Primers for CDK6, CTNNBIP1, SMAD1 and HDAC4 used in RT reactions are listed in Additional file 3. Final results have been analyzed working with Step 1 Program v2. 1 and statistical significances had been calculated by a college students t test, unpaired. Transfections of USSC for Western blots and practical analyses To analyze improvements in endogenous protein abundance dur ing osteogenesis, USSC have been cultivated on six very well plates and induced with DAG as described over. Proteins have been isolated at days 0, seven, 9, and 12. To assess the im pact of miRNAs on endogenous target proteins, USSC have been cultivated on 6 very well plates and transfected with 40pmol of appropriate miRNA mimic/well applying Dharmafect1 reagent according to the suppliers directions, proteins had been isolated 48h after transfection. Native, osteo differentiated and transfected USSC have been washed with PBS and lysed utilizing RIPA Buffer supplemented with protease inhibitor cocktail tablets.
Up to 50ug of proteins/lane have been separated by SDS PAA electro phoresis, blotted onto nitrocellulose membranes, and membranes were blocked with 3% Milk/PBS, incubated with main antibodies in 3% milk/PBS followed by incubation with selleckchem Bortezomib the appropiate secondary anti entire body in 3% milk/PBS/0,15% Tween twenty. Membranes have been washed in PBS T/0. 1% Tween twenty, dried, and scanned with the LI COR Odyssey Infrared Imager. Visible false colour signals were quan tified employing Odyssey 2. one program and normalized against signals from quantified housekeeping proteins Tubulin or Actin. The next antibodies have been made use of in this study. CDK6, sc 177, HDAC4, sc 11418, SMAD1, sc 7965, Tubu lin, B five 1 2.Actin, ab34731. IRDye 680LT mouse and IRDye 800CW rabbit. For practical analyses, USSC had been cultivated on 24 properly plates and transfected with 10pmol miRNA mimic/well using Dharmafect1 reagent according to the manu facturers directions.
Transfected cells had been induced to osteogenic differentiation 24 h submit transfection as described above. Differentiation was analyzed at day seven by alizarin red stainig and calcium released as described over. Ethics statement Function with USSC was authorized through the Ethics Commit tee within the Health-related Faculty, University D?sseldorf, selleck chemicals aurora inhibitor research numbers 3436 and 2975. Breast tumor initiating cells are functionally de fined by their unlimited renewal possible and capability to re create tumor heterogeneity, attracting attention as therapeutic targets. There may be growing proof that molecular pathways required for usual stem cell func

tions are deregulated in BT ICs. As occurs with regular organogenesis and cell differentiation, the selective activa tion and repression of these pathways can be mediated by microRNAs. These short non coding RNAs in hibit gene expression by mRNA degradation or transla tional inhibition.

Fish immunogenetics has received consid erable attention as a result of its necessary purpose in understand ing the origin and evolution of immune systems. Additional, it is actually also useful within the creation of immune based treatment of significant fish illnesses. Wonderful progress in bioinformatics and genome projects in model organisms, as well as human, mouse, frog, chicken, and zebrafish, has led to your emergence of stu dies concentrating on the identification and characterization of immune linked genes in teleost fish dependant on com parative genomics. These have offered preliminary observations on fish “additional reading “ immunogenetics and evolutionary historical past of immune programs from lower vertebrates to mammals. On the other hand, substantial scale identification of immune related genes with the genome or transcriptome amounts in fish was witnessed in limited species as a consequence of the inadequate quantity of higher throughput deep sequencing technologies on the market.
This is certainly an even more troublesome trouble in non model fish species with totally unknown genome sequences. Lately formulated RNA deep sequencing technolo gies, just like Solexa/Illumina RNA seq selelck kinase inhibitor and Digital gene expression, have substantially modified the way immune associated genes in fish are recognized because these technologies facilitate the investigation on the functional complexity of transcriptomes. RNA Seq refers to complete transcriptome shotgun sequencing wherein mRNA or cDNA is mechanically fragmented, leading to overlapping short fragments that cover the complete transcriptome. DGE is usually a tag based mostly transcriptome sequencing approach where brief raw tags are generated by endonuclease. The expression level of almost all genes inside the sample is measured by counting the num ber of person mRNA molecules generated from just about every gene.
Compared with DGE evaluation, the RNA Seq strategy is more highly effective for unraveling transcriptome complexity,

