Influence of dexamethasone-induced matrices on the TM transcriptome.

Pathologic bidirectional interactions between the extracellular matrix (ECM) and cells within the human trabecular meshwork (hTM) contribute to ocular hypertension. An in vitro model is needed to study these cell-matrix interactions and their effect on outflow homeostasis. This study aimed to determine whether pathogenic ECM derived from dexamethasone (DEX)-treated hTM cultures induces clinically relevant glaucoma-like changes in healthy hTM cells at the transcriptional level. Corneoscleral rims from non-glaucoma donors were used to isolate primary hTM cells after validation according to the consensus recommendations for TM culture. Normal hTM cells (n = 5) were plated on a coverslip and treated with 100 nM DEX or ethanol for four weeks. These cultures were then decellularized, plated with primary hTM cells, and allowed to grow for another 72 h. RNA was extracted from these hTM cells for stranded total RNA-Seq. Sequencing libraries prepared using the Zymo-Seq RiboFree Total RNA library kit were pooled and sequenced using Illumina NovaSeq 6000. After quality control, sequence reads were aligned to the human genome build hg19. Differential expression (DE) analyses were performed using paired multi-factorial ANOVA. The expression of several DE genes associated with glaucoma (ANGPTL2, PDE7B, C22orf23, COL4A1, ADAM12, IFT122, SEMA6C) was validated using EvaGreen-based Droplet Digital PCR (ddPCR) assays. Gene ontology analyses of the DE genes were performed using the PANTHER and NDEx IQA databases, and functional analyses were performed with the DAVID Bioinformatics software. Using a cutoff of p-value <0.05 and fold change ≥2.0, our differential analysis identified 267 up- and 135 down-regulated genes in DEX-induced ECM-treated cells compared to the control. These differentially expressed genes were found to play a significant role in pathways such as cytokine and oxidative stress-induced inflammation, integrin signaling, matrix remodeling, and angiogenesis. These findings were further supported by previously performed proteomics studies using the same model. Using ddPCR, we validated the expression of seven genes associated with the risk of primary open-angle glaucoma. These results not only provide support for the pathogenic ECM model of steroid-induced glaucoma, but also demonstrate that the pathologic changes induced by this model are indeed found at the transcriptional level. These findings further demonstrate that matrix changes significantly influence cell expression profiles, which enable further understanding of the molecular mechanisms underlying glaucomatous changes in the TM. However, future studies with a larger and more diverse set of samples and longer time points are needed to confirm the utility of this model for mechanistic studies.

Characterization of extracellular matrix deposited by segmental trabecular meshwork cells.

PURPOSE: Biophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models (in vitro or ex vivo) to study this phenomena. METHODS: Primary trabecular meshwork cells were isolated from segmental flow regions, and cells were cultured for 4 weeks in the presence or absence or dexamethasone to obtain cell derived matrices (CDM). The biomechanical attributes of the CDM, composition of the matrisome, and incidence of crosslinks were determined by atomic force microscopy and mass spectrometry. RESULTS: Data demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. However, no correlations were observed between elastic moduli and presence or absence of mature and immature crosslinks in the CDMs. CONCLUSION: Regardless of a direct correlation between matrix stiffness and crosslinks, we observed distinct differences in the composition and mechanics of the matrices deposited by segmental flow cells. These results suggest distinct differences in cellular identify and likely a basis for mechanical memory post isolation and culture. Nevertheless, we conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

Endogenous expression of Notch pathway molecules in human trabecular meshwork cells.

PURPOSE: Cells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-ß), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity. METHODS: Primary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2-6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment. RESULTS: Innate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions. CONCLUSIONS: We demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.

Circumferential trabecular meshwork cell density in the human eye.

