Specialized Podosome- or Invadopodia-like Structures (PILS) for Focal Trabecular Meshwork Extracellular Matrix Turnover

Glaucoma is a major blinding disease, affecting well over 67 million people worldwide.1,2 Intraocular pressure (IOP) elevation, resulting from a poorly understood obstruction to the outflow of aqueous humor through the trabecular meshwork (TM), is a major risk factor for primary open-angle glaucoma.1,3,4 The fluid resistance to aqueous humor outflow appears to reside within the cribriform or juxtacanalicular (JCT) region of the TM in both normal and glaucomatous eyes.5–7 However, definite identification of the actual site or of the specific component(s) responsible for the normal or the glaucomatous resistance remains uncertain.8–11 Evidence has been presented that both the trabecular ECM and cellular cytoskeleton contribute to the outflow resistance.8,10–20 Because of the tight coupling that exists between cells and their ECMs, separation of their individual contributions has been difficult. Homeostatic IOP regulation by the TM has been reported.21–23 One hypothetical mechanism involves TM cells' sensing the mechanical distortion and stretching of the JCT region produced by increased IOP and responding by initiating ECM turnover.11,21 TM cells have been shown to sense and respond to mechanical stretching.21–32 In anterior segment organ culture, increasing the levels or activity of MMPs within the TM increases outflow facility while inhibiting the endogenous MMP activity decreases outflow facility.14 This fact argues strongly that ongoing ECM turnover is necessary to maintain the outflow resistance and that changing ECM turnover can modify the resistance. In addition to increasing MMP activity and thus initiating ECM degradation, biosynthetic replacement of the degraded ECM with new ECM components with a slightly modified composition seems necessary to facilitate modest homeostatic adjustment of the outflow resistance.11 This process would have to be highly controlled and accurately directed to keep from disorganizing the outflow resistance and disrupting the structure and function of the JCT region. Intuitively, it seems likely that ECM turnover would occur at or near the JCT cells' surface and in a very focal and controlled manner. To date, no specific mechanism for such focal ECM turnover control in the TM has been postulated. Structures involved in focal ECM degradation has been identified on several types of cells.33 These structures, commonly referred to as podosomes or invadopodia, are specialized cell adhesion sites that are also involved in ECM degradation. They are central to macrophage ECM degradation, osteoclast bone resorption, metastatic cell invasion, diapedesis, cell motility, and migration.34–40 Similar structures have been observed under some conditions in fibroblasts, endothelial cells, smooth muscle cells, and epithelial cells.37,38 The size, shape, and molecular organization of these structures in various cell types are relatively diverse.36–38,41 Simple podosomes, as are commonly found on the ventral surface of macrophages, are approximately 500 nm in diameter, with a vertical F-actin core and numerous actin-binding proteins and are surrounded by a ring of talin, paxillin, and vinculin.37,38 Their formation is thought to be associated with cortactin clustering at discrete microdomains on the ventral surface of cells, perhaps recruited directly or indirectly by cdc42 and sometimes near sites where stress fibers insert into adhesion plaques or focal adhesions. N-WASP (neural-Wiscott-Aldrich syndrome protein) and Arp 2/3 (actin-related protein) are involved in branching of F-actin microfilaments and play important roles in podosome or invadopodia formation.42,43 Invadopodia have longer lives and may evolve from simple podosomes,36 which normally last only minutes to an hour. In some types of cells, 5-μm circular rosettes and larger complex aggregates or clusters of podosomes with less uniform structures are observed.36–38,41 Components common to PILS include: cortactin, α-actinin, caldesmon, gelsolin, dynamin, F-actin, ARP 2/3, N-WASP, MMP-2, MMP-14, MMP-9, TIMP-2, phosphotyrosine, Src, cdc42, cofilin, vinculin, talin, paxillin, tubulin, and in some cases, integrin-αVβ3. All these components are also found in other cellular or cell-associated structures, but their confluence is generally considered indicative of podosomes or invadopodia.34–40 However, because of the rapidly evolving structures of podosomes and invadopodia, the relative temporal distributions of these components is not well defined and is probably variable. The ECM degradation component of the IOP homeostatic mechanism involving mechanical stretching of the JCT region is mediated at least in part by MMP-2 and -14.14,21,22 MMP-2 (also known as gelatinase A or type IV collagenase) is different from the other MMPs, in that its activation involves recruitment to the cell surface by TIMP-2 where it is activated by MMP-14 (also known as membrane type 1 MMP).44–49 While evaluating modulation of MMP-2 and -14 by agents that affect outflow facility, we noted that these two proteinases colocalized on the ventral surface of TM cells. These regions of colocalization were unusual but very distinctive, often appearing as a circle, uneven oval or a skewed irregular triangle or rhombus. They were common on TM cells and, subjectively, appeared to be increased by mechanical stretching or the addition of TNFα, TGFβ, or serum (not shown). Somewhat similar structures had been reported for podosomes and invadopodia.37,50–53 We therefore investigated whether these MMP-2 and -14 colocalization regions could be podosome- or invadopodia-like structures (PILS) and whether they exhibited dynamic properties that could be related to their putative ECM turnover function in the JCT.