and for identification of genes, construction of transcripts, option splicing, non coding RNAs, and new transcription units. In contrast, the DGE protocol is a lot more ideal and very affordable for comparative gene expression research simply because it enables direct transcript profiling with out compromise and potential bias, therefore making it possible for for any even more delicate and accurate profiling of your transcriptome that more closely resembles the biol ogy of the cell. These two technologies happen to be used in transcriptome profiling research for various applications, which include cellular improvement, cancer, and immune defence of numerous organisms. How ever, they have not been used in immunogenetic analy sis of marine fish species. Japanese sea bass is an eco nomically important marine species extensively cultured in fisheries throughout the world. Numerous illnesses brought about by bacterial and viral pathogens plague this species. Large mortal ity is associated with infection with Vibri harveyi, a typi cal gram adverse pathogen of the wide choice of marine animals. o

However, concentrating all efforts on the NGS tech nology could possibly hamper the evaluation of numerous animals and samples as essential for eQTL scientific studies and genetic genomics. We are rather convinced that the NGS technological innovation and very well annotated DNA chips will stay complementary for a while in domestic species. The SLA RNRSP8 13K chip reported right here represents an accu rately annotated chip devoted towards the pig immune sys tem and can provide a worthwhile device for diagnostics and investigation. Alternative within the in vitro models to examine immune response activation PMA, also called twelve O tetradecanoylphorbol 13 ace tate, is known as a potent tumor promoter regularly used in bio health care research. Ionomycin is definitely an ionophore generated by Streptomyces conglobatus. PMA in conjunc tion with ionomycin is acknowledged to CUDC-101 HDAC inhibitor activate T and B cells and is used in several immune associated studies.
LPS is actually a major structural part of the outer membrane of gram adverse bacteria and it is a very well referenced PAMP. LPS stimulation of mammalian cells occurs via a series of interactions with proteins which includes LPS binding protein, CD14, MD 2 and their explanation TLR4. LPS is amongst the perfect studied immunostimulatory parts of bacteria and might induce systemic inflam mation and sepsis if excessive signals happen. LPS stimulation mimics a bacterial infection and is extensively utilized to research innate immune response. Two current scientific studies in pig have reported tran scriptome modifications in mesenteric lymph node or spleen after infection by Salmonella enterica serovar Choleraesuis and Haemophilus para suis, respectively. S. Choleraesuis and H. parasuis are each gram negative bacteria. Our success on LPS stimulation reveal that several genes by now identi fied immediately after in vivo infection by S. Choleraesuis and H.
para suis are up or down regulated confirming that in vitro LPS activation of PBMCs is actually a good model to research innate immune response to infection with gram negative bacte ria in pig. Indeed, LPS and PMA/ionomycin stimulations had been chosen since they’re extensively utilised as gold traditional in vitro designs to measure cytokines launched from the medium by

PBMCs in lots of species. A distinctive time stage was studied and we’re mindful that the many outcomes reported here correspond to this one of a kind time level i. e. 24 hours immediately after stimulation. It’s been reported that time points earlier than 24 hrs are far more pertinent to decipher the onset with the response to stimulus as proven in kinetics scientific studies in cow, pig, mouse or human. Moreover, kinetics studies have exposed that many genes return to their basal expression level by 48 hours of stim ulation, suggesting that homeostasis is restored at that time. Within this report, we had been keen on examine ing the PBMC transcriptome at the time when cytokines released within the medium are effectively measured.