The cells residing in the trabecular meshwork (TM) fulfill important roles in the maintenance of the tissue and the regulation of intraocular pressure (IOP). Here we examine (i) TM cell distribution along the circumference of the human eye, (ii) differences in TM cell density between regions of high and low outflow, and (iii) whether TM cell distribution in eyes from donors with primary open angle glaucoma (POAG) differs from that of normal eyes. Toward this end, the TM cell density from 12 radial segments around the circumference of the TM of human donor eyes (n = 6) with and without POAG was determined using histochemical methods. Areas of high, median, and low outflow were mapped in a different set of human donor eyes that were perfused in organ culture, and TM cell densities in these areas were determined in normal (n = 11) and POAG eyes (n = 6). Our analysis of 1380 tissue sections taken from the first set of six eyes shows that the average TM cell density of these six eyes ranges from 15.5 to 23.7 cells/100 µm and is negatively correlated to the maximum IOP recorded for each donor eye (R2 = 0.91). Considerable differences in TM cell density exist among sections taken from the same segment of an individual eye (average standard deviation = 2.35 cells/100 µm). Less variability is observed among the segment averages across the eye's circumference (average standard deviation = 1.03 cells/100 µm). Variations in cell density are similar between normal and POAG eyes and are not correlated with the anatomic position of examined segments (p = 0.745). The analysis of the second set of eyes shows that TM regions of high outflow display a TM cell density similar to regions of median or low outflow in both normal and POAG eyes. Together these findings demonstrate that (i) statistically significant differences in TM cell density exist along the circumference of each eye (ii) TM cellularity is not correlated with segmental flow and (iii) eyes with POAG, while displaying reduced TM cellularity, do not exhibit higher TM cell variability than normal eyes. Finally, statistical analysis of sections and segments indicates that measurements from 12 sections taken from 2 segments provide a reliable and cost-effective estimate of a human eye's TM cell density.

Elevated pressure influences relative distribution of segmental regions of the trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma and is the only treatable feature of the disease. There is a correlation between elevated pressure and homeostatic reductions in the aqueous humor outflow resistance via changes in the extracellular matrix of the trabecular meshwork. It is unclear how these extracellular matrix changes affect segmental patterns of aqueous humor outflow, nor do we understand their causal relationship. The goal of this study was to determine whether there are changes in the segmental outflow regions with perfusion in normal eyes, and whether these regions change during the IOP homeostatic response to elevated pressure. Using human anterior segment perfusion organ culture, we measured the amount of high flow (HF), intermediate flow (MF), and low flow (LF) regions before and after 7 days of perfusion at either physiologic pressure ("1x") or at elevated pressure ("2x"). We found a small but significant decrease in the amount of HF regions over 7 days perfusion at 1x pressure, and a twofold increase in the amount of MF regions over 7 days perfusion at 2x pressure. Small positional differences, or shifts in the specific location of HF, MF, or LF, occurred on a per eye basis and were not found to be statistically significant across biological replicates. Differences in the amount of segmental flow regions of contralateral eyes flowed at 1x pressure for 7 days were small and not statistically significant. These results demonstrate that perfusion at physiologic pressure had little effect on the distribution and amount of HF, MF and LF regions. However, the overall amount of MF regions is significantly increased in response to perfusion at elevated pressure during IOP homeostatic resistance adjustment. The amount of both HF and LF regions was decreased accordingly suggesting a coordinated response in the TM to elevated pressure.

Isolation and Characterization of primary human trabecular meshwork cells from segmental flow regions: New tools for understanding segmental flow.

Segmental flow in the human trabecular meshwork is a well-documented phenomenon but in depth mechanistic investigations of high flow (HF) and low flow (LF) regions are restricted due to the small amount of tissue available from a single donor. To address this issue we have generated and characterized multiple paired HF and LF cell strains. Here paired HF and LF cell strains were generated from single donors. Cells were characterized for growth and proliferation, as well as gene and protein expression of potential segmental region markers. Cells isolated from HF and LF regions have similar growth and proliferation rates. Gene expression data reveals vascular cell adhesion protein 1 (VCAM1), thrombospondin 2 (THBS2), and tissue inhibitor of metalloproteinase 1 (TIMP1) are potential markers of LF cells in vitro. Protein expression of VCAM1, THBS2 and TIMP1 are complex and may reflect the dynamic nature of the TM. Initial protein expression levels of these genes is either similar between HF and LF cells (VCAM1, THBS2), or higher in HF compared to LF in some strains (TIMP1). However, after long term culture LF cells express higher levels of VCAM1, TIMP1 and THBS2 protein compared to HF cells. HF and LF cell strains are a powerful new tool that enable understanding segmental flow allowing for multiple experiments on the same genetic background.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Pressure-induced expression changes in segmental flow regions of the human trabecular meshwork.

