MmuEX0019422 @ mm9

The study examined the cell-specific expression of two fibronectin isoforms, EIIIA (contains MmuEX0019422) and EIIIB (contains MmuEX0019423), during experimental hepatic fibrosis induced by ligation of the biliary duct. AT the mRNA level, EIIIA and EIIIB were undetectable in normal liver but expressed early injury, preceding fibrosis. The cellular sources of these changes were determined by fractionating the liver at various time points after bile duct ligation into its constituent cell populations and extracting RNA from the fresh isolates. EIIIA-containing fibronectin mRNA was undetectable in normal sinusoidal endothelial cells but increased rapidly within 12 h of injury. By contrast, the EIIIB form was restricted to hepatic lipocytes (Ito or fat-storing cells) and appeared only after a lag of 12-24 h: it was minimal in sinusoidal endothelial cells. Both forms were minimal in hepatocytes. At the protein level, EIIIA-containing fibronectin was markedly increased within two days of injury and exhibited a sinusoidal distribution. Secretion of this form by endothelial cells was confirmed in primary culture. Matrices deposited in situ by endothelial cells from injured liver accelerated the conversion (activation) of normal lipocytes to myofibroblast-like cells, and pretreatment of matrices with monoclonal antibody to the EIIIA segment blocked this response. Finally, recombinant fibronectin peptide containing the EIIIA segment was stimulatory to lipocytes in culture.

To study the function of extra domain A (EDA) (contains MmuEX0019422) of fibronectin 1 (FN) in vivo, the authors generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

In atherosclerotic lesions, fibronectin 1 (FN) including the EIIIA domain (EIIIA-FN) (also called EDA)(MmuEX0019422), is abundant, whereas FN in the flanking vessel wall lacks EIIIA. Lesional EIIIA-FN is localized with endothelial cells and macrophage foam cells. To directly test the function of EIIIA-FN, the study generated EIIIA-null (EIIIA(-/-)) mice that lack the EIIIA exon and crossed them with apolipoprotein E (ApoE)-null (ApoE(-/-)) mice that develop arterial wall lesions. Compared with ApoE(-/-) controls, EIIIA(-/-)ApoE(-/-) mice had significantly smaller lesions throughout the aortic tree. EIIIA-FN was increased in ApoE(-/-) plasma, and total plasma cholesterol was reduced in EIIIA(-/-)ApoE(-/-) mice, specifically in large lipoprotein particles, suggesting a functional role for plasma EIIIA-FN. To assess a role for macrophage EIIIA-FN in the vessel wall, in vitro foam cell assays were conducted. EIIIA(-/-)ApoE(-/-) macrophages accumulated significantly less intracellular lipid than control ApoE(-/-) cells. These results provide genetic evidence that suggests roles for EIIIA-FN in plasma lipoprotein metabolism and in foam cell formation.

[Negative results]. Fibronectin 1 (FN) splice variants containing the EIIIA (MmuEX0019422) and/or EIIIB (MmuEX0019423) exons are prominently expressed in the vasculature of a variety of human tumors but not in normal adult tissues. To understand the functions of these splice variants in physiological and tumor angiogenesis, thus study used EIIIB-null and EIIIA-null strains of mice to examine neovascularization of mouse retinas, pancreatic tumors in Rip-Tag transgenic mice, and transplanted melanomas. Contrary to expectations, physiological and tumor angiogenesis was not significantly affected by the absence of either EIIIA or EIIIB splice variants. Tumor growth was also not affected. In addition, the expression levels of smooth muscle alpha actin, believed to be modulated by EIIIA-containing fibronectins, were not affected either.

The fibronectins (FN) comprise a family of adhesive extracellular matrix proteins thought to mediate important functions in cutaneous wounds. Plasma fibronectin (pFN) extravasates for days from intact hyperpermeable vessels following injury whereas mRNAs encoding the cellular fibronectins (cFN) that include two segments, termed EIIIA (EDA) (MmuEX0019422) and EIIIB (EDB) (MmuEX0019423), are expressed by wound cells. Wounds in mice null for pFN appear to heal normally whereas those in EIIIA null mice exhibit defects, suggesting that cFN may play a role when pFN is missing. Integrin alpha9beta1, a receptor for several extracellular matrix proteins as well as the EIIIA segment, is expressed normally in the basal layer of squamous epithelia. The study reports results from immunohistochemistry on healing wounds demonstrating that EIIIA-containing cFN are deposited abundantly but transiently from day 4 to 7 whereas EIIIB-containing cFN persist at least through day 14. Elevated expression of alpha9beta1 is seen in basal and suprabasal epidermal keratinocytes in wounds. The spatial expression patterns of cFN and alpha9beta1 are distinct, but overlap in the dermal-epidermal junction, and both are expressed contemporaneously. These observations suggest a role for alpha9beta1-EIIIA interactions in wound keratinocyte function.

