Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

and protein were also increased in astrocytes exposed to HP (see Fig. 4B). A pull-down assay confirmed and quantified the increase of GTP-bound Rho GTPase in astrocytes exposed to HP for 10–60 min (see Fig. 4A). Taken together, our data indicate that morphological changes in astrocyte cell shape and motility induced by HP are mediated by the Rho signaling pathway (87).

Ras Signaling Pathway

Microarray data suggested that molecular cascades leading to the activation of Ras p21 might be involved in responses to pressure (87). Pressure can activate astrocyte membrane receptors such as growth factors, G proteins, and integrin receptors to initiate the signal transduction involving the Ras signaling pathway (74). Alternatively, it is possible that HP-induced changes in fluidity of cellular membrane lipid bilayer may affect the conformation and/or interaction of membrane proteins that can be transduced into Ras activation (91). HP induced Ras activity in ONH astrocytes as detected by a pull-down assay 10 min after exposure to HP. Ras p21 activates the extracellular signal-regulated protein kinase 1/2 (ERK1/2), a member of the mitogen-activated kinase family (MAPK) in reactive astrocytes in brain injury and in cultured astrocytes exposed to various forms of mechanical injury (92,93). Ras p21 activation mediates iNOS in primary astrocytes (94). Reactive astrocytes express iNOS in the ONH with glaucoma in vivo and after exposure to HP in vitro (95,96). Microarray analysis indicates that RASA2 mRNA, a member of the GAP1 family of GTPase-activating proteins (97), is upregulated under pressure (86). RASA2 gene product stimulates the GTPase activity of normal Ras p21 but not of its oncogenic counterpart. Acting as a suppressor of Ras function, the protein enhances the weak intrinsic GTPase activity of Ras proteins, resulting in the inactive GDP-bound form of Ras, thereby allowing regulation of cell proliferation and differentiation. In addition, microarray data indicated increased expression of GRP1, a Ras GDP/GTP exchange factor, suggests the generation of strong Ras pathway signaling in response to HP (98).

FUNCTIONS OF REACTIVE ASTROCYTES

Reactive Astrocytes are Migratory

Migration of astrocytes occurs during normal development, in neurodegenerative diseases, after injury, and during tumor invasion in the CNS. Cell migration occurs in response to soluble molecules such as growth factors or cytokines or in response to ECM molecules such as fibronectin, laminin, and hyaluronan Interactions between growth factors and ECM molecules, such as those between heparin-binding EGFlike growth factor (52,99) or FGF (53), TGF- (100), and TGF- (101,102) as well as interactions between growth factor receptors and cell adhesion molecules such as NCAM affect cell mobility and can be modeled in vitro (103,104). In glaucoma, reactive astrocytes migrate from the cribriform plates into the nerve bundles (105) and synthesize neurotoxic mediators such as nitric oxide (NO) and TNF- , which may be released near the axons causing neuronal damage (106,107). However, the interactions that lead to astrocyte migration in glaucoma are complex and need to be modeled in vitro. Previous work in our laboratory demonstrates that human ONH

synthesize integrin 1. As a fibronectin receptor, integrin 1 causes astrocytes to migrate toward where the extracellular fibronectin is located, leaving the areas without extracellular fibronectin as cell-free cavities. Astrocytes form stress fibers in the cell processes extending along the extracellular fibronectin. Reorganization of the ECM, including fibronectin, is observed in glaucomatous optic nerves, especially in the lamina cribrosa region (108). The redistributed extracellular fibronectin may be the scaffold for migration of reactive astrocytes. Many other genes related with cell migration are rapidly upregulated by the EGFR pathway in astrocytes. These genes include fibroblast growth factor receptor 3, Unc-5, myosin, paxillin, cadherin, microtubuleassociated protein-1b and microtubule-associated protein-6, and nesting (62). The morphology of the disordered astrocytes in vivo may result from the guided migration of reactive astrocytes regulated by the EGFR pathway to remodel the extracellular matrices.

Cell Adhesion

Reactive astrocytes express several cell adhesion molecules including cadherins, integrins, and neural cell adhesion molecules (NCAM) in CNS degenerations (109,110). Three NCAM isoforms result from alternative splicing of a single gene: two major transmembrane isoforms, denoted NCAM140 and NCAM180 and NCAM120 a glycosyl- phosphatidylinositol-linked isoform (111). The adhesive properties of NCAM depend on alternative splicing of the primary transcript or posttranslational modifications, such as polysialylation (111,112). Activation by extracellular cues of cellular signaling pathways regulate alternative splicing (113). Studies suggest that c-Jun, a transcription factor, regulates alternative splicing of NCAM pre-mRNA and the synthesis of NCAM140 (114). In the adult human ONH, quiescent astrocytes and lamina cribrosa cells express NCAM140 as the predominant isoform (115). NCAM120 and NCAM180 are not expressed by quiescent ONH astrocytes in vivo or in vitro (45,115). In the human glaucomatous ONH, reactive astrocytes differentially express NCAM180 mRNA and protein (115). Injury to the mouse optic nerve increases expression of NCAM180 in the axons and in reactive astrocytes (116).