Elevated intraocular pressure (IOP) is thought to create distortion or stretching of the juxtacanalicular and Schlemm's canal cells and their extracellular matrix (ECM) leading to a cascade of events that restore IOP to normal levels, a process termed IOP homeostasis. The ECM of the trabecular meshwork (TM) is intricately involved in the regulation of outflow resistance and IOP homeostasis, as matrix metalloproteinase (MMP)-initiated ECM turnover in the TM is necessary to maintain outflow facility. Previous studies have shown ECM gene expression and mRNA splice form differences in TM cells in response to sustained stretch, implicating their involvement in the dynamic process of IOP homeostasis. The observation that outflow is segmental around the circumference of the eye adds another layer of complexity to understanding the molecular events necessary to maintaining proper outflow facility. The aim of this work was to identify molecular expression differences between segmental flow regions of the TM from anterior segments perfused at either physiological or elevated pressure. Human anterior segments were perfused in an ex vivo model system, TM tissues were extracted and quantitative PCR arrays were performed. Comparisons were made between high flow and low flow regions of the TM from anterior segments perfused either at normal (8.8 mmHg) or at elevated (17.6 mmHg) perfusion pressure for 48 h. The results are presented here as independent sets: 1) fold change gene expression between segmental flow regions at a single perfusion pressure, and 2) fold change gene expression in response to elevated perfusion pressure in a single flow region. Multiple genes from the following functional families were found to be differentially expressed in segmental regions and in response to elevated pressure: collagens, ECM glycoproteins including matricellular proteins, ECM receptors such as integrins and adhesion molecules and ECM regulators, such as matrix metalloproteinases. In general, under normal perfusion pressure, more ECM genes were enriched in the high flow regions than in the low flow regions of the TM, whereas more ECM genes were found to be enriched in low flow regions of the TM in response to elevated perfusion pressure. Thus it appears that a limited subset of ECM genes is differentially regulated in both high and low flow regions and in response to elevated pressure. Some of these same ECM genes have previously been shown to be involved in the pressure response of stretched TM cells supporting their central role in IOP homeostasis. In general, different ECM gene family members are called upon to produce the response to elevated pressure in different segmental regions of the TM.

Biological role of prolyl 3-hydroxylation in type IV collagen.

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.

Extracellular matrix in the trabecular meshwork: intraocular pressure regulation and dysregulation in glaucoma.

The trabecular meshwork (TM) is located in the anterior segment of the eye and is responsible for regulating the outflow of aqueous humor. Increased resistance to aqueous outflow causes intraocular pressure to increase, which is the primary risk factor for glaucoma. TM cells reside on a series of fenestrated beams and sheets through which the aqueous humor flows to exit the anterior chamber via Schlemm's canal. The outer trabecular cells are phagocytic and are thought to function as a pre-filter. However, most of the outflow resistance is thought to be from the extracellular matrix (ECM) of the juxtacanalicular region, the deepest portion of the TM, and from the inner wall basement membrane of Schlemm's canal. It is becoming increasingly evident that the extracellular milieu is important in maintaining the integrity of the TM. In glaucoma, not only have ultrastructural changes been observed in the ECM of the TM, and a significant number of mutations in ECM genes been noted, but the stiffness of glaucomatous TM appears to be greater than that of normal tissue. Additionally, TGFß2 has been found to be elevated in the aqueous humor of glaucoma patients and is assumed to be involved in ECM changes deep with the juxtacanalicular region of the TM. This review summarizes the current literature on trabecular ECM as well as the development and function of the TM. Animal models and organ culture models targeting specific ECM molecules to investigate the mechanisms of glaucoma are described. Finally, the growing number of mutations that have been identified in ECM genes and genes that modulate ECM in humans with glaucoma are documented.

