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laminin to sense changes in stress and strain within the prelaminar region and the lamina cribrosa. Vascular endothelial cell stress may be mediated by integrins a3ab1, a6b1, and a6b4, along with a5b1 and aVb1 (Morrison, 2006). In glaucomatous ONH astrocytes there are low expression levels of a-integrins, suggesting that other adhesion mechanisms may facilitate migration in glaucoma (Hernandez et al., 2002).

Connective tissue changes in the glaucomatous optic nerve head

Extracellular matrix synthesis by ONH astrocytes

CNS axons fail to regenerate beyond a lesion site, even in the absence of a recognizable glial scar, suggesting that reactive astrocytes establish a local biochemical barrier (Zhang et al., 2006; Galtrey and Fawcett, 2007). Tenascin expressed by reactive astrocytes in the glaucomatous ONH may represent such a barrier (Pena et al., 1999b). Also, proteoglycans, such as neurocan and phosphacan secreted by reactive astrocytes, inhibit neurite outgrowth from different populations of neurons and sequester growth factors such as TGFb, preventing neurite growth-promoting effects (McKeon et al., 1999).

There is substantial evidence that ONH astrocytes are responsible for the normal maintenance of the ECM in normal and that reactive astrocytes remodel the ECM in response to elevated IOP in human and experimental glaucoma (Hernandez, 2000; Tezel et al., 2001). Reactive astrocytes in the ONH express large amounts of elastin, leading to elastotic degeneration of the ECM in glaucoma and loss of resiliency and deformability in response to elevated IOP (Hernandez et al., 2000; Pena et al., 2001). Synthesis of elastin by astrocytes has not been reported in other regions of the normal CNS. However, recent reports indicate that astrocytomas, the most common form of primary brain tumors, express elastin and elastin binding protein (EBP) or elastin laminin receptors (ELRs) in vivo and in vitro, indicating that synthesis of new elastin participates in tumor proliferation and invasion (Lapis and Timar, 2002) and may facilitate ONH astrocyte migration in glaucoma (Varela and Hernandez, 1997).

We recently reported that astrocytes from African American (AA) normal donors expressed high levels of elastin mRNA and protein, and decreased levels of LOXL2 (Urban et al., 2007). Patients of Scandinavian ancestry with pseudoexfoliation glaucoma exhibit variations in the LOXL1 gene sequence that may predispose this group to elevated IOP and glaucomatous optic neuropathy due to accumulation of pseudoexfoliation material (Thorleifsson et al., 2007). We previously published marked elastosis in the ONHs of patients with pseudoexfoliation glaucoma (Netland et al., 1995). The downregulation of LOXL2 in AA astrocytes may confer a similar susceptibility to elevated IOP in this population. Synthesis of elastin and related ECM proteins by ONH astrocytes makes ONH astrocytes unique among glia and demonstrate the plasticity of this cell type to perform diverse functions in the CNS.

Extracellular matrix degradation by reactive astrocytes

MMPs, or matrixins, degrade ECM components such as collagens, proteoglycans, elastin, laminin, fibronectin, and glycoproteins in normal and pathologic conditions (Clark et al., 2007). Some MMPs are expressed constitutively in most cell types, and others are inducible and tissue-specific. Specific proteins known as tissue inhibitors of MMP (TIMP 1-4) are the physiologic regulators of these enzymes. Expression of MMPs and TIMPs by reactive astrocytes in the CNS depends on the type of injury or disease (Clark et al., 2007). Reactive astrocytes express MMP3 and MMP9 in neural inflammation in response to cytokine stimulation. In mice, deficiency in MMP9 protects against RGC death after optic nerve ligation (Chintala et al., 2002), whereas, in mice deficient in MMP2 there was no neuroprotection of RGC (Asahi et al., 2001) Reactive astrocytes are the main source of MMP9 activity, and ERK and p38 MAP kinases mediate secretion of MMP9 after mechanical injury (Wang et al., 2002). In the glaucomatous optic nerve, reactive astrocytes express increased MMP1 and MT1-MMP but do not express MMP9, MMP3, or MMP7, suggesting

highly regulated proteolytic activity (Hernandez et al., 2002; Agapova et al., 2003a).

TGFb signaling in ONH astrocytes in glaucoma

TGFb signaling has long been associated with fibrous scar formation in the injured CNS by enhancing the deposition of laminin, fibronectin, and CSPGs (Logan et al., 1999a, b; McKeon et al., 1999; Asher et al., 2000). Furthermore, although TGFb1 and -b2 are believed to signal through the same molecular pathways (Hartsough and Mulder, 1997), a role for scar production after spinal cord injury (SCI) has been attributed specifically to TGFb2 that is expressed in multiple cell types including astrocytes in the spinal lesion during scar formation (Lagord et al., 2002). TGFb2 which acts via TGFBR1 and TGFBR2 receptors and downstream signaling proteins (SMADs), is the primary form of TGFb produced by ONH astrocytes (Pena et al., 1999a). The receptors TGFBR1 and TGFBR2, are upregulated in glaucomatous astrocytes from Caucasian donors (Fig. 7). Similarly, TGFBR3, another TGFb receptor, is also upregulated (2.1-fold) in glaucomatous astrocytes. TGFBR3 serves as a co-receptor that alters the ligand binding properties of TGFb receptors so as to favor interaction with TGFb2 (Esparza-Lopez et al., 2001). Thus, in glaucomatous astrocytes, there may be increased sensitivity to TGFb2. Downstream to the TGFBR2 are SMAD2 and SMAD3 which function as transcriptional regulators in ONH astrocytes (Fuchshofer et al., 2005) and other cells in the CNS (Flanders et al., 1998). These proteins are

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also coupled to Rho, Cdc42, and Rac1/Rac2 signaling pathways that control astrocyte polarity and migration (Kalman et al., 1999; EtienneManneville and Hall, 2001). Analysis of microarray data from glaucomatous ONH astrocytes compared to controls indicated that LM04, a LIM domain protein that modulates SMAD3 transcriptional activity (Lu et al., 2006), is upregulated 1.8-fold in glaucomatous astrocytes. SMAD3 was upregulated in ONH astrocytes exposed to hydrostatic pressure in vitro suggesting that pressure modulates the TGFb pathway (Yang et al., 2004). Countering SMAD signaling is ubiquitin-linked degradation by SMURF2. Although SMURF2 expression is not altered in glaucomatous astrocytes, it is downregulated by an increase in hydrostatic pressure (Yang et al., 2004). Thus, there may be a potentiation of TGFb signaling in glaucomatous astrocytes with changes in IOP. Although there are reports of increased levels of TGFb2 in the aqueous humor collected from POAG eyes (reviewed in LutjenDrecoll, 2005), our data suggests that changes in TGFb signaling occurs at the level of the receptors and associated signaling molecules in astrocytes from glaucomatous donors.

Expression of several TGFb2-regulated genes associated with extracellular matrix in glaucomatous astrocytes has been explored by several groups including ours. In lamina cribrosa cells TGFb1 induced expression and release of ECM components (Kirwan et al., 2004, 2005), elastin deposition and gene expression is upregulated in astrocytes in experimental glaucoma in monkeys (Pena et al., 2001) and in human ONH astrocytes