Search

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 vitroorex 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.

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 (TGFβ2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGFβ2 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 TGFβ2, VehMs and GIMs activate Smad and non-Smad TGFβ2 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-TGFβ2-induced changes being mostly more pronounced. This suggests dual glaucomatous insults potentiate profibrotic signaling/phenotypes. Lastly, we demonstrate type I TGFβ receptor kinase inhibition abrogates VehM-/GIM- and/or TGFβ2-induced upregulation of α-SMA and CTGF. Collectively, pathological TM microenvironments are sufficient to elicit adverse cellular responses that may be ameliorated by targeting TGFβ2 pathway.

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-β), 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 (6h and 24h), or (iii) cultured on 3kPa and 80kPa 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 6h 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.

Purpose: GPR158 is a newly characterized family C G-protein-coupled receptor, previously identified in functional screens linked with biological stress, including one for susceptibility to ocular hypertension/glaucoma induced by glucocorticoid stress hormones. In this study, we investigated GPR158 function in the visual system. Methods: Gene expression and protein immunolocalization analyses were performed in mouse and human brain and eye to identify tissues where GPR158 might function. Gene expression was perturbed in mice, and in cultures of human trabecular meshwork cells of the aqueous outflow pathway, to investigate function and mechanism. Results: GPR158 is highly expressed in the brain, and in this study, we show prominent expression specifically in the visual center of the cerebral cortex. Expression was also observed in the eye, including photoreceptors, ganglion cells, and trabecular meshwork. Protein was also localized to the outer plexiform layer of the neural retina. Gpr158 deficiency in knockout (KO) mice conferred short-term protection against the intraocular pressure increase that occurred with aging, but this was reversed over time. Most strikingly, the pressure lowering effect of the acute stress hormone, epinephrine, was negated in KO mice. In contrast, no disruption of the electroretinogram was observed. Gene overexpression in cell cultures enhanced cAMP production in response to epinephrine, suggesting a mechanism for intraocular pressure regulation. Overexpression also increased survival of cells subjected to oxidative stress linked to ocular hypertension, associated with TP53 pathway activation. Conclusions: These findings implicate GPR158 as a homeostatic regulator of intraocular pressure and suggest GPR158 could be a pharmacological target for managing ocular hypertension.

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 β-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/β-catenin and YAP/TAZ pathways were determined. Results At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-e(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 β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-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 β-catenin. Conclusions Increased cytoplasmic TAZ may inhibit β-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.

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.

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 multi-factorial 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.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Ocular hypertension is a causal risk-factor to developing glaucoma. This is associated with stiffening of the trabecular meshwork (TM), the primary site of resistance to aqueous-humor-outflow. The mechanisms underlying this stiffening or how pathologic extracellular matrix (ECM) affects cell function are poorly understood. It is recognized that mechanotransduction systems allow cells to sense and translate the intrinsic biophysical properties of ECM into intracellular signals to control gene transcription, protein expression, and cell behavior. Using an anterior segment perfusion model, we document that there are significantly more low flow regions that are much stiffer, and fewer high flow regions that are less stiff in glaucomatous TM (GTM) when compared to non-glaucomatous TMs (NTM). GTM tissue also has fewer cells overall when compared with NTM tissue. In order to study the role of pathologic ECM in glaucoma disease progression, we conducted studies using cell derived matrices (CDM). First, we characterized the mechanics, composition and organization of fibronectin in ECM deposited by GTM and NTM cells treated with glucocorticosteroids. Then, we determined that these GTM-derived ECM are able to induce stiffening of normal NTM cells, and alter their gene/protein expression to resemble that of a glaucomatous phenotype. Further, we demonstrate that GTM-derived ECM causes endoplasmic reticular stress in NTM. They also became resistant to being reorganized by these NTM cells. These phenomena were exacerbated by ECMs obtained from steroid treated glaucoma model groups. Collectively, our data demonstrates that CDMs represent a novel tool for the study of bidirectional interactions between TM cells and their immediate microenvironment. Statement of Significance Extracellular matrix (ECM) changes are prevalent in a number of diseases. The precise mechanisms by which changes in the ECM contribute to disease progression is unclear, primarily due to absence of appropriate models. Here, using glaucoma as a disease model, we document changes in cell derived matrix (CDM) and tissue mechanics that contribute to the pathology. Subsequently, we determine the effect that ECMs from diseased and healthy individuals have on healthy cell behaviors. Data emanating from this study demonstrate that CDMs are a potent tool for the study of cell-ECM interactions.

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.

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.

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.

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.