Posttranslational modifications in type I collagen from different tissues extracted from wild type and prolyl 3-hydroxylase 1 null mice.

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.

Mutation in cyclophilin B that causes hyperelastosis cutis in American Quarter Horse does not affect peptidylprolyl cis-trans isomerase activity but shows altered cyclophilin B-protein interactions and affects collagen folding.

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.

Characterization of extracellular matrix deposited by segmental trabecular meshwork cells.

Biophysical and biochemical attributes of the extracellular matrix are major determinants of cell fate in homeostasis and disease. Ocular hypertension and glaucoma are diseases where the trabecular meshwork tissue responsible for aqueous humor egress becomes stiffer accompanied by changes in its matrisome in a segmental manner with regions of high or low flow. Prior studies demonstrate these alterations in the matrix are dynamic in response to age and pressure changes. The underlying reason for segmentation or differential response to pressure and stiffening are unknown. This is largely due to a lack of appropriate models ( in vitro or ex vivo ) to study this phenomena. In this study, we characterize the biomechanical attributes, matrisome, and incidence of crosslinks in the matrix deposited by primary cells isolated from segmental flow regions and when treated with glucocorticosteroid. Data demonstrate that matrix deposited by cells from low flow regions are stiffer and exhibit a greater number of immature and mature crosslinks, and that these are exacerbated in the presence of steroid. We also show a differential response of high or low flow cells to steroid via changes observed in the matrix composition. We conclude that although a mechanistic basis for matrix stiffness was undetermined in this study, it is a viable tool to study cell-matrix interactions and further our understanding of trabecular meshwork pathobiology.

Valve-Like Outflow System Behavior With Motion Slowing in Glaucoma Eyes: Findings Using a Minimally Invasive Glaucoma Surgery-MIGS-Like Platform and Optical Coherence Tomography Imaging.

Purpose: This study aimed to investigate anatomic relationships and biomechanics of pressure-dependent trabecular meshwork and distal valve-like structure deformation in normal and glaucoma eyes using high-resolution optical coherence tomography (HR-OCT). Methods: We controlled Schlemm's canal (SC) pressure during imaging with HR-OCT in segments of three normal (NL) and five glaucomatous (GL) ex vivo eyes. The dissected limbal wedges were studied from 15 locations (5 NL and 10 GL). A minimally invasive glaucoma surgery (MIGS)-like cannula was inserted into the SC lumen, whereas the other end was attached to a switch between two reservoirs, one at 0, the other at 30 mm Hg. A steady-state pressure of 30 mm Hg was maintained to dilate SC and collector channels (CC) during 3D volume imaging. The resulting 3D lumen surface relationships were correlated with internal structural features using an image mask that excluded tissues surrounding SC and CC. While imaging with HR-OCT, real-time motion responses in SC and CC areas were captured by switching pressure from 0 to 30 or 30 to 0 mm Hg. NL vs. GL motion differences were compared. Results: Lumen surface and internal relationships were successfully imaged. We identified SC inlet and outlet valve-like structures. In NL and GL, the mean SC areas measured at the steady-state of 0 and 30 mm Hg were each significantly different (p < 0.0001). Synchronous changes in SC and CC lumen areas occurred in <200 ms. Measured SC area differences at the steady-state 0 and 30 mmHg, respectively, were larger in NL than GL eyes (p < 0.0001). The SC motion curves rose significantly more slowly in GL than NL (p < 0.001). Pressure waves traveled from the cannula end along the SC lumen to CC and deep intrascleral channels. Conclusion: HR-OCT provided simultaneous measurements of outflow pathway lumen surfaces, internal structures, and biomechanics of real-time pressure-dependent dimension changes. We identified SC inlet and outlet valve-like structures. GL tissues underwent less motion and responded more slowly than NL, consistent with increased tissue stiffness. A MIGS-like shunt to SC permitted pulse waves to travel distally along SC lumen and into CC.

Prolyl 3-hydroxylase 1 null mice display abnormalities in fibrillar collagen-rich tissues such as tendons, skin, and bones.

Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans.

Normal and glaucomatous outflow regulation.

Glaucoma remains only partially understood, particularly at the level of intraocular pressure (IOP) regulation. Trabecular meshwork (TM) and Schlemm's canal inner wall endothelium (SCE) are key to IOP regulation and their characteristics and behavior are the focus of much investigation. This is becoming more apparent with time. We and others have studied the TM and SCE's extracellular matrix (ECM) extensively and unraveled much about its functions and role in regulating aqueous outflow. Ongoing ECM turnover is required to maintain IOP regulation and several TM ECM manipulations modulate outflow facility. We have established clearly that the outflow pathway senses sustained pressure deviations and responds by adjusting the outflow resistance correctively to keep IOP within an appropriately narrow range which will not normally damage the optic nerve. The glaucomatous outflow pathway has in many cases lost this IOP homeostatic response, apparently due at least in part, to loss of TM cells. Depletion of TM cells eliminates the IOP homeostatic response, while restoration of TM cells restores it. Aqueous outflow is not homogeneous, but rather segmental with regions of high, intermediate and low flow. In general, glaucomatous eyes have more low flow regions than normal eyes. There are distinctive molecular differences between high and low flow regions, and during the response to an IOP homeostatic pressure challenge, additional changes in segmental molecular composition occur. In conjunction with these changes, the biomechanical properties of the juxtacanalicular (JCT) segmental regions are different, with low flow regions being stiffer than high flow regions. The JCT ECM of glaucomatous eyes is around 20 times stiffer than in normal eyes. The aqueous humor outflow resistance has been studied extensively, but neither the exact molecular components that comprise the resistance nor their exact location have been established. Our hypothetical model, based on considerable available data, posits that the continuous SCE basal lamina, which lies between 125 and 500 nm beneath the SCE basal surface, is the primary source of normal resistance. On the surface of JCT cells, small and highly controlled focal degradation of its components by podosome- or invadopodia-like structures, PILS, occurs in response to pressure-induced mechanical stretching. Sub-micron sized basement membrane discontinuities develop in the SCE basement membrane and these discontinuities allow passage of aqueous humor to and through SCE giant vacuoles and pores. JCT cells then relocate versican with its highly charged glycosaminoglycan side chains into the discontinuities and by manipulation of their orientation and concentration, the JCT and perhaps the SCE cells regulate the amount of fluid passage. Testing this outflow resistance hypothesis is ongoing in our lab and has the potential to advance our understanding of IOP regulation and of glaucoma.

Generating cell-derived matrices from human trabecular meshwork cell cultures for mechanistic studies.

Ocular hypertension has been attributed to increased resistance to aqueous outflow often as a result of changes in trabecular meshwork (TM) extracellular matrix (ECM) using in vivo animal models (for example, by genetic manipulation) and ex vivo anterior segment perfusion organ cultures. These are, however, complex and difficult in dissecting molecular mechanisms and interactions. In vitro approaches to mimic the underlying substrate exist by manipulating either ECM topography, mechanics, or chemistry. These models best investigate the role of individual ECM protein(s) and/or substrate property, and thus do not recapitulate the multifactorial extracellular microenvironment; hence, mitigating its physiological relevance for mechanistic studies. Cell-derived matrices (CDMs), however, are capable of presenting a 3D-microenvironment rich in topography, chemistry, and whose mechanics can be tuned to better represent the network of native ECM constituents in vivo. Critically, the composition of CDMs may also be fine-tuned by addition of small molecules or relevant bioactive factors to mimic homeostasis or pathology. Here, we first provide a streamlined protocol for generating CDMs from TM cell cultures from normal or glaucomatous donor tissues. Second, we document how TM cells can be pharmacologically manipulated to obtain glucocorticoid-induced CDMs and how generated pristine CDMs can be manipulated with reagents like genipin. Finally, we summarize how CDMs may be used in mechanistic studies and discuss their probable application in future TM regenerative studies.

Glucocorticoid-induced cell-derived matrix modulates transforming growth factor ß2 signaling in human trabecular meshwork cells.

