Vascular endothelial growth factor receptors 1 and 2 were also evaluated using immunohistochemistry techniques. Functional assays evaluated cell proliferation, cytokine production, tubule formation, and cell migration using the modified Boyden chamber technique. RESULTSEndothelial cells derived from AVMs expressed high levels of vascular endothelial growth factor A and significantly overexpressed the vascular endothelial growth factor receptors 1 and 2 (P < 0.05), as compared with control endothelial cells. In addition, comparison to control brain endothelial cells demonstrated that AVM brain endothelial cells proliferated faster, migrated more quickly, and produced aberrant tubule-like structures. CONCLUSIONEndothelial cells derived from cerebral AVMs are highly activated cells overexpressing proangiogenic growth factors and exhibiting abnormal functions consistent with highly activated endothelial cells.

Inactivation of Smad4 leads to impaired ocular development and cataract formation.

PURPOSE Signaling by members of the TGFβ superfamily of molecules is essential for embryonic development and homeostasis. Smad4, a key intracellular mediator in TGFβ signaling, forms transcriptional activator complexes with Activin-, BMP-, and TGFβ-restricted Smad proteins. However, the functional role of Smad4 in controlling different visual system compartments has not been fully investigated. METHODS Using the Pax6 promoter-driven Cre transgenic, smad4 was conditionally inactivated in the lens, cornea and ectoderm of the eyelids. Standard histological and molecular analytical approaches were employed to reveal morphological and cellular changes. RESULTS Inactivation of Smad4 in the lens led to microphthalmia and cataract formation in addition to the persistent adhesion of the retina to the lens and the iris to the cornea. Inactivation of Smad4 from the ectoderm of the eyelid and cornea caused disruption to eyelid fusion and proper development of the corneal epithelium and corneal stroma. CONCLUSIONS Smad4 is required for the development and maintenance of the lens in addition to the proper development of the cornea, eyelids, and retina.

Ocular Surface Reconstitution

1.1 The ocular surface – anatomy and pathology The corneal epithelium, conjunctival epithelium, and the lacrimal system constitute the ocular surface. A healthy corneal epithelium is essential for corneal health and visual function. The corneal epithelium is a 5to 6-cell-thick layer that provides a defensive barrier against pathologic organisms. It exists in a dynamic equilibrium, with superficial cells being constantly shed into the tear film. Populations of pluripotent stem cells reside in the palisades of Vogt at the human corneoscleral limbus and generate transient amplified cells that centripetally migrate toward the central cornea. These transient amplified cells undergo terminal differentiation into epithelial cells and repopulate the corneal epithelium, i.e. the XYZ hypothesis (Thoft et al., 1983). Severe ocular surface disorders, such as infection, keratoconjunctivitis sicca, Stevens-Johnson syndrome, ocular cicatricial pemphigoid or chemical/thermal injuries, can progress to corneal scarring, conjunctivalization, neovascularization, or stromal melts. Depletion of the limbal stem cells may follow, resulting in impaired vision or eventual corneal blindness. According to the World Health Organization, corneal disorders, e.g. trachoma or onchocerciasis, constitute a significant cause of loss of vision and blindness in the world (Thylefors et al., 1995). The conjunctiva is a thin, transparent, mucus membrane, overlying a thin vascular stroma. It is divided into three geographic zones: bulbar, forniceal, and palpebral. The conjunctival nonkeratinized stratified epithelium contains mucin-producing goblet cells, which are important for tear film stability. Additionally, the conjunctiva participates in the ocular surface antimicrobial defense via the conjunctiva-associated lymphoid tissue, as well as secretory antimicrobial peptides, such as defensins (Haynes et al., 1999). Disorders of the conjunctiva include elastotic changes, fibrovascular proliferation, malignancies, and autoimmune conditions such as Stevens-Johnson syndrome or cicatricial pemphigoid. Complications include dysfunctional tear syndrome, keratinization, symblepharon formation, eyelid disfigurement, and eyelash misalignment. Patient discomfort, cosmetic imperfection, increased risk of infection, and visual impairment are some notable concerns. A normal tear film is essential for maintenance of the corneal and conjunctival epithelia. Composed of three layers, mucin, aqueous and lipid layers, the human tripartite tear film has antimicrobial, epitheliotrophic, mechanical, and optical properties. A wide range of physiologic or pathologic conditions, such as biologic aging, hormonal changes, chemical or thermal injuries, chronic inflammation, or autoimmune disorders, may disrupt the tear film and trigger a deleterious cascade, injuring ocular surface epithelia. Furthermore, suboptimal