Abstract
Primary open-angle glaucoma (POAG) is a leading cause of blindness due to chronic degeneration of retinal ganglion cells and their optic nerve axons. It is associated with disturbed regulation of intraocular pressure, elevated intraocular levels of TGF-β2, aberrant extracellular matrix (ECM) deposition and increased outflow resistance in the trabecular meshwork (TM). The mechanisms underlying these changes are not fully understood. Cell-matrix interactions have a decisive role in TM maintenance and it has been suggested that TGF-β-induced inhibition of matrix metalloproteases may drive aberrant ECM deposition in POAG. Invadopodia and podosomes (invadosomes) are distinct sites of cell-matrix interaction and localized matrix-metalloprotease (MMP) activity. Here, we report on the effects of TGF-β2 on invadosomes in human trabecular meshwork cells. Human TM (HTM) cells were derived from donor tissue and pretreated with vehicle or TGF-β2 (2 ng/ml) for 3d. Invadosomes were studied in ECM degradation assays, protein expression and MMP-2 activity were assessed by western blot and zymography and ECM protein transcription was detected by RT-qPCR. HTM cells spontaneously formed podosomes and invadopodia as detected by colocalization of Grb2 or Nck1 to sites of gelatinolysis. Pretreatment with TGF-β2 enhanced invadosomal proteolysis and zymographic MMP-2 activity as well as MMP-2, TIMP-2 and PAI-1 levels in HTM cell culture supernatants. Rho-kinase inhibition by H1152 blocked the effects of TGF-β2. Concomitant transcription of fibronectin and collagens-1, -4 and -6 was increased by TGF-β2 and fibrillar fibronectin deposits were observed in areas of invadosomal ECM remodelling. In contrast to a current hypothesis, our data indicate that TGF-β2 induces an active ECM remodelling process in TM cells, characterized by concurrent increases in localized ECM digestion and ECM expression, rather than a mere buildup of material due to a lack of degradation. Invadosomal cell adhesion and signaling may thus have a role in POAG pathophysiology.
Highlights
The trabecular meshwork (TM) serves as a dynamic resistor to control the outflow of intraocular fluid and regulate intraocular pressure
The formation of podosome- or invadopodia-like structures (PILS) has been suggested in trabecular meshwork cells based on the colocalization of structural proteins and matrix metalloprotease (MMP)-2 and -14 [15]
Human TM (HTM) cells spontaneously formed characteristic ring-shaped structures, which stained for the podosomal marker Grb2 and colocalized with rings of gelatinolytic activity
Summary
The trabecular meshwork (TM) serves as a dynamic resistor to control the outflow of intraocular fluid and regulate intraocular pressure. The extent of ECM deposition correlates with the severity of optic nerve damage [3]. The cause of these changes and the subsequent increase in outflow resistance are not fully elucidated, but strong evidence points to transforming growth factor beta (TGF-b) as having a major role. Mounting evidence indicates that cell-matrix interactions in the TM have a crucial role in POAG pathophysiology. Actin modulating drugs such as Rho-kinase (ROCK) inhibitors have been shown to lower intraocular pressure [7] and are currently being tested in clinical trials to treat glaucoma patients
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