Akira Ooshima

Smad3 Signaling Is Required for Epithelial-Mesenchymal Transition of Lens Epithelium after Injury

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

Stretch-Induced Collagen Synthesis in Cultured Smooth Muscle Cells from Rabbit Aortic Media and a Possible Involvement of Angiotensin II and Transforming Growth Factor-β

Mechanical strain reportedly stimulates the synthesis of collagen in vascular smooth muscle cells (SMCs). The present study was designed to investigate a possible involvement of angiotensin II (Ang II) and transforming growth factor (TGF)-β in stretch-induced collagen synthesis of cultured SMCs derived from the rabbit aortic media. SMCs were cyclically stretched at a rate of 10% elongation and 30 cycles/min for 24 h using the Flexercell® strain unit (Flexcell International Corp., McKeesport, Pa.). A two-fold increase in collagen synthesis and a concurrent increase in total protein synthesis were noted in stretched SMCs. Concentration of immunoreactive Ang II in the conditioned medium was elevated under the mechanical strain. Stretch-induced collagen and total protein synthesis were inhibited by either a selective antagonist to Ang II (saralasin), an angiotensin I-converting enzyme inhibitor (captopril) or an antisense oligonucleotide for angiotensinogen mRNA. An elevated secretion of TGF-β, both active and latent forms, was found in the medium of stretched SMCs. Saralasin inhibited the stretch-induced secretion of TGF-β from SMCs. Stretch-induced collagen and total protein synthesis was further inhibited by either an anti-TGF-β1 neutralizing antibody or an adenovirus-mediated transfer of a truncated TGF-β type II receptor. Elevated expression of collagen α1(III) chain and TGF-β1 mRNAs, and its reversal by saralasin were also demonstrated in stretched SMCs. Results indicate that the stretch-induced collagen and total protein synthesis appears to be mediated via an autocrine-paracrine mechanism of Ang II and TGF-β released from SMCs.

Keloid‐derived fibroblasts show increased secretion of factors involved in collagen turnover and depend on matrix metalloproteinase for migration

Background   A keloid is a specific skin lesion that expands beyond the boundaries of the original injury as it heals. Histologically, it is characterized by the excessive accumulation of collagen. However, the reasons for the expansion and the invasive nature of keloids remain unknown.

Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice

Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial–mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-β (TGF-β) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-β and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of α-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-β signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR.

Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali.

Damage to the cornea from chemical burns is a serious clinical problem that often leads to permanent visual impairment. Because transforming growth factor (TGF)-beta has been implicated in the response to corneal injury, we evaluated the effects of altered TGF-beta signaling in a corneal alkali burn model using mice treated topically with an adenovirus (Ad) expressing inhibitory Smad7 and mice with a targeted deletion of the TGF-beta/activin signaling mediator Smad3. Expression of exogenous Smad7 in burned corneal tissue resulted in reduced activation of Smad signaling and nuclear factor-kappaB signaling via RelA/p65. Resurfacing of the burned cornea by conjunctival epithelium and its differentiation to cornea-like epithelium were both accelerated in Smad7-Ad-treated corneas with suppressed stromal ulceration, opacification, and neovascularization 20 days after injury. Introduction of the Smad7 gene suppressed invasion of monocytes/macrophages and expression of monocyte/macrophage chemotactic protein-1, TGF-beta1, TGF-beta2, vascular endothelial growth factor, matrix metalloproteinase-9, and tissue inhibitors of metalloproteinase-2 and abolished the generation of myofibroblasts. Although acceleration of healing of the burned cornea was also observed in mice lacking Smad3, the effects on epithelial and stromal healing were less pronounced than those in corneas treated with Smad7. Together these data suggest that overexpression of Smad7 may have effects beyond those of simply blocking Smad3/TGF-beta signaling and may represent an effective new strategy for treatment of ocular burns.

Therapeutic Effect of Topical Administration of SN50, an Inhibitor of Nuclear Factor-κB, in Treatment of Corneal Alkali Burns in Mice

We evaluated the therapeutic efficacy of topical administration of SN50, an inhibitor of nuclear factor-kappaB, in a corneal alkali burn model in mice. An alkali burn was produced with 1 N NaOH in the cornea of C57BL/6 mice under general anesthesia. SN50 (10 microg/microl) or vehicle was topically administered daily for up to 12 days. The eyes were processed for histological or immunohistochemical examination after bromodeoxyuridine labeling or for semi-quantification of cytokine mRNA. Topical SN50 suppressed nuclear factor-kappaB activation in local cells and reduced the incidence of epithelial defects/ulceration in healing corneas. Myofibroblast generation, macrophage invasion, activity of matrix metalloproteinases, basement membrane destruction, and expression of cytokines were all decreased in treated corneas compared with controls. To elucidate the role of tumor necrosis factor (TNF)-alpha in epithelial cell proliferation, we performed organ culture of mouse eyes