We also observed protein phosphatase 1 regulatory subunit 15B plus the elongation initiation component two subunit two to become among the upregulated genes. PP1rs15B is often a constitutively expressed inhibitory subunit of PP1, one from the key eukaryotic serine/threonine phosphatases. PP1rs15B dephosphorylates the subunit of eIF2. The microarray data verify an upregulation of both PP1rs15B and eIF2s2 indicating that these pro teins may perhaps be associated with the handle on the STAT3 overex pression, irrespective of whether these genes possess a function in upkeep of pluripotency is still unclear. Within the second group we identified hexokinase II to become enhanced in cells overexpressing STAT3, this confirms the importance of this isozyme for embryo viability and indi cates that a correct energetic balance is really impor tant inside the late phases of preimplantation and in the starting of postimplantation in the embryos.
The third group of differentially expressed genes com prises molecules associated with the maintenance of pluripo tency and cell viability. Some of the recognized genes have been previously correlated with pluripotency or with embryo viability in somatic nuclei derived cloned blasto cysts. Lefty2 is regulated by pathways this kind of as Smad2/3 and WNT and by OCT 3/4, which help stemness. Lefty can be induced on exit from your state selleck of stemness, includ ing forced in vitro differentiation and LIF withdrawal. When LIF is withdrawn, the expression of Lefty increases inside 48 hours of cytokine withdrawal. Similarly, retinoic acid that induces differentiation leads to improved expression of Lefty in mouse embryonic carci noma cells. Differentiation of stem cells to embryoid bodies also prospects to greater expression of Lefty in vitro. For this reason, Lefty could possibly be essential each towards the stemness and differentiation events that stick to the exit from this state.
Murine Pem/Rhox5 is surely an X linked homeobox containing gene, selleckchem whose homeodomain shares essential structural capabilities with two other homeobox genes, that are expressed in extra embryonic lineages and throughout sper matogenesis. The Pem/Rhox5 protein is expressed while in the late morula stage, in TE and ICM of blastocyst and just after implantation in further embryonic tissues, inside the pari etal and visceral endoderm, but not during the primitive ecto derm derivatives. Pem/Rhox5 can also be expressed in ES cells, in primordial germ cells and in teratocarcinoma cell lines. Overexpression of Pem/Rhox5 had no phenotype in ES cells, but entirely inhibited

differentiation in to the 3 major cell lineages, when ES cells had been cultured as embryoid bodies in suspension with out LIF.

In contrast, Ifit2 could not shield mice from neuropathogenesis caused by encephalomyocarditis virus, a picornavirus. Therefore, we’ve uncovered a virus certain, tissue specific and ISG exact antiviral effect with the IFN system. Generation of Ifit2/ISG54 and Ifit1/ISG56 knockout mice Ifit2 gene knockout mice have been generated by deleting the entire protein encoding region of the gene, which was accomplished by flanking exons 2 and three with frt recombinase internet sites in C57BL/6 embryonic stem cells and excising the flanked region with Flp recombinase. Ifit22/2 mice were bred to homozygosity, and deficiency for induced expression of Ifit2 protein was confirmed in lysates of IFN b handled main murine embryonic fibroblasts. Mice deficient for Ifit1 were derived from C57BL/6 embryonic stem cells lacking the whole Ifit1 coding area. Genotypic homozygosity in the Ifit12/2 mice and deficiency for Ifit1 protein induction were confirmed.
Both knockout mouse lines have been wholesome and fertile. Additionally, deletion of one particular gene within the Ifit locus didn’t alter the pattern of induction of other adjacent gene family members, as when compared to wild type mice. Ifit2 protects mice from lethal intranasal VSV infection To find out the influence of Ifit2 around the selleck chemical DOT1L inhibitor outcome of viral infections in vivo, we in contrast susceptibilities of Ifit22/2 and wt mice to VSV infection, employing IFNAR2/2 mice as favourable controls of enhanced susceptibility. Virus was administered at a reduced dose, intranasally, reflecting a pure route of infection for VSV. As viewed previously, 100% of IFNAR2/2 mice swiftly succumbed to VSV infection within 2 days, right after struggling signs of lethargy. On the other hand, 79% of wt mice survived, the remaining selleck 21% succumbed to VSV, and this occurred later, at 7?ten days submit infection.
In contrast, 100% of Ifit22/2 mice died by 7 d. p. i. with most succumbing

by 6 d. p. i. ; so, we observed uniform and even more quickly happening death of Ifit22/2 in comparison with wt mice soon after VSV infection. Inside 24 h prior to death, both wt and Ifit22/2 mice created neurological indications such as ataxia, hind limb paralysis, and hyper excitability. Ifit2+/2 mice displayed an intermediate survival curve, demonstrating a gene dosage effect. Up coming, the part of the connected gene, Ifit1, in VSV pathogenesis was evaluated by infecting Ifit12/2 mice. Contrary to the results observed with Ifit22/2 mice, no statistically significant maximize in mortality was observed in Ifit12/2 mice. Constant with this, survival kinetics of Ifit12/2 and wt mice have been equivalent. Rising the virus dose by ten,000 fold didn’t appreciably alter the survival curves of wt, Ifit12/2, or Ifit22/2 mice. These benefits demonstrate functional variations between the two closely connected proteins encoded by Ifit1 and Ifit2.

