Stanislav I. Tomarev

Neuroprotective effects of intravitreal mesenchymal stem cell transplantation in experimental glaucoma.

Purpose. Retrograde neurotrophic factor transport blockade has been implicated in the pathophysiology of glaucoma. Stem cell transplantation appears to ameliorate some neurodegenerative conditions in the brain and spinal cord, in part by neurotrophic factor secretion. The present study was conducted to determine whether local or systemic bone marrow-derived mesenchymal stem cell (MSC) transplantation can confer neuroprotection in a rat model of laser-induced ocular hypertensive glaucoma. Methods. MSCs were isolated from the bone marrow of adult wild-type and transgenic rats that ubiquitously express green fluorescent protein. MSCs were transplanted intravitreally 1 week before, or intravenously on the day of, ocular hypertension induction by laser photocoagulation of the trabecular meshwork. Ocular MSC localization and integration were determined by immunohistochemistry. Optic nerve damage was quantified by counting axons within optic nerve cross-sections 4 weeks after laser treatment. Results. After intravitreal transplantation, MSCs survived for at least 5 weeks. Cells were found mainly in the vitreous cavity, though a small proportion of discrete cells migrated into the host retina. Intravitreal MSC transplantation resulted in a statistically significant increase in overall RGC axon survival and a significant decrease in the rate of RGC axon loss normalized to cumulative intraocular pressure exposure. After intravenous transplantation, MSCs did not migrate to the injured eye. Intravenous transplantation had no effect on optic nerve damage. Conclusions. Local, but not systemic, transplantation of MSCs was neuroprotective in a rat glaucoma model. Autologous intravitreal transplantation of MSCs should be investigated further as a potential neuroprotective therapy for glaucoma.

The transcription factor Prox1 is a marker for lymphatic endothelial cells in normal and diseased human tissues.

Detection of lymphatic endothelal cells (LECs) has been problematic because of the lack of specific markers. The homeobox transcription factor Prox1 is expressed in LECs of murine and avian embryos. We have studied expression of Prox1 in human tissues with immunofluorescence. In 19‐wk‐old human fetuses, Prox1 and vascular endothelial growth factor receptor‐3 (VEGFR‐3) are coexpressed in LECs of lymphatic trunks and lymphatic capillaries. Prox1 is located in the nucleus, and its expression is mutually exclusive with that of the blood vascular marker PAL‐E. Prox1 is a constitutive marker of LECs and is found in tissues of healthy adults and lymphedema patients. Blood vascular endothelial cells (BECs) of hemangiomas express CD31 and CD34, but not Prox1. A subset of these cells is positive for VEGFR‐3. Lymphatics in the periphery of hemangiomas express Prox1 and CD31, but not CD34. In lymphangiomas, LECs express Prox1, CD31, and VEGFR‐3, but rarely CD34. In the stroma, spindle‐shaped CD34‐positive cells are present. We show that Prox1 is a reliable marker for LECs in normal and pathologic human tissues, coexpressed with VEGFR‐3 and CD31. VEGFR‐3 and CD34 are less reliable markers for LECs and BECs, respectively, because exceptions from their normal expression patterns are found in pathologic tissues.

Intraocular pressure in genetically distinct mice: an update and strain survey

BackgroundLittle is known about genetic factors affecting intraocular pressure (IOP) in mice and other mammals. The purpose of this study was to determine the IOPs of genetically distinct mouse strains, assess the effects of factors such as age, sex and time of day on IOP in specific strain backgrounds, and to assess the effects of specific candidate gene mutations on IOP.ResultsBased on over 30 studied mouse strains, average IOP ranges from approximately 10 to 20 mmHg. Gender does not typically affect IOP and aging results in an IOP decrease in some strains. Most tested strains exhibit a diurnal rhythm with IOP being the highest during the dark period of the day. Homozygosity for a null allele of the carbonic anhydrase II gene (Car2n) does not alter IOP while homozygosity for a mutation in the leptin receptor gene (Leprdb) that causes obesity and diabetes results in increased IOP. Albino C57BL/6J mice homozygous for a tyrosinase mutation (Tyrc-2J) have higher IOPs than their pigmented counterparts.ConclusionsGenetically distinct mouse strains housed in the same environment have a broad range of IOPs. These IOP differences are likely due to interstrain genetic differences that create a powerful resource for studying the regulation of IOP. Age, time of day, obesity and diabetes have effects on mouse IOP similar to those in humans and other species. Mutations in two of the assessed candidate genes (Lepr and Tyr) result in increased IOP. These studies demonstrate that mice are a practical and powerful experimental system to study the genetics of IOP regulation and disease processes that raise IOP to harmful levels.