Integrin receptors cluster at focal adhesion complexes, where cells anchor into the ECM. Binding at these sites can activate intracellular signaling pathways to modify cell behavior. Microarray analysis of gene expression showed downregulation of - integrins by reactive astrocytes in glaucoma provided evidence of profound changes in the adhesion state, matrix specificity, and integrin signaling of glaucomatous astrocytes (72). Integrins affect important astrocyte functions including cell migration, differentiation, adhesion to substrates, and target recognition (72).

As part of the changes from quiescent astrocytes to reactive astrocytes, glaucomatous ONH astrocytes exhibit differential upregulation of ephrinB1 and ephrinB2 in human glaucomatous astrocytes compared with normal age-matched controls (117). Activation of the EphB1/ephrinB1 pathway in glaucomatous astrocytes represents a recapitulation of a developmental program that may play a protective role during optic nerve degeneration by preserving the blood–nerve barrier and preventing invasion of non-neural cells into areas of axonal loss, as an attempt to provide boundaries to the axonal damage.

ECM Synthesis

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. Tenascin expressed by reactive astrocytes in the glaucomatous ONH may represent such a barrier (118). 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 TGF- , preventing neurite growthpromoting effects (119).

There is substantial evidence that astrocytes are responsible for the normal maintenance of the ECM in normal ONH and that reactive astrocytes remodel the ECM in response to elevated IOP in human and experimental glaucoma (45,120–123). 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 (121,124). Previous studies indicate that abnormal elastin synthesis in experimental glaucomatous optic neuropathy in the monkey is specific to elevated IOP and not secondary to axonal loss (121). The mechanisms by which elevated IOP induces enhanced elastin synthesis in laminar astrocytes are unknown but differ from those involved in acute axonal injury such as transection, where inflammation and breakdown of the blood–nerve barrier occur (121). In vitro models of elevated HP demonstrate that ONH astrocytes increase synthesis and secrete soluble elastin into the media (125).

Proteoglycans are secreted from astrocytes to form the organized ECM that surrounds neurons and axons. The expression and metabolism of neural proteoglycans change during development, after brain injury, and under certain pathological conditions (126). Aggrecan 1 (chondroitin sulfate proteoglycan 1) is a member of the chondroitin sulfate proteoglycan (CSPG1) family. The members of this family are widely expressed in most tissues including the CNS (127,128). Aggrecan 1 can interact with various molecules including growth factors, cell adhesion molecules, and ECM molecules. In the CNS, CSPG1 family members are involved in various events such as cell proliferation, cellular differentiation, axonal growth, pathfinding, and synaptogenesis. Microarray analysis showed that aggrecan 1 mRNA was expressed abundantly in normal astrocytes and was markedly downregulated in glaucomatous ONH astrocytes (see Fig. 6). Aggrecan 1 is the core protein of chondroitin sulfate proteoglycans, which are present in the ECM of the normal human lamina cribrosa (129–131). The decrease of aggrecan 1 expression by astrocytes in the glaucomatous ONH may lead to changes in structure and loss of axonal support at the level of the lamina cribrosa in vivo.

Microarray analysis revealed that the expression profiles of some ECM protein mRNAs were different in glaucomatous ONH astrocytes compared with normal ONH astrocytes (72). The expression level of collagen type XI 1 mRNA (COL11A1) was higher in astrocytes from glaucomatous eyes than from normal eyes. Quantitative RTPCR confirmed the expression differences for COL11A1 between cell cultures from normal and glaucomatous eyes (see Fig. 6) (72). Collagen type XI was localized in the nerve bundles and in association with blood vessels in the normal ONH. In the glaucomatous ONH, increased deposition of collagen type XI was observed in the ECM. Colocalization of GFAP with collagen type XI in ONH astrocytes confirmed that the source of collagen type XI is the reactive astrocyte. Collagen type XI is a fibrillar

Fig. 6. Level of mRNA expression in normal and glaucomatous optic nerve head (ONH) astrocytes by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). (A) Collagen type XI -1 chain expression level was low in all normal samples and increased in glaucoma samples. (B) Aggrecan 1 expression decreased in glaucoma samples compared with that in normal samples in agreement with GeneChip data. The relative expression level of each gene was determined in four normal (N1–N4) and four glaucoma (G1–G4) cell cultures. The expression level of each mRNA is indicated relative to the N1 sample. Standard curves were used to calculate relative expression levels of collagen type XI, aggrecan 1, and GAPDH mRNA in the samples. Glyceraldehyde phosphode hydrogenase (GAPDH) mRNA served as internal control for normalization of the mRNA amount in each sample (from Hernandez MR, Agapova OA, Yang P, et al. Glia 2002;38:45–64, with permission).

collagen, which plays a key role in regulating collagen fibril diameter (132). Collagen type II 1 mRNA (COL2A1) was also increased in glaucomatous ONH astrocytes. Collagen types II and XI are usually expressed together and are present in cartilage and in the vitreous body of the eye (133).