In contrast, we observed major variations from the expression of MRFs between placebo and losartan handled mice at later on phases from the muscle remodeling procedure. Expression of Pax7 and MyoD remained elevated 19 days after CT damage from the placebo treated animal but returned to baseline amounts from the losartan taken care of animal. Conversely, myogenin and p21 expression have been considerably improved while in the losartan handled animals as when compared with the placebo treated animals at 19 days following CT. With each other, these information suggest that aged regenerating skeletal muscle was unable to transition from a proliferation stage of satellite cells in to the differentiation approach of muscle fibers. Modulation with the canonical supplier Cediranib and noncanonical TGF B signaling cascades restored the necessary down regulation of Pax7 and MyoD and up regulation of p21 and myogenin with the muscle differentiation stage, which permitted effective remodeling of muscle injury.
Losartan prevents disuse atrophy in sarcopenic mice Past evidence suggests that skeletal muscle atrophy triggered by disuse is exaggerated while in aging. In addition, immobilization employing distinctive approaches is related with selleck transient alterations with the canonical and noncanonical TGF B signaling pathways. We therefore evaluated whether losartan would have useful results on skeletal muscle of 21 month old mice subjected to immobilization with the TA muscle for 21 days implementing a surgical staple process. Immobilization caused a significant 16% lessen inside the moist TA excess weight of your placebo handled group as when compared to the contralateral TA that was not observed when evaluating the immobilized and con tralateral TA of your losartan taken care of groups. His tological analyses of immobilized muscular tissues revealed tiny regions of fibrosis during the placebo taken care of mice.
Unexpectedly, detailed morphometric analyses on the

minimal Ferets diameter did not reveal a big difference in muscle fiber dimension in either TA in the control, placebo taken care of, or losartan handled mice. This end result suggests that real reduction of muscle fibers as opposed to basic atrophy, commonly observed in youthful mice, may possibly be accountable for the distinctions in moist muscle bodyweight. For that reason, we assessed the cross sectional place on the intact TA and the total myofiber amount in our animals. Each CSA and complete myofiber amount had been appreciably reduced in placebo taken care of mice by 35 and 33%, respectively. In contrast, losartan remedy prevented the reduce of CSA, and mice exhibited a total myo fiber count just like that of the nonimmobilized manage group. In summary, losartan protected aged mice towards disuse atrophy by preventing reduction of muscle fibers as opposed to avoiding atrophy within the personal myofibers.

RA increases histone acetylation on the enhancer I region containing the NF AT Smad3 binding web-sites Provided the stringent dependence within the RA enhancing impact on Smad3 action along with the proximity of your RAR RXR binding website to the Smad3 binding web page it appeared probably the mechanism within the enhancing result may possibly be as a result of a direct impact of RA on Smad3 transcriptional action. To test this hypothesis we initial determined if RAR RXR physically interacted with Smad3. Accordingly, we subjected CD4 cells subjected to TCR TGF B stimulation inside the presence of RA to co immunoprecipitation scientific studies utilizing a number of pertinent antibodies inside a assortment of combinations but could discover no proof of RAR RXR Smad3 interaction. Subsequent we sought to established irrespective of whether RAR RXR could increase the accessibility from the enhancer I to transcription components.
Considering that this region is made up of no CpG sequences we chose to carry out this by carrying out ChIP research to assess the level of histone acetylation in these regions rather then by doing methylation studies. As proven in Figure 7A, with respect to the enhancer I area, stimulation of CD4 cells with anti CD3 CD28 was associated with tiny or no histone acetylation, stimulation selleck inhibitor with TCR TGF B was connected using a mild level of histone acetylation and stimulation with TGF B plus RA was related by using a large level of histone acetylation. In contrast, RA stimulation or TGF B plus RA enhanced acetylation only somewhat in the promoter area. These data indicate the major result of RA for the accessibility of transcription factors to gene target web-sites was within the enhancer one region. AP one and RA enrich whereas IL 27 inhibits pSmad3 binding to Enhancer I In subsequent series of research we initial sought to find out if your enhanced accessibility with the Smad3 binding area induced by RAR RXR noted above final results in an actual improved binding of pSmad3 to this region.
To this finish we carried out Smad3 unique ChIP assays on TCR TGF B stimulated CD4 cells stimulated while in the presence and absence of RA. As shown in Figure selleck 7B, RA stimulation of cells was connected with markedly