Chicken homeobox gene prox 1 related to Drosophila prospero is expressed in the developing lens and retina

Prox 1 is the vertebrate homolog of Drosophila prospero, a gene known to be expressed in the lens‐secreting cone cells of fly ommatidia. Chicken Prox 1 cDNAs were isolated from 14 day embryonic chicken lenses, and a complete open reading frame encoding an 83 kDa protein was elucidated. The homeodomains of chicken and mouse Prox 1 are identical at the amino acid level and are 65–67% similar to the homeodomains of Drosophila and C. elegans prospero. The homology between these proteins extends beyond the homeodomain. There is 56% identity between chicken Prox 1 and Drosophila prospero in the C‐terminal region downstream of the homeodomain, whereas there is little similarity upstream of the homeodomain. Prox 1 is expressed most actively in the developing lens and midgut and at lower levels in the developing brain, heart, muscle, and retina. cDNA sequencing has established that there are alternatively spliced forms of the single Prox 1 gene, which probably account for the two abundant RNAs of about 2 and 8 kb and two less abundant RNAs close to 3.5 kb in length in the lens. In the lens fibers, only the shortest mRNA was present, whereas, in the epithelial cells, both short and long mRNAs were detected. By using in situ hybridization, expression of the Prox 1 gene was first detected at stage 14 in the early lens placode and slightly preceded the expression of δ1‐crystallin, the first crystallin gene expressed in the developing chicken lens. At later stages of development, Prox 1 mRNA was observed throughout the lens, but it appeared more abundant around the bow region of the equator than in the anterior epithelium or the fibers. In the retina, expression of the Prox 1 gene was detected mainly in the inner nuclear layer during later stages of histogenesis. The conserved pattern of Prox 1/prospero gene expression in vertebrates and Drosophila suggests that Prox 1, like Pax‐6, may be essential for eye development in different systematic groups.

Expression of Mutated Mouse Myocilin Induces Open-Angle Glaucoma in Transgenic Mice

We developed a genetic mouse model of open-angle glaucoma by expression of mutated mouse myocilin (Myoc) in transgenic (Tg) mice. The Tyr423His point mutation, corresponding to the severe glaucoma-causing Tyr437His mutation in the human MYOC gene, was introduced into bacterial artificial chromosome DNA encoding the full-length mouse Myoc gene and long flanking regions. Both wild-type (Wt) and Tg animals expressed Myoc in tissues of the irido-corneal angle and the sclera. Expression of mutated Myoc induced the accumulation of Myoc in cell cytoplasm and prevented its secretion into the extracellular space. The levels of ATPase-1 were reduced in the irido-corneal angle of Tg mice compared with Wt animals. Tg mice demonstrated a moderate elevation of intraocular pressure, the loss of ∼20% of the retinal ganglion cells (RGCs) in the peripheral retina, and axonal degeneration in the optic nerve. RGC depletion was associated with the shrinkage of their nuclei and DNA fragmentation in the peripheral retina. Pathological changes observed in the eyes of Tg mice are similar to those observed in glaucoma patients.

Overexpression of Optineurin E50K Disrupts Rab8 Interaction and Leads to a Progressive Retinal Degeneration in Mice

Glaucoma is one of the leading causes of bilateral blindness affecting nearly 8 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure (IOP). However, patients with normal tension glaucoma (NTG), a subtype of primary open-angle glaucoma (POAG), develop the disease without IOP elevation. The molecular pathways leading to the pathology of NTG and POAG are still unclear. Here, we describe the phenotypic characteristics of transgenic mice overexpressing wild-type (Wt) or mutated optineurin (Optn). Mutations E50K, H486R and Optn with a deletion of the first (amino acids 153–174) or second (amino acids 426–461) leucine zipper were used for overexpression. After 16 months, histological abnormalities were exclusively observed in the retina of E50K mutant mice with loss of RGCs and connecting synapses in the peripheral retina leading to a thinning of the nerve fiber layer at the optic nerve head at normal IOP. E50K mice also showed massive apoptosis and degeneration of entire retina, leading to approximately a 28% reduction of the retina thickness. At the molecular level, introduction of the E50K mutation disrupts the interaction between Optn and Rab8 GTPase, a protein involved in the regulation of vesicle transport from Golgi to plasma membrane. Wt Optn and an active GTP-bound form of Rab8 complex were localized at the Golgi complex. These data suggest that alternation of the Optn sequence can initiate significant retinal degeneration in mice.