Members of the TGF-ß family of growth factors are powerful regulators of synthesis and degradation of ECM in many tissues. TGF-ß1 and TGF-ß2 expression by ONH astrocytes increased in human glaucoma (134). TGF-ß2 treatment of cultured human ONH astrocytes leads to elevated expression of the basement membrane collagen type 4 1, collagen type 1 1 and thrombospondin-1 (TSP1) suggesting that this growth factor may be involved in the remodeling of the ECM in the ONH in glaucoma (135).

ECM Degradation by Reactive Astrocytes

Matrix metalloproteinases (MMPs), or matrixins, degrade ECM components such as collagens, proteoglycans, elastin, laminin, fibronectin, and glycoproteins in normal and pathologic conditions (136). 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 (126). 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 (137); whereas, in mice deficient in MMP2, there was no neuroprotection of RGC (138). Reactive astrocytes are the main source of MMP9 activity, and ERK and p38 MAP kinases mediate secretion of MMP9 after mechanical injury (139). In the glaucomatous optic nerve, reactive astrocytes express increased MPP1 and MT1-MMP but do not express MMP9, MMP3, or MMP7, suggesting highly regulated proteolytic activity. MT1-MMP was upregulated in reactive ONH astrocytes in experimental glaucoma compared with the control untreated eyes; in contrast, MT1-MMP was not upregulated in ONH astrocytes after optic nerve transection (see Fig. 7) suggesting that the effects of chronic elevated IOP in ONH astrocytes are specific and different from those events caused by acute trauma. Our published data suggest that elevated IOP in glaucoma can induce increased expression of MT-MMP1 leading to detachment of astrocytes from the underlying basement membranes and migration through the ECM. Subsequently, MMP1 permits migration of astrocytes throughout the ECM of the lamina cribrosa into the nerve bundles, where MMP1, if not counterbalanced by TIMP1, will continue to degrade the scant ECM around the axons and interfere with axon survival. Our data suggest that feedback regulation of MMP activity or expression in reactive astrocyte occurs by interactions between MMPs, TIMPs, growth factors/cytokines, and ECM substrates with astrocyte membrane or intracellular components (140,141).

Transcriptional Regulation in ONH Astrocytes

In experimental eyes with chronic ocular hypertension, stress associated with elevated IOP induced localization of c-Jun and c-Fos transcription factors into the nucleus of astrocytes but not of other cell types present in the ONH (Hashimoto et al., 2005).Transcription factors are associated with the regulatory pathways that convert extracellular mechanical stimuli into a cellular response Expression of the c-Fos and c-Jun was increased after exposure to elevated HP as demonstrated earlier in responses to mechanical stress (142). c-Fos is a leucine zipper protein that forms dimers with proteins of the Jun family in the nucleus, thereby forming the transcription factor complex AP-1. Western blot analysis showed that under elevated pressure, 43 kDa c- Fos protein levels decreased in the cytoplasm and increased in nuclear extracts of ONH astrocytes, suggesting that HP induces c-Fos protein translocation from the cytoplasm into the nuclear compartment (see Fig. 8). The activation of the immediate early genes c-Jun and c-Fos in astrocytes in the glaucomatous ONH may be part of responses that affect the axons of the RGCs. Known gene products of reactive astrocytes that are regulated at the level of transcription by c-Fos and c-Jun may be protective or

Fig. 7. MT-MMP1 expression in monkey optic nerves from eyes with experimental glaucoma and with optic nerve transection. (A–D) Double immunostaining for MT1-MMP (red) and glial fibrillary acidic protein (GFAP) (green) in the optic nerve head. (A) Weak immunoreactivity of MT1-MMP in the normal optic nerve head. (B) MT1-MMP immunoreactivity markedly increased in astrocytes of the lamina cribrosa (LC) (arrows) in the contralateral eye with experimental glaucoma. (C and D) Low immunoreactivity for MT1-MMP is present in the LC of normal and transected contralateral eye. NB, nerve bundles; CP, cribriform plates; V, blood vessels; Fb, fibroblasts; Ma, macrophages. Scale bars 20 μ. (For color version of this figure please see the accompanying CD). (from Agapova OA, Kaufman PL, Lucarelli MJ, et al. Brain Res 2003;967:132–143, with permission).

