Ursula M. Dietrich

Repulsive axon guidance molecule Slit3 is a novel angiogenic factor.

Slits are large, secreted repulsive axon guidance molecules. Recent genetic studies revealed that the Slit3 is dispensable for neural development but required for non-neuron-related developmental processes, such as the genesis of the diaphragm and kidney. Here we report that Slit3 potently promotes angiogenesis, a process essential for proper organogenesis during embryonic development. We observed that Slit3 is expressed and secreted by both endothelial cells and vascular smooth muscle cells in vasculature and that the Slit cognate receptors Robo1 and Robo4 are universally expressed by endothelial cells, suggesting that Slit3 may act in paracrine and autocrine manners to regulate endothelial cells. Cellular function studies revealed that Slit3 stimulates endothelial-cell proliferation, promotes endothelial-cell motility and chemotaxis via interaction with Robo4, and accelerates endothelial-cell vascular network formation in vitro with a specific activity comparable with vascular endothelial growth factor. Furthermore, Slit3 stimulates neovessel sprouting ex vivo and new blood vessel growth in vivo. Consistent with these observations, the Slit3 knockout mice display disrupted angiogenesis during embryogenesis. Taken together, our studies reveal that the repulsive axon guidance molecule Slit3 is a novel and potent angiogenic factor and functions to promote angiogenesis in coordinating organogenesis during embryonic development.

Effects of antifungal drugs and delivery vehicles on morphology and proliferation of equine corneal keratocytes in vitro

OBJECTIVE To evaluate the effects of topical antifungal drugs and delivery vehicles on the morphology and proliferation rate of cultured equine keratocytes. STUDY POPULATION 16 corneas obtained from 8 apparently ophthalmologically normal horses < 0.5 hours after euthanasia for reasons unrelated to the study. PROCEDURES Primary cultures of equine keratocytes were obtained from corneal stroma and were exposed to several concentrations of 3 commonly used, topically applied antifungals: natamycin, itraconazole, and miconazole. In addition, effects of drug delivery vehicles DMSO, benzalkonium chloride, and carboxymethylcellulose and a combination vehicle composed of polyethylene glycol, methylparaben, and propylparaben were also evaluated. Morphological changes and cellular proliferation were assessed 24, 48, and 72 hours after application. RESULTS At the highest concentrations tested, all antifungals caused marked cellular morphological changes and inhibited proliferation. At low concentrations, natamycin and miconazole induced rounding, shrinking, and detaching of the cells with inhibition of cellular proliferation. Natamycin caused the most severe cellular changes. Itraconazole, at the low concentrations, caused minimal morphological changes and had a minimal effect on proliferation. All vehicles tested had significantly less effects on cellular morphology and proliferation when compared with the antifungals, except for the combination vehicle, which caused severe morphological changes and inhibited proliferation, even at low concentrations. The DMSO had minimal effects on cellular morphology and proliferation, even at high concentrations. CONCLUSIONS AND CLINICAL RELEVANCE Itraconazole had significantly less cytotoxic effects on equine keratocytes in culture than did natamycin or miconazole. Natamycin had severe cytotoxic effects in vitro.

Immunohistochemical study of matrix metalloproteinases-2 and -9, macrophage inflammatory protein-2 and tissue inhibitors of matrix metalloproteinases-1 and -2 in normal, purulonecrotic and fungal infected equine corneas.