greater pSmad3 binding from the enhancer I area which incorporates the pSmad3 binding web page. We then sought to find out if binding of pSmad3 to your enhancer I region in CD4 cells is impacted by IL 27 signaling which inhibits the two TGF B and TGF B plus RA induced up regulation of Foxp3 expression by way of activation of Stat3 and, in turn, the subsequent activation of your gene silencing website in enhancer II. Accordingly, Smad3 distinct ChIP assays have been performed on CD4 cells stimulated with TGF B or TGF B plus RA from the presence and absence of IL 27.

diabetic ApoE null mice. Furthermore, as SMCs were the main cell form expressing ROCK1 during the aorta, we isolated SMCs in the aortas of wild style and RAGE null mice and taken care of them with RAGE ligand S100B. Although primary aortic SMCs from wild type mice displayed greater ROCK1 action upon incubation with RAGE ligand, S100B, SMCs from RAGE null mice failed to boost ROCK1 activity under these disorders. Our data reveal that the observed changes within the Tgf B pathway are common of changes in transcription associated with atherogenesis accompanying the onset of diabetes in ApoE null mice, as well as impact of RAGE deletion in diabetic ApoE null mice. On-line TableII delivers the numbers of differentially expressed one of a kind genes for each comparison which have Entrez Gene symbols and the numbers with beneficial and detrimental log fold adjustments.
Additionally, this table gives the numbers of genes resulting from Boolean operations you can check here on these genelists. Online TablesIIIVII give the lists of genes selleck chemicals whose numbers are offered in On the internet TableII. On line Figure represents a Venn diagram showing the intersection of comparison one, diabetic ApoE null relative to non diabetic ApoE null, with comparison four, diabetic ApoE null RAGE null relative to diabetic ApoE null. While one can find 53 genes that are statistically considerably differentially expressed in diabetic ApoE null relative on the non diabetic ApoE null state, and 216 genes that are statistically appreciably differentially expressed in diabetic ApoE null RAGE null relative to diabetic ApoE null, only 15 of these genes are statistically appreciably differentially expressed in each comparisons. There may be extremely tiny overlap within the genes that are differentially expressed each within the onset of diabetes in ApoE null mice and inside the impact of RAGE deletion in diabetic ApoE null mice.
Subsequent, to particularly link RAGE to SMC proliferation and migration, we performed studies in principal SMCs retrieved from RAGE expressing or RAGE deficient mouse aortas. As illustrated in figure 6A and 6B, incubation of wild type SMCs with RAGE ligand S100B resulted in significantly

elevated proliferation and migration, but S100B failed to stimulate proliferation and migration in RAGE deficient SMCs. Note that in wild sort and RAGE deficient SMCs, incubation with Tgf B2 or perhaps a non RAGE ligand PDGF enhanced proliferation and migration, suggesting that Tgf B2 and PDGF are not direct ligands of RAGE, that RAGE deficient SMCs are capable of proliferation and migration, and that exogenous addition of Tgf B2 to RAGE deficient cells restores proliferation and migration. Eventually, to set up that RAGE ligand stimulated SMC proliferation and migration needed Tgf B2 and ROCK1 action, we treated wild form SMCs with S100B within the presence or absence of Tgf B2 or ROCK1 inhibitors.