may be part of the injury response to elevated IOP in RGCs. Inhibition of c-Fos in models of cerebral ischemia reduces neuronal apoptosis; however, these studies did not focus on astrocytes (143,144), and in astrocytes, c-Fos activation leads to astrogliosis which is modulated by insulin growth factor I in response to neurotoxic stimulus (145). Increased expression and activation of c-Jun in astrocytes is part of the responses to neural degeneration (146). Astrocyte TGFsignaling through the c-Jun–AP1 pathway is neuroprotective to neurons (147) suggesting that induction of c-Jun in ONH astrocytes under elevated IOP may be neuroprotective for axons through the activity of TGF- . Stimulation of endothelin B receptors by endothelin-1 (ET-1) in astrocytes induced increased expression of c-Fos mRNA in astrocytes that results in activation of the ERK–MAPK pathway (148).

Microarray analysis of ONH astrocytes responses to HP demonstrated the upregulation of several transcription factors (86). Smad3 is phosphorylated by activated activin/TGF-ß receptors (149). Smad3 signaling pathway is essential for the increase in Rho activity, induction of stress fiber formation, and increased production of all collagen types in response to TGF-ß1 stimulation (150). Smad3 interacts with c-Jun and c-Fos to mediate TGF-ß-induced immediate early transcriptional activation of

target gene promoters (151). Expression of Smurf2 was decreased under exposure to HP. Smurf2 is a Smad-ubiquitin ligase that induces the ubiquitination and degradation of the TGF-ß–Smad2/3 pathway to regulate TGF-ß signaling (152).

Egr2 (Krox-20) is upregulated at 24 and 48 h in astrocytes exposed to pressure. Egr2 belongs to the Egr family of zinc-finger transcription factors, which is involved in myelination in the CNS and PNS, and mutations on this gene produce various neuropathies (153). Egr-2 can stimulate the transcriptional activity of c-Fos and c-Jun; however, the downstream events of these interactions are unknown. The induction of c-Fos, c-Jun, and Egr-2 in response to HP suggests that a cocktail of immediate early genes might be required for the responses of ONH astrocytes to pressure in vivo and in vitro.

Among the transcription factors that may be involved in response to chronic stress is NFkB, a well-studied transcription factor, which is expressed constitutively in the CNS and activated under pathological conditions and in neurological diseases (154). We demonstrated that NFkB expression is upregulated in glaucomatous ONH astrocytes in culture and that in vivo NFkB was localized to the nucleus of ONH astrocytes in control and ExpG eyes by immunohistochemistry (see Fig. 9) (155). Constitutive nuclear localization of NFkB has been observed in neurons in vivo. The basis for constitutive expression in the CNS is unclear, but NFkB appears to play a role in neuroprotection (156). NFkB is activated by ROS, and positively regulates anti-oxidative enzymes like superoxide dismutase (SOD2) (157) and glutamate-cysteine ligase (GCL) (158).

Role of Androgens in Glaucomatous Optic Neuropathy

Previous data from our laboratory demonstrate that expression and activity of 3-alpha dihydrosteroid dehydrogenases (HSD) isoforms; AKR1C1, AKR1C2 and AKR1C3 are upregulated in glaucomatous astrocytes ONH in vitro and in vivo. Increased 3-alpha HSD expression was further demonstrated in human ONH with POAG and in the ONH of monkeys with experimental glaucoma in vivo (see Fig. 10) (159). Peroxidation of polyunsaturated fatty acids, particularly arachidonic acid and the metabolism of steroid hormones, leads to the generation of reactive aldehydes, such as 4-hydroxy-2-trans- nonenal (HNE), which is an aldehydic product of membrane lipid peroxidation and a powerful agent of lipid peroxidation. Our data in glaucomatous astrocytes indicate increased expression of prostaglandin D2 and prostaglandin E2 synthases in these cells, which also leads to HNE production (72). It has been shown that HNE is metabolized through AKR1C1 (160); thus, this enzyme may play an unrecognized role in a response mounted to counteract oxidative stress in the glaucomatous ONH. AKR1C1 represent alternative GSH-independent/NADPH-dependent routes for the reductive elimination of HNE or other ROS. Of these, AKR1C1 provides an inducible cytosolic barrier to HNE following ROS exposure (161).

A recent study found that normal ONH astrocytes exhibit a strong antioxidant response to HNE treatment by inducing the transcription factors cFOS, NFkB, and Nrf2, which can all upregulate the expression of GCLC, to produce more GSH in the cell (161). AKR1C1 is also upregulated after HNE treatment to reduce HNE to DHN, independent of GSH availability in the cells. Oxidative stress has been identified as a component of the pathogenesis of glaucoma (162,163). Moreno et al. (164) analyzed