Restricted expression of the homeobox gene prox 1 in developing zebrafish

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Genetically Increasing Myoc Expression Supports a Necessary Pathologic Role of Abnormal Proteins in Glaucoma

ABSTRACT Despite the importance of MYOC for glaucoma, the proteins normal function(s) and the pathogenic mechanism(s) of MYOC mutations are not clear. Elevated intraocular pressure (IOP) and glaucoma are sometimes induced by corticosteroids, and corticosteroid use can result in substantially increased MYOC expression. It has been suggested, therefore, that steroid-induced MYOC protein levels cause steroid-induced glaucoma and that protein level-increasing mutations in MYOC contribute to glaucoma not associated with steroid use. A causative role of elevated MYOC levels in steroid-induced glaucoma is controversial, however, and it is not clear if elevated MYOC levels can result in IOP elevation. To directly test if increased levels of MYOC can cause IOP elevation and glaucoma, we generated bacterial artificial chromosome transgenic mice that overexpress Myoc at a level similar to that induced by corticosteroid use. These mice do not develop elevated IOP or glaucoma. Our present findings, along with the absence of glaucoma in mice completely lacking MYOC, show that changing the level of MYOC is not pathogenic (from absent to approximately 15 times normal). These findings suggest that noncoding sequence variants are unlikely to influence glaucoma and that disease pathogenesis in primary open-angle glaucoma patients is dependent upon the expression of abnormal mutant proteins. This work does not support a causative role for increased MYOC levels or the MYOC gene in steroid-induced glaucoma.

Rodent models of glaucoma

Glaucoma is a progressive, age-related optic neuropathy and a leading cause of irreversible blindness in the world. Animal models of glaucoma are essential to our continued efforts of elucidating the natural course of the disease and to developing therapeutic interventions to halt or reverse the progression of the condition. Over the past 10-15 years, rodents have become a popular model organism to study glaucoma, because of their high degree of availability, relatively low cost, short life-span, and amenability to experimental and genetic manipulation. In this review, we examine the numerous in vivo and in vitro rodent models of glaucoma, discuss the methods used to generate them, summarize some of the major findings obtained in these models, and identify individual strengths and weaknesses for the various systems.

Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts.

The lymphatic endothelium has mostly been thought to be derived by sprouting from specialized veins. Recently it has been shown that mice deficient for the homeobox transcription factor Prox1 are practically devoid of lymphatics. We have studied the expression of Prox1 mRNA and protein in chick embryos and human fetuses. In the chick, Prox1 is expressed in specific compartments of all germ layers. In the ectoderm, it is found in the neural tube, trigeminal, spinal and sympathetic ganglia and the retina, and also in placodal structures such as the lens, olfactory, otic, facial, glossopharyngeal and vagal placodes, and the apical ectodermal ridge. In the endoderm, Prox1 is a marker of hepatocytes, bile duct and pancreatic epithelium. In the mesoderm, weak expression is observed in cardiomyocytes, and strong expression in lymphatic endothelium. Identical expression domains are found in 19-week-old human fetuses. In day 6.5 chick embryos, there are several sites of contact of lymphatics with the jugular vein, which has a mixed endothelium of Prox1-positive and -negative cells. The only non-lymphatic endothelial cells expressing Prox1 are found on the concave side of the cardiac valves. To further analyse development of lymphatics, we studied early chick embryos and observed scattered Prox1-positive cells in the dermatome, giving rise to Prox1-positive lymphatic networks during subsequent development. Furthermore, the anlagen of the posterior lymph sacs and the paired thoracic duct can already be observed in day-4 chick embryos. Our studies show that lymphatics develop much earlier than previously described, and they mostly do not seem to be derived by sprouting from veins. In contrast, lymphangioblasts are present in the deep and superficial compartments of the early mesoderm, independently giving rise to the deep and superficial lymphatics.