Vaccination strategies based on the in vivo targeting of antigens (Ags) to dendritic cells (DCs) are needed to improve the induction of specific T cell immunity against tumors and infectious agents. In this study, the authors used a recombinant protein encompassing the extra domain A from fibronectin (EDA) (MmuEX0019422), an endogenous ligand for TLR4, to deliver Ags to TLR4-expressing DC. The purified EDA protein was shown to bind to TLR4-expressing HEK293 cells and to activate the TLR4 signaling pathway. EDA also stimulated the production by DC of proinflammatory cytokines such as IL-12 or TNF-alpha and induced their maturation in vitro and in vivo. A fusion protein between EDA and a cytotoxic T cell epitope from ovalbumin (OVA) efficiently presented this epitope to specific T cells and induced the in vivo activation of a strong and specific cytotoxic T cell (CTL) response. Moreover, a fusion protein containing EDA and the full OVA also improved OVA presentation by DC and induced CTL responses in vivo. These EDA recombinant proteins protected mice from a challenge with tumor cells expressing OVA. These results strongly suggest that the fibronectin extra domain A may serve as a suitable Ag carrier for the development of antiviral or antitumoral vaccines.

The activation of mast cells by extra domain A of fibronectin (FN-EDA) (contains HsaEX0026101), an endogenous ligand of TLR4, and its contribution to the pathogenesis of rheumatoid arthritis (RA) in vivo were examined. FN-EDA, but no other domain of the fibronectin fragment, III(11) (FN-III(11)) and III(12) (FN-III(12)), stimulated bone marrow-derived murine mast cells (BMMCs) dose-dependently to secret cytokines (TNF-alpha, IL-6, and IL-1beta), similar to the pattern produced by LPS. FN-EDA-induced cytokine production was mediated by TLR4, as cytokine production by FN-EDA was absent in TLR4-deficient (TLR4-/-) BMMCs. The study also examined the roles of TLR4-mediated mast cell activation by this form of fibronectin fragment in the pathogenesis of RA in vivo. The injection of FN-EDA, but not FN-III(11)and FN-III(12), to joints resulted in joint swelling of mice in vivo. Genetically mast cell-deficient WBB6F(1)-W/W(v) mice exhibited significantly less swelling and cytokine production compared with mast cell-sufficient +/+ mice, suggesting that swelling and inflammatory cytokine production were partially dependent on tissue mast cells. Reduced swelling and cytokine production were recovered by the reconstitution of tissue mast cells by the injection of BMMCs from wild-type mice but not from TLR4-/- mice. Altogether, these results suggest that the TLR4-mediated activation of mast cells by endogenous ligand FN-EDA might contribute to the pathogenesis of RA through proinflammatory cytokine production.

Mice constitutively expressing extra domain A-containing FN (EDA(+)FN) (contains MmuEX0019422) have a significant decrease of FN levels in plasma and most tissues. Hepatocytes modified to produce EDA(+)FN have normal extracellular matrix-FN levels but secrete less soluble FN. In a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN.

Alternatively spliced variants of fibronectin (FN) containing exons EIIIA (MmuEX0019422) and EIIIB (MmuEX0019423) are expressed around newly forming vessels in development and disease but are downregulated in mature vasculature. The sequences and patterns of expression of these splice variants are highly conserved among vertebrates, suggestive of their biological importance; however the functions of EIIIA and EIIIB-containing FNs are unknown. To understand the role(s) of these splice variants, the authors deleted both EIIIA and EIIIB exons from the FN gene and observed embryonic lethality with incomplete penetrance by embryonic day 10.5. Deletion of both EIIIA and EIIIB exons did not affect synthesis or cell surface deposition of FN, indicating that embryonic lethality was due specifically to the absence of EIIIA and EIIIB exons from FN. EIIIA/EIIIB double-null embryos displayed multiple embryonic cardiovascular defects, including vascular hemorrhage, failure of remodeling embryonic and yolk sac vasculature, defective placental angiogenesis and heart defects. In addition, they observed defects in coverage and association with dorsal aortae of alpha-smooth-muscle-actin-positive cells. These studies indicate that the presence or absence of EIIIA and EIIIB exons alters the function of FN and demonstrate the requirement for these alternatively spliced exons in cardiovascular development.

