T. Borras

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

Processing and Transport of Matrix γ-Carboxyglutamic Acid Protein and Bone Morphogenetic Protein-2 in Cultured Human Vascular Smooth Muscle Cells EVIDENCE FOR AN UPTAKE MECHANISM FOR SERUM FETUIN

Matrix γ-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 γ-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-γ-carboxylated MGP and fully γ-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 receptor-mediated 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.

Processing and transport of matrix Gla protein (MGP) and bone morphogenetic protein-2 (BMP-2) in cultured human vascular smooth muscle cells: Evidence for an uptake mechanism for serum fetuin

Matrix γ-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 γ-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-γ-carboxylated MGP and fully γ-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 receptor-mediated 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.

Advances in Glaucoma Treatment and Management: Gene Therapy

Glaucoma is characterized by the death of retinal ganglion cells (RGCs) and loss of vision. It is the second most frequent cause of irreversible blindness in the world and affects primarily the older population. 1 Projections of prevalence show that by 2020 there will be an estimated 79.6 million people worldwide with glaucoma, with a higher proportion of women than men. 1 Glaucoma can be treated but is not yet curable. Treatments currently available, such as the daily administration of eye drops, lead to high levels of noncompliance, especially in the aged population. In several laboratories, including ours, intense research is ongoing to search for alternative treatments of the disease. Using genes, we seek to develop gene drug therapies that can be many times more efficient than conventional drugs and allow less frequent administration—perhaps just once every 1 or 2 years. In cases of inherited glaucoma caused by either recessive or dominant genes, we seek gene replacement and gene knockdown strategies that could reverse the undesired outcome of the mutation. Technology for this new state-of-the-art treatment is currently available and has been successfully applied in other diseases, including conditions in the eye. 2 Most glaucomas are induced by dysfunction of the trabecular meshwork (TM) tissue, which in turn leads to increased resistance to aqueous humor outflow and elevated intraocular pressure (IOP). The mechanism by which elevated IOP damages the posterior RGCs is not fully understood. Whether the elevated IOP first damages the RGC axons and then causes the cell body to die or it damages the cell body first, which then causes the axons to die is a subject of intense deliberation. Although elevated IOP is not the only factor causing RGC death, it is the most common. All currently available drugs that lower IOP slow the progression of all glaucomas, even the form referred to as normal-tension glaucoma. Because of this particular physiology, there are two main avenues to follow in approaching research in gene therapy for glaucoma. The first entails targeting the RGCs with the goal of promoting their survival and protecting them against glaucomatous insults (neuroprotection). The second entails targeting the TM with the goal of lowering IOP. Gene therapy vectors deliver the genetic material to the inside of the cells. Because viruses have been selected during evolution to cleanly and efficiently enter living cells, they have emerged as excellent carriers of any desired genetic material. Given the tools of molecular biology and recombinant DNA that are now available, a virus can be specially engineered both to remove the potentially deleterious sequences from its genome and to insert the beneficial foreign sequences to be delivered to the target cell.

Exfoliation syndrome: assembling the puzzle pieces

To summarize various topics and the cutting edge approaches to refine XFS pathogenesis that were discussed at the 21st annual Glaucoma Foundation Think Tank meeting in New York City, Sept. 19–20, 2014.