OBJECTIVE Determine the effects of matrix metalloproteinases (MMPs)-2, -9, macrophage inflammatory protein-2 (MIP-2), tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 by immunohistochemical expression in fungal affected and purulonecrotic corneas. PROCEDURE Paraffin-embedded equine corneal samples; normal (n = 9), fungal affected (FA; n = 26), and purulonecrotic without fungi (PN; n = 41) were evaluated immunohistochemically for MMP-2, -9, MIP-2, TIMP-1 and -2. The number of immunoreactive inflammatory cells was counted and statistics analyzed. Western blot was performed to detect MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins. RESULTS Matrix metalloproteinases-2, -9, MIP-2, TIMP-1 and -2 immunoreactivity was identified in corneal epithelium of normal corneas, and in corneal epithelium, inflammatory cells, keratocytes, and vascular endothelial cells of both FA and PN samples. Inflammatory cell immunoreactivity was significantly higher in FA and PN samples than in the normal corneas. There was positive correlation between MMP-2 and MIP-2, MMP-9 and MIP-2, and MMP-9 and TIMP-1 in inflammatory cell immunoreactivity in FA samples. There was positive correlation between MMP-9 and MIP-2, MMP-9 and TIMP-2, MIP-2 and TIMP-1, and MIP-2 and TIMP-2 in inflammatory cell immunoreactivity in PN samples. Western blot confirmed the presence of all four proteins in equine corneal samples. CONCLUSION Increased immunoreactivity of MMP-2 and -9 in FA and PN samples is indirectly related to MIP-2 through its role in neutrophil chemo-attraction. Tissue inhibitors of matrix metalloproteinase-1 and TIMP-2 are up-regulated in equine purulonecrotic and fungal keratitis secondary to MMP-2 and MMP-9 expression. The correlation between MMPs -2 and -9, MIP-2, TIMPs -1 and -2 suggests that these proteins play a specific role in the pathogenesis of equine fungal keratitis.

Biocompatibility of a Novel Microfistula Implant in Nonprimate Mammals for the Surgical Treatment of Glaucoma

PURPOSE The purpose of this study was to evaluate the ocular safety of a novel microfistula implant and its composite materials in an animal model. METHODS The anterior chambers of 12 rabbit eyes were injected with either glutaraldehyde cross-linked porcine gelatin extract or balanced salt solution and were followed by serial slit lamp examinations over 3 days. The eyes of 18 canines underwent microfistula implantation or a sham procedure. The animals were monitored over the subsequent 12 months, using serial slit lamp examinations, indirect ophthalmoscopy, tonometry, specular microscopy, and high-resolution ultrasonography. Ocular tissues were examined histopathologically on postoperative days 7, 30, 90, 180, and 365. RESULTS Glutaraldehyde cross-linked porcine gelatin did not induce significant intraocular inflammation in the rabbit model. The microfistula implant was well tolerated and did not stimulate significant tissue response in the canine eye. The microfistula tube did not undergo structural change or degradation over the course of the study. CONCLUSIONS In nonprimate mammals, the material composing the microfistula implant and the implant itself do not induce significant inflammation or tissue reaction.

Establishing a reproducible method for the culture of primary equine corneal cells.

OBJECTIVE To establish a reproducible method for the culture of primary equine corneal epithelial cells, keratocytes, and endothelial cells and to describe each cells morphologic characteristics, immunocytochemical staining properties and conditions required for cryopreservation. PROCEDURES Corneas from eight horses recently euthanized for reasons unrelated to this study were collected aseptically and enzymatically separated into three individual layers for cell isolation. The cells were plated, grown in culture, and continued for several passages. Each cell type was characterized by morphology and immunocytochemical staining. RESULTS All three equine corneal cell types were successfully grown in culture. Cultured corneal endothelial cells were large, hexagonal cells with a moderate growth rate. Keratocytes were small, spindloid cells that grew rapidly. Epithelial cells had heterogeneous morphology and grew slowly. The endothelial cells and keratocytes stained positive for vimentin and were morphologically distinguishable from one another. The epithelial cells stained positive for cytokeratin. Keratocytes and endothelial cells were able to be cryopreserved and recovered. The cryopreserved cells maintained their morphological and immunocytochemical features after cryopreservation and recovery. DISCUSSION This work establishes reproducible methods for isolation and culture of equine corneal keratocytes and endothelial cells. Cell morphology and cytoskeletal element expression for equine corneal epithelial cells, keratocytes, and endothelial cells are also described. This has not previously been reported for equine corneal cells. This report also demonstrates the ability to preserve equine keratocytes and endothelial cells for extended periods of time and utilize them long after the primary-cell collection, a feature that has not been reported for veterinary corneal cell culture.