To analyze the role of fibronectin (FN) isoforms (with/without extra domain A (EDA) (MmuEX0019422)) in atherosclerotic lesion formation the authors utilized mouse strains devoid of EDA exon regulated splicing, which constitutively include (EDA(+/+)) or exclude (EDA(-/-)) the exon. Both mutant mice had a 40% reduction in atherosclerotic lesions after the atherogenic-diet treatment (mean+/-S.E., microm(2); 22969+/-2185; 13660+/-1533; 14260+/-2501 for EDA(wt/wt), EDA(+/+) and EDA(-/-), respectively; p< or =0.01 ANOVA test) associated to a lower capacity of macrophages to uptake modified LDL and undergo foam-cell formation. Lesions in control mice were more numerous and bigger, with augmented and deeper macrophage infiltration, and increased FN expression in the sub-endothelial area. Previous experiments have shown that apoE(-/-)EDA(-/-) mice have a decreased number and size of atherosclerotic lesions and, on this basis, it has been proposed that the EDA domain has a pro-atherogenic role. These data with the EDA(+/+) mice rules out this hypothesis and suggest that regulated splicing of the EDA exon of the FN gene is involved in progression of atherosclerosis, highlighting the importance of alternative splicing in regulating cellular processes.

Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury. FN containing Extra Domain A (EDA+ FN) (MmuEX0019422) is absent from normal plasma, but elevated plasma levels of EDA+ FN are found in several pathological conditions. The authors hypothesized that EDA+ FN plays a special role in thrombosis. They used mouse strains constitutively including (EDA+/+) or excluding (EDA-/-) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model the authors found that EDA+ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA+/+ mice thrombi (>30 microm) grew faster when compared with EDA(WT/WT) (6.6+/-0.2 minutes versus 8.3+/-0.6 minutes, P

Idiopathic pulmonary fibrosis lung fibroblasts produced markedly more extra domain A (EDA)-containing fibronectin (containing MmuEX0019422) and alpha-smooth muscle actin (alpha-SMA) than control fibroblasts. EDA-null mice failed to develop significant fibrosis 21 days after bleomycin challenge, whereas wild-type controls developed the expected increase in total lung collagen. Histologic analysis of EDA-null lungs after bleomycin showed less collagen and fewer alpha-SMA-expressing myofibroblasts compared with that observed in wild-type mice. Failure to develop lung fibrosis in EDA-null mice correlated with diminished activation of latent transforming growth factor (TGF)-beta and decreased lung fibroblast responsiveness to active TGF-beta in vitro. The data show that EDA-containing fibronectin is essential for the fibrotic resolution of lung injury through TGF-beta activation and responsiveness, and suggest that EDA-containing fibronectin plays a critical role in tissue fibrogenesis.

Fibronectin (FN) is a major component of the extracellular matrix and occurs in two main forms: plasma and cellular FN. The latter includes the alternatively spliced domain A (EDA) (MmuEX0019422). Although EDA-containing cellular fibronectin (EDA-FN) is associated with fibroblast differentiation, how EDA-FN promotes differentiation is incompletely understood. This study investigated the mechanism by which EDA-FN contributes to fibroblast differentiation with emphasis on the characterization of the EDA-FN receptor. EDA-FN increases a-SMA expression (immunofluorescence), collagen deposition, cell contractility, and focal adhesion kinase (FAK) activation (immunoblotting); whereas plasma FN, a form lacking EDA, shows no effect. Primary lung fibroblasts constitutively express a(4)beta(7) integrin receptor (FACS and RT-PCR). Blocking of a(4)beta(7) reduces fibroblast adhesion to EDA-FN and inhibits a-SMA expression, collagen deposition, and FAK activation induced by EDA-FN. Using recombinant EDA-containing peptides, it demonstrated that the EDA segment is sufficient to induce fibroblast differentiation via binding to a(4)beta(7). EDA-FN induces MAPK-Erk1/2 activation and inhibition of MEK1/2 attenuates EDA-FN-induced a-SMA expression. These findings demonstrate that EDA-FN induces fibroblast differentiation by a mechanism that involves binding of EDA to a(4)beta(7) integrin followed by activation of FAK and MAPK-associated signaling pathways

