Adenoviral reporter gene transfer to the human trabecular meshwork does not alter aqueous humor outflow. Relevance for potential gene therapy of glaucoma

The Journal of Gene Medicine

Wang

Choi

et al. 2006

Section: Viral Vectorsmentioning

confidence: 99%

“…Their eyes were dissected within 30 to 40 h of death and anterior segment organ cultures were prepared as described previously [27][28][29]. Briefly, eyes were bisected at the equator and the lens, iris, and vitreous were removed.…”

Section: Perfused Human Anterior Segment Organ Culturesmentioning

confidence: 99%

Mechanisms of AAV transduction in glaucoma‐associated human trabecular meshwork cells

The Journal of Gene Medicine

Wang

Choi

et al. 2006

“…All procedures were in accordance with the Tenets of the Declaration of Helsinki. Whole eye globes within 8 -40 h of death were dissected at the equator, cleaned away from vitreous, iris, and lens, and then mounted on the custom-made perfusion chambers as described previously (14,15,35) (Fig. 1).…”

Section: Methodsmentioning

confidence: 99%

Individual molecular response to elevated intraocular pressure in perfused postmortem human eyes

Comes

2009

Physiological Genomics

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma. In the clinic, the response to elevated pressure and thus the risk for development of glaucoma differs among individuals. We took advantage of our ability to subject postmortem human eyes from the same individual to physiological and elevated pressure in a perfused outflow model and compared individual patterns of gene expression under pressure. The architecture of the trabecular meshwork, tissue responsible for the maintenance of IOP, was conserved. We performed two sets of experiments. The first set (n ϭ 5, 10 eyes) used Affymetrix GeneChips, identified the 20 most pressure-altered genes in each individual, and compared their pressure response in the other four. The second set (n ϭ 5, 10 eyes) selected 21 relevant trabecular meshwork genes and examined, by real-time TaqMan-PCR, the rank of their abundance and of their pressure differential expression in each individual. The majority of the up-and downregulated top-changers of each individual showed an individual response trend. Few genes were general responders. Individual responders included STATH, FBN2, TF, OGN, IL6, IGF1, CRYAB, and ELAM1 (marker for glaucoma). General responders included MMP1, MMP10, CXCL2, and PDPN. In addition, we found that although the relative abundance of selected genes was very similar among nonstressed individuals, the response to pressure of those same genes had a marked individual component. Our results offer the first molecular insight on the variation of the individual response to IOP observed in the clinical setting. individual response; trabecular meshwork; microarrays THE INTRAOCULAR PRESSURE (IOP) of the human eye under normal physiological conditions is 10 -22 mmHg (mean 16 Ϯ 6 mmHg) (58), while the pressure of the episcleral veins, site of the aqueous humor drainage outside the eye, is 8 -9 mmHg (24). This pressure differential is essential to maintain the ocular globe inflated. Increased changes in IOP create a mechanical stress situation that, in a not well understood manner, is transmitted to the back of the eye and exerts damage to the retinal ganglion cells (RGC) and to their axons (optic nerve). Death of RGCs impedes transmission of the visual signal to the brain and results in progressive loss of visual field and irreversible blindness. The resulting disease is glaucoma, the second leading cause of blindness worldwide (49). Elevated IOP is the major risk factor for glaucoma, and lowering IOP is the only currently available treatment for all types of glaucoma.The tissue responsible for maintaining the differential pressure in the eye is the outflow tissue, formed by the trabecular meshwork and the single cell layer lining the Schlemm's canal. This outflow tissue (from now on referred to as the "trabecular meshwork") is avascular, it is localized at the angle formed by the cornea and the iris, and, in humans, is the main route for the outflow of aqueous humor (9). The trabecular meshwork consists of layers with different cell types (uveal, cor...

“…High titer viral stocks were obtained by propagation in 293 cells and purification by CsCl density centrifugation as described (24,25) Purified viruses were titered by spectrophotometric reading at A 260 and stored in aliquots at Ϫ80°C. The absence of contaminant wildtype viruses was tested in each stock by PCR amplification with E1A-specific primers 5Ј-TCGAAGAGGTACTGGCTGAT-3Ј and 5Ј-TGACAAGACCTGCAACCGTG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells

Wajih¹,

Journal of Biological Chemistry

Wang

et al. 2004

Matrix ␥-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent ␥-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-␥-carboxylated MGP and fully ␥-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptormediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.