Purpose The extracellular matrix (ECM) of the trabecular meshwork (TM) modulates resistance to aqueous humor outflow, thereby regulating IOP. Glaucoma, a leading cause of irreversible blindness worldwide, is associated with changes in the ECM of the TM. The elastic modulus of glaucomatous TM is larger than age-matched normal TM; however, the biomechanical properties of segmental low (LF) and high flow (HF) TM regions and their response to elevated pressure, are unknown. Methods We perfused human anterior segments at two pressures using an ex vivo organ culture system. After extraction, we measured the elastic modulus of HF and LF TM regions by atomic force microscopy and quantitated protein differences by proteomics analyses. Results The elastic modulus of LF regions was 2.3-fold larger than HF regions at physiological (1×) pressure, and 7.4-fold or 3.5-fold larger than HF regions at elevated (2×) pressure after 24 or 72 hours, respectively. Using quantitative proteomics, comparisons were made between HF and LF regions at 1× or 2× pressure. Significant ECM protein differences were observed between LF and HF regions perfused at 2×, and between HF regions at 1× compared to 2× pressures. Decorin, TGF-β-induced protein, keratocan, lumican, dermatopontin, and thrombospondin 4 were common differential candidates in both comparisons. Conclusions These data show changes in biomechanical properties of segmental regions within the TM in response to elevated pressure, and levels of specific ECM proteins. Further studies are needed to determine whether these ECM proteins are specifically involved in outflow resistance and IOP homeostasis.

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. Not only have ultrastructural changes been observed in the ECM of the TM in glaucoma, and a significant number of mutations in ECM genes are known to be associated with glaucoma, but the stiffness of glaucomatous TM appears to be greater than that of normal tissue. Additionally, TGFβ2 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.

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.

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.

Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm’s canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

SummaryProlyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap−/− bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.

Purpose The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements. Methods Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward. Results The elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glaucomatous hTM stiffness (2.75 ± 1.19 kPa). None of these differences were statistically significant. TM in HF wedges was softer than that in LF wedges for both normal and glaucomatous eyes based on the inverse FEM approach but not by AFM. Outflow facility was significantly correlated with TM stiffness estimated by FEM in six human eyes (P = 0.018). Conclusions TM stiffness is higher, but only modestly so, in glaucomatous patients. Outflow facility in both normal and glaucomatous human eyes appears to associate with TM stiffness. This evidence motivates further studies to investigate factors underlying TM biomechanical property regulation.

Mutations in the cartilage oligomeric matrix protein (COMP) gene result in pseudoachondroplasia (PSACH), which is a chondrodysplasia characterized by early-onset osteoarthritis and short stature. COMP is a secreted pentameric glycoprotein that belongs to the thrombospondin family of proteins. We have identified a novel missense mutation which substitutes a glycine for an aspartic acid residue in the thrombospondin (TSP) type 3 calcium-binding domain of COMP in a patient diagnosed with PSACH. Immunohistochemistry and immunoelectron microscopy both show abnormal retention of COMP within characteristically enlarged rER inclusions of PSACH chondrocytes, as well as retention of fibromodulin, decorin and types IX, XI and XII collagen. Aggrecan and types II and VI collagen were not retained intracellularly within the same cells. In addition to selective extracellular matrix components, the chaperones HSP47, protein disulfide isomerase (PDI) and calnexin were localized at elevated levels within the rER vesicles of PSACH chondrocytes, suggesting that they may play a role in the cellular retention of mutant COMP molecules. Whether the aberrant rER inclusions in PSACH chondrocytes are a direct consequence of chaperone-mediated retention of mutant COMP or are otherwise due to selective intracellular protein interactions, which may in turn lead to aggregation within the rER, is unclear. However, our data demonstrate that retention of mutant COMP molecules results in the selective retention of ECM molecules and molecular chaperones, indicating the existence of distinct secretory pathways or ER-sorting mechanisms for matrix molecules, a process mediated by their association with various molecular chaperones.

Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP).Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC(-/-), and tenascin X (TNX(-/-)) knockout mice were measured.TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemm's canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC(-/-) or TNX(-/-) and wild-type mice.TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase.

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. Background: 3-Hydroxylation of proline residues in type I collagen is rare but important. Results: 3-Hyp sites have been identified in both chains of mouse type I collagen in wild type and P3H1 null mice. Conclusion: The absence of 3-Hyp does not alter the D-period of collagen fibrils, but alters the lateral growth of the fibrils. Significance: Type I collagen prolyl 3-hydroxylation is tissue-specific.