Wild-type mice and mice lacking the extra domain A (EDA) (MmuEX0019422) of fibronectin (fn), referred to as EDA(-/-), underwent permanent ligation of the left anterior coronary artery. Despite equal infarct size between groups (38.2?4.6% versus 38.2?2.9% of left ventricle; P=0.985), EDA(-/-) mice exhibited less left ventricular dilatation and enhanced systolic performance compared with wild-type mice as assessed by serial cardiac MRI measurements. In addition, EDA(-/-) mice exhibited reduced fibrosis of the remote area without affecting collagen production, cross-linking, and deposition in the infarct area. Subsequently, ventricular contractility and relaxation was preserved in EDA(-/-). At tissue level, EDA(-/-) mice showed reduced inflammation, metalloproteinase 2 and 9 activity, and myofibroblast transdifferentiation. Bone marrow transplantation experiments revealed that myocardium-induced EDA and not EDA from circulating cells regulates postinfarct remodeling. Finally, the absence of EDA reduced monocyte recruitment as well as monocytic Toll-like receptor 2 and CD49d expression after infarction. This study demonstrated that parenchymal fn-EDA plays a critical role in adverse cardiac remodeling after infarction. Absence of fn-EDA enhances survival and cardiac performance by modulating matrix turnover and inflammation via leukocytes and fibroblasts after infarction.

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here the authors report that the isoform characterized by the presence of extradomain A (encoded by HsaEX0026101) activates alpha4beta1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B (encoded by HsaEX0026102) enhances the binding of fibronectin through the RGD sequence to beta3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. These study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation.

The authors perform quantitative proteomic analysis of the matrisome of murine carotid arteries in mice deficient in the production of FN splice isoforms containing alternative exons EIIIA and EIIIB (FN-EIIIAB null) after exposure to low and disturbed flow in vivo. They also examine serum-derived and endothelial-cell contributions to the matrisome in a simplified in vitro system. Flow-induced differences in the carotid artery matrisome that were impaired in FN-EIIIAB null mice. One of the most interesting differences was reduced recruitment of FBLN1 (fibulin-1), abundant in blood and not locally produced in the intima. This defect was validated in these in vitro assay, where FBLN1 recruitment from serum was impaired by the absence of these alternatively spliced segments.

This paper reports that extra domain A (ED-A) (HsaEX0026101)-containing fibronectin (FN) deposition precedes alpha-smooth muscle (alpha-SM) actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFbeta1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of alpha-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se alpha-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFbeta1-triggered enhancement of alpha-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. These findings indicate that ED-A-containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFbeta1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.

Because EDA-containing (containing HsaEX0026101) fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), this study examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. It found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.

Dysfunction of lymphatic valves underlies human lymphedema, yet the process of valve morphogenesis is poorly understood. This study shows that during embryogenesis, lymphatic valve leaflet formation is initiated by upregulation of integrin-alpha9 expression and deposition of its ligand fibronectin-EIIIA (FN-EIIIA) (containing HsaEX0026101) in the extracellular matrix. Endothelial cell-specific deletion of Itga9 (encoding integrin-alpha9) in mouse embryos results in the development of rudimentary valve leaflets characterized by disorganized FN matrix, short cusps, and retrograde lymphatic flow. Similar morphological and functional defects are observed in mice lacking the EIIIA domain of FN. Mechanistically, they demonstrate that in primary human lymphatic endothelial cells, the integrin-alpha9-EIIIA interaction directly regulates FN fibril assembly, which is essential for the formation of the extracellular matrix core of valve leaflets.

The objective of this study was to investigate whether tumor derived fibronectin alternatively spliced EDA domain has a lymphangiogenic potency on human lymphatic endothelial cells (LECs) in tumor generation to facilitate tumor lymphatic metastasis. LECs were cultured in three-dimentional culture system and treated with SW480 supernant which was highly rich in EDA, the result demonstrated that SW480 supernant could facilitate tube-like formations of LECs evidently when compared with controls. Integrinalpha9 was identified by immunofluorescence to be a specific receptor for EDA because the authors found co-locozation of EDA and integrinalpha9 on LECs as well as significant upregulation of integrinalpha9 in SW480 supernant treated group. Western blot and immunofluorescence revealed that EDA also had important roles accommodating the expressions of some key regulators of lymphangiogenesis such as Prox1 and F-actin so as to facilitate motility and sprouting of LECs. In addition, it had been confirmed that all of these effects could be inhibited markedly by EDA antibody (IST-9). Based on these findings, EDA derived from tumor cells has an important role in facilitating lymphangiogenesis of malignant tumor.