Aberrant remodeling of trabecular meshwork (TM) extracellular matrix (ECM) may induce ocular hypertensive phenotypes in human TM (hTM) cells to cause ocular hypertension, via a yet unknown mechanism. Here, we show that, in the absence of exogenous transforming growth factor-beta2 (TGFß2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGFß2 signaling in hTM cells, correlated with overexpression/activity of structural ECM genes (fibronectin, collagen IV, collagen VI, myocilin), matricellular genes (connective tissue growth factor [CTGF], secreted protein, acidic and rich in cysteine), crosslinking genes/enzymes (lysyl oxidase, lysyl oxidase-like 2-4, tissue transglutaminase-2), and ECM turnover genes/enzymes (matrix metalloproteinases-MMP2,14 and their inhibitors-TIMP2). However, in the presence of exogenous TGFß2, VehMs and GIMs activate Smad and non-Smad TGFß2 signaling in hTM cells, associated with overexpression of α-smooth muscle actin (α-SMA), and differential upregulation of aforementioned ECM genes/proteins with new ones emerging (collagen-I, thrombospondin-I, plasminogen activator inhibitor, MMP1, 9, ADAMTS4, TIMP1); with GIM-TGFß2-induced changes being mostly more pronounced. This suggests dual glaucomatous insults potentiate profibrotic signaling/phenotypes. Lastly, we demonstrate type I TGFß receptor kinase inhibition abrogates VehM-/GIM- and/or TGFß2-induced upregulation of α-SMA and CTGF. Collectively, pathological TM microenvironments are sufficient to elicit adverse cellular responses that may be ameliorated by targeting TGFß2 pathway.

Crosslinked Extracellular Matrix Stiffens Human Trabecular Meshwork Cells Via Dysregulating ß-catenin and YAP/TAZ Signaling Pathways.

Purpose: The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating ß-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways. Methods: Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/ß-catenin and YAP/TAZ pathways were determined. Results: At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold (P < 0.001) stiffer, total ß-catenin was unchanged, pß-catenin was elevated, and pGSK3ß was suppressed. Although 10% XCDM had no effect on cytoplasmic ß-catenin levels, it reduced nuclear ß-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear ß-catenin. Conclusions: Increased cytoplasmic TAZ may inhibit ß-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.

Expression of prolyl 3-hydroxylase genes in embryonic and adult mouse tissues.

Collagen requires hydroxylation of its proline residues to achieve proper assembly, structure, and function. Prolyl 4-hydroxylase catalyzes formation of 4-hydroxyproline, which is essential for collagen triple helix formation and stability. Prolyl 3-hydroxylase catalyzes formation of 3-hydroxyproline, which is far less abundant in collagens and whose function remains unclear. Recently mutations in prolyl 3-hydroxylase 1 have been associated with osteogenesis imperfecta, yet the temporal and spatial expression patterns of the prolyl 3-hydroxylase family members during development and in adult tissues remain undefined. By northern blot analysis distinct differences in transcript sizes of the three prolyl 3-hydroxylase genes were detected. Quantitative RTPCR demonstrated tissue-specific differences in prolyl 3-hydroxylase expression, most notable of which were high levels of prolyl 3-hydroxylase 2 in kidney and prolyl 3-hydroxylase 1 expression in embryonic tissues. Finally, in situ hybridization was used to assess spatio-temporal distribution of three prolyl 3-hydroxylases at embryonic days 11-15. Importantly, prolyl 3-hydroxylase 1 was expressed within cartilage condensations of the vertebral bodies and in the aortic arch of the developing heart, whereas prolyl 3-hydroxylase 2 was expressed in developing lens capsule. The prolyl 3-hydroxylase 3 gene showed more generalized expression overlapping somewhat with the other two genes. This report characterizes expression of the three prolyl 3-hydroxylase genes in embryonic and adult mice. Overall these data demonstrate tissue specific prolyl 3-hydroxylase gene expression in both fetal and adult tissues indicating a developmental role for prolyl 3-hydroxylase activity.