As proven in Fig. 5A and constant with all the benefits of Fig. 3A utilizing rapamycin, expression of management or RAPTOR focusing on shRNA in AKR 2B fibroblasts has no impact for the morphological improvements induced by TGF B. Nevertheless, fibroblasts expressing a RICTOR targeting shRNA exhibit a substantial attenuation in TGF B mediated formation of spindle shaped cells. Thus, mTORC2 could be associated with TGF B mediated morphological changes which are insensitive to rapamycin. The obtaining that rapamycin does not have an effect on TGF B mediated morphological transformation whereas RICTOR knockdown attenuates this process suggests that mTORC2 is just not appreciably inhibited by rapamycin in AKR 2B cells. To investigate the sensitivity of mTORC2 in AKR 2B cells to rapamycin, we handled serum starved AKR 2B cells with motor vehicle or rapamycin for 24 hours just before TGF B stimulation. As shown in Fig.
5B, prolonged rapamycin treatment method did not attenuate TGF B mediated Akt S473 phosphorylation though it entirely inhibited S6K1 T389 phosphorylation. Though this may well seem to vary from the research by Sarbassov et al. these investigators also reported the sensitivity of mTORC2 to prolonged rapamycin treatment varied substantially selleck inhibitor amongst diverse cell lines with some exhibiting nearly total reduction of Akt S473 phosphorylation from the presence of 10% serum when other people showed no attenuation. As such, in order to further define the sensitivity of mTORC2 in fibroblasts, AKR 2B, Swiss3T3, and IMR 90 fibroblasts were taken care of with either EtOH or rapamycin in the presence of selelck kinase inhibitor 10% serum for 24 hours. Fig. 5C demonstrates that whereas rapamycin thoroughly abrogates S6K1 phosphorylation, it has no affect around the phosphorylation of Akt Ser473.
These success indicate that mTORC2 expressed inside a subset of human and murine fibroblast lines is rapamycin insensitive, as has been described for other cell sorts. Up coming, we investigated the function of both mTOR complexes in TGF B mediated AIG. Offered that cells can exhibit variability within the extent of growth in soft agar, we

carried out transient transduction with lentiviruses expressing shRNA molecules to avoid differences in development thanks to clonal choice. Fig. 6A demonstrates shRNA expressing lentiviruses had been helpful at decreasing the expression of RAPTOR, RICTOR, and mTOR without the need of influencing the expression of other mTOR complex parts. These AKR 2B cultures were then employed to determine the ability of TGF B to induce soft agar colony formation.

The glaucomatous ONH displays characteristic cupping and excavation with the optic disc, collapse and remodeling of the LC, and activation of ONH astrocytes. The LC area with the ONH includes a characteristic sieve like framework by way of which RGC axons exit the eye. These laminar plates consist of extracellular matrix proteins including elastin and collagens. Right organization and assembly of your collagen and elastin fibers during the LC offers the two a supportive framework and elasticity to the ONH, that’s believed to protect RGC axons from mechanical pressure. Major cell sorts existing while in the human ONH include ONH astrocytes and LC cells. These cells help RGC axons by synthesizing growth components and extracellular matrix proteins. Remodeling of the ECM, including changes in fibrillar collagens, basement membrane components, and elastin composition, is characteristic with the glaucomatous ONH.
The extracellular matrix improvements include backward bowing of the laminar selleck chemical plates with elevated quantities of collagen I, IV, and VI. Altered elastin deposition in LC is imagined to alter the elastic properties from the ONH. Elevated synthesis and deposition of ECM proteins from the LC area could disrupt nutritional and mechanical support to RGC axons, leading to RGC atrophy. Quite a few scientific studies propose that ONH astrocytes and LC cells reply to elevated IOP by raising transforming development component B2 synthesis within the LC area, which in flip brings about altered ECM protein expression. TGF B2 belongs on the TGF B superfamily and plays a basic position in the biology of your ECM. In fibrotic illnesses, elevated TGF B2 levels cause the pathological deposition of ECM proteins. TGF B2 seems to be concerned from the pathogenesis of POAG.
Sufferers with glaucoma have higher amounts of TGF B2 inside their aqueous original site humor, and TGF B2 is proven to increase ECM protein in human trabecular meshwork cells. Additionally, TGF B2 improved IOP in cultured human perfused

anterior eye segments. In addition, adenoviral gene transfer of active TGF B2 elevates IOP in mice and rats and decreases outflow facility in mice. Robertson et al. also reported that gene transfer of TGF B1 to the anterior chamber of rats elevated IOP. A similar pathophysiology is observed in glaucomatous ONH as well as elevated TGF B2 and greater deposition of ECM proteins. From the glaucomatous ONH, elevated TGF B2 is associated with ECM remodeling. Fuchshofer and colleagues demonstrated that TGF B2 treatment of cultured ONH astrocytes upregulates mRNA and protein expression of collagen I, collagen IV, fibronectin, connective tissues growth factor, tissue transglutaminase, and thrombospondin 1. These observations recommend that TGF B2 could possibly be an initiation element in ECM remodeling during the glaucomatous ONH.