A Novel Mgp-Cre Knock-In Mouse Reveals an Anticalcification/Antistiffness Candidate Gene in the Trabecular Meshwork and Peripapillary Scleral Region.

Glaucoma is a complex optic neuropathy that results in irreversible blindness. It is well-established that elevated IOP is the principal risk factor associated with the development of the disease.1 Physiological and/or elevated IOP is determined by the resistance offered to aqueous humor flow by the trabecular meshwork (TM). The TM is a spongiform soft tissue formed by different types of endothelial-like cells, which use a variety of functions to regulate IOP. One of its most relevant functions is that which controls extracellular matrix (ECM) composition and deposition levels, which has been shown to have a direct correlation with increased flow resistance and glaucoma.2 Collagen and elastic fibers are key components of this extracellular space and contribute to the formation of the scaffold that helps maintaining the TM's unique architecture.3 Other tissues of the anterior segment assist the TM on its IOP regulatory function and contribute to modulations of IOP. Thus, the ciliary muscle, which intertwines posteriorly with the TM, affects IOP by contracting and pulling on the trabecular beams consequently opening spaces in the outflow pathway.4,5 Another tissue, the ciliary body, can modulate the outcome of total IOP by increasing or decreasing secretion of aqueous humor, which would then flow through the TM and cause decrease or increase of IOP. The elevated IOP generated in the anterior segment is transmitted to the back of the eye where the sclera senses the pressure fluctuations and exerts a biomechanical strain on the optic nerve (ON), contributing to the death of retinal ganglion cells (RGCs).6–,8 It has been reported that the TM tissue from postmortem glaucoma patients is stiffer than that of controls.9 Also, it has been proposed that the degree of stiffness of the sclera might be an important determinant of the extent of susceptibility of ON damage in glaucoma.10,11 Identification of genes, which are involved in the function and pathology of any of the glaucoma associated eye tissues would not only provide further understanding of the mechanisms of the disease but will open the door to new therapeutics. Early studies from our laboratory had identified the Matrix Gla gene (MGP) among the 10 most highly expressed genes in the human TM.12,13 Transcription of the gene was altered by the mechanical forces of pressure and by other IOP-inducing agents such as TGF-β and dexamethasone.13,14 We had also shown that not only the gene, but the encoded MGP protein, was present in the TM tissue in its active conformation and that the activity of the enzyme responsible for its activation, γ-Carboxylase, was very high in the TM tissue.15 Matrix Gla binds calcium and is a potent inhibitor of calcification.16 While mineralization of bone and tooth is a physiological process, soft tissue calcification is a pathological condition. In cartilage, calcification leads to loss of plasticity and bone formation. In the cartilage, MGP is highly secreted by chondrocytes and is thought to be responsible for keeping the tissue from calcifying. In the arteries, where MGP is highly abundant in the vascular smooth muscle cells (VSMCs) of the tunica media, calcification leads to formation of atherosclerotic plaques and calcified cartilaginous lesions.17,18 In patients, MGP polymorphisms have been linked to coronary artery calcification.19 An autosomal recessive rare disease, Keutel syndrome, has been associated with several mutations in the MGP gene.20–,22 Patients develop abnormal soft tissue calcification and stenosis of pulmonary arteries. Although very few ophthalmologic exams have been described, in one case a 6-year-old patient had sudden loss of vision in both eyes at the age 3 and had bilateral ON atrophy.21 Matrix Gla–deficient mice die between 5 and 6 weeks of age due to massive arterial calcification. The mechanisms by which MGP inhibits calcification in the arteries are not totally understood. Earlier, it had been demonstrated that after binding to calcium, MGP binds and sequesters bone morphogenetic protein 2 (BMP2), a potent stimulator of osteochondrogenic differentiation, and thus prevents chondrogenic transformation of the VSMCs.15,23,24 More recently, some investigators have shown that MGP may be inhibiting calcification by a direct effect on elastin. During calcification, the elastic lamina (dense layer of elastic fibers of the vessel wall) of large arteries is the first site of mineral deposition, with elastin, a major elastic lamina protein, acting as a mineral containing scaffold.25,26 Matrix Gla colocalizes with elastin in the arterial elastic lamina.25,26 It is known that calcium overload and calcification of elastic fibers lead to increase arterial stiffness as it was determined by measuring their elastic modulus and isobaric elasticity.27 It was also shown that such stiffness results in higher systolic blood pressure.27 Although gene expression studies by mRNA quantification had identified high expression of MGP in the human TM, attempts to localize the expression of this gene in the eye had met with little success. In situ hybridization and immunohistochemistry have been unable to detect endogenous MGP in the eye (our own unpublished results). To be able to elucidate a temporal and spatial pattern of expression of this gene in the eye, we made use of a set of mouse genetic tools. Engineered mouse lines carrying the Cre-recombinase bacteriophage gene under the control of a eukaryotic gene's regulatory sequences express the Cre enzyme in the eukaryotic gene's specific cell-type. When these mice are crossed with an engineered mouse line containing loxP sites flanking a STOP signal for a reporter gene, Cre recombines the two loxP sites to remove the STOP signal, which results in expression of the reporter gene. Histologic assessment of the reporter in the offspring of these crosses can reveal expression of the eukaryotic gene and accurately distinguish expression in neighboring cells. Thus, the generation of Cre-expressing mouse lines is extremely valuable for the determination of the desired gene expression in cell types, its temporal distribution during embryonic and adult development and its regulation under disease conditions. An accurate determination of these parameters is essential to establish a mechanistic link between MGP and glaucoma. Therefore, to elucidate the expression pattern of Mgp in mice, we generated knock-in mice, in which an IRES-Cre recombinase expression cassette was inserted in between the translational Mgp STOP signal and its polyA (pA). This knock-in allele MgpCre was designed to express Cre recombinase only in cells where Mgp is expressed. Here, we describe the expression pattern of the MgpCre allele in the mouse eye.