[Disease association]. Thoracic aortic aneurysm is a common complication in patients with bicuspid aortic valve (BAV). Alternatively spliced extra domain A (EDA) (HsaEX0026101) of fibronectin (FN) has an essential role in tissue repair. This study analyzes the expression of FN spliceforms in dilated and nondilated ascending aorta of tricuspid aortic valve (TAV) and BAV patients. The mRNA expression was analyzed in the ascending aorta by Affymetrix Exon arrays in patients with TAV (n=40) and BAV (n=69). EDA and extra domain B (EDB) (HsaEX0026102) expression was increased in dilated aorta from TAV patients compared with nondilated aorta (P

There is increasing evidence of a role for Toll-like receptors (TLRs) in inflammatory arthritis. The extra domain A (ED-A)-containing isoform (HsaEX0026101) of fibronectin is generated under pathologic conditions such as rheumatoid arthritis, and ED-A has been identified as an endogenous TLR-4 ligand. Leukotriene B4 (LTB4) and polymorphonuclear neutrophils (PMNs) play a critical role in murine models of inflammatory arthritis. The aim of this study was therefore to investigate the putative effects of ED-A on leukotriene biosynthesis and PMN migration through TLR signaling. The effect of recombinant human ED-A (rhED-A) on leukotriene biosynthesis was evaluated in isolated human blood PMNs and monocytes by high-performance liquid chromatography. The capacity of rhED-A to stimulate PMN migration was evaluated using a transendothelial/matrix migration assay in vitro and the mouse air-pouch model in vivo. Recombinant human ED-A efficiently primed the biosynthesis of LTB4 in PMN and monocyte suspensions. This priming effect was dependent on TLR-4 activation, since the TLR-4-signaling inhibitor CLI-095 completely blocked the effect of rhED-A but not that of other TLR ligands (R-848, Pam2 CSK4) or cytokines. Moreover, rhED-A stimulated transendothelial migration of PMNs in vitro, which was inhibited by 50-60% with the LTB4 receptor 1 (BLT1) antagonist CP105,696 or the cytosolic phospholipase A2 alpha inhibitor pyrrophenone. In vivo, rhED-A induced a significant PMN recruitment into the air pouch of C3H/HeOuJ mice (expressing functional TLR-4), but not in C3H/HeJ mice (expressing nonsignaling TLR-4). These results demonstrate the ability of rhED-A to promote LTB4 biosynthesis and PMN migration through TLR-4 activation, thus providing new insights on TLR-dependent mechanisms of regulation of LTB4 biosynthesis and PMN infiltration in inflammatory joint diseases.

Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. The authors now report using human dermal fibroblast cultures that the EDA domain (encoded by a single exon, HsaEX0026101) of fibronectin or EDA-derived peptides modeled after the C-C? loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the alpha4 integrin. These data indicate that the interaction between the alpha4beta1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition. Note, uses the exon of interest (the EDA) as a separate peptide.

EDA(+)FN overexpression increases cell motility, whereas knockout of EDA(+)FN using CRISPR decreases cell motility.

Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage, which leads to impaired aqueous humor outflow. Here, the authors explore a novel molecular mechanism involved in glaucomatous TM damage. The authors investigated the role of an endogenous Toll-like receptor 4 (TLR4) ligand, fibronectin-EDA (FN-EDA), in TGFbeta2-induced ocular hypertension in mice. The authors utilized transgenic mouse strains that either constitutively express only FN containing the EDA isoform (contains cassette exon HsaEX0026101, which encodes the EDA) or contain an EDA-null allele and express only FN lacking EDA, with or without a mutation in Tlr4, in these inducible mouse model of ocular hypertension by injection of Ad5.TGFbeta2. IOP was measured over time and eyes accessed by immunohistochemistry for total FN and FN-EDA expression. Constitutively active EDA caused elevated IOP starting at 14 weeks of age. Ad5.TGFbeta2 induced ocular hypertension in wildtype C57BL/6J mice and further amplified the IOP in constitutively active EDA mice. TLR4 null and EDA null mice blocked Ad5.TGFbeta-induced ocular hypertension. Total FN and FN-EDA isoform expression increased in response to Ad5.TGFbeta2. These data suggest that both TLR4 and FN-EDA contribute to TGFbeta2 induced ocular hypertension.