PURPOSE. Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican’s potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated. METHODS. Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNAsilencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility. RESULTS. Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture. CONCLUSIONS. Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes. (Invest Ophthalmol Vis Sci. 2011;52:5049 –5057) DOI

This chapter reviews the current knowledge of collagens, a protein family important for the extracellular matrix of vertebrate animals. The 28 types of collagens are discussed in terms of their structure, their distribution in tissues, and their involvement in human diseases. The biosynthesis of collagens with their numerous posttranslational modifications is reviewed. The current views on chain selection, trimerization, and folding of collagens and collagen-like peptides are described and recent advances in the determination of the atomic structure of the triple helix are also discussed.

The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

The rough endoplasmic reticulum-resident FK-506-binding protein FKBP65 can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. The peptidylprolyl cis-trans-isomerase activity of FKBP65 was previously shown to have only marginal effects on the rate of triple helix formation (Zeng, B., MacDonald, J. R., Bann, J. G., Beck, K., Gambee, J. E., Boswell, B. A., and Bachinger, H. P. (1998) Biochem. J. 330, 109-114). Here we show that FKBP65 is a monomer in solution and acts as a chaperone molecule when tested with two classic chaperone assays: FKBP65 inhibits the thermal aggregation of citrate synthase and is active in the denatured rhodanese refolding and aggregation assay. The chaperone activity is comparable to that of protein-disulfide isomerase, a well characterized chaperone. FKBP65 delays the in vitro fibril formation of type I collagen, indicating that FKBP65 is also able to interact with triple helical collagen, and acts as a collagen chaperone.

The collagen prolyl hydroxylases are enzymes that are required for proper collagen biosynthesis, folding, and assembly. They reside within the endoplasmic reticulum and belong to the group of 2-oxoglutarate and iron-dependent dioxygenases. Although prolyl 4-hydroxylase has been characterized as an alpha2beta2 tetramer in which protein disulfide isomerase is the beta subunit with two different alpha subunit isoforms, little is known about the enzyme prolyl 3-hydroxylase (P3H). It was initially characterized and shown to have an enzymatic activity distinct from that of prolyl 4-hydroxylase, but no amino acid sequences or genes were ever reported for the mammalian enzyme. Here we report the characterization of a novel prolyl 3-hydroxylase enzyme isolated from embryonic chicks. The primary structure of the enzyme, which we now call P3H1, demonstrates that P3H1 is a member of a family of prolyl 3-hydroxylases, which share the conserved residues present in the active site of prolyl 4-hydroxylase and lysyl hydroxylase. P3H1 is the chick homologue of mammalian leprecan or growth suppressor 1. Two other P3H family members are the genes previously called MLAT4 and GRCB. In this study we demonstrate prolyl 3-hydroxylase activity of the purified enzyme P3H1 on a full-length procollagen substrate. We also show it to specifically interact with denatured collagen and to exist in a tight complex with other endoplasmic reticulum-resident proteins. Immunohistochemistry with a monoclonal antibody specific for chick P3H1 localizes P3H1 specifically to tissues that express fibrillar collagens, suggesting that other P3H family members may be responsible for modifying basement membrane collagens.

Extracellular matrix homeostasis is dependent in part upon a family of matrix metalloproteinases and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Recently, gene defects in TIMP-3 have been identified in the affected individuals of several families with Sorsby's fundus dystrophy (SFD). Very little information is available regarding TIMP-3 function or even its existence in the retina or choroid.We used reverse transcription-polymerase chain reaction and Northern analysis to evaluate the expression of TIMP mRNA and Western immunoblots to evaluate TIMP protein produced by select cells of the human retina and choroid. We also used these methods and immunohistochemistry to localize the TIMPs in the retina and choroid.TIMP-3 transcripts are found in cultured human retinal pigment epithelium (RPE), choroidal microcapillary endothelium and pericytes. RPE cells also express and secrete TIMP-3 protein, which is localized to the extracellular matrix and is not found in culture medium; TIMP-1 and -2 are found almost exclusively in the medium. Immunohistochemistry of human retina/choroid sections shows pronounced TIMP-3 immunostaining in Bruch's membrane, particularly near the surface of the RPE and endothelial cells, presumably in their basement membranes, with minimal staining in other portions of the retina. Immunostaining for TIMP-1 is absent and for TIMP-2 is much less prevalent, but detectable in Bruch's membrane.TIMP-1, -2 and -3 exhibit distinctive expression patterns in the retina and choroid. This distribution and expression pattern partially explains why TIMP-3 mutations result in SFD, rather than other retinal pathologies, such as those associated with proliferative diabetic retinopathy.

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.

Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S. cerevisiae. Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene. In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent. The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA. Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences. The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations. When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity. Deletion of the CEN region from the clones increases the copy number to 10-20/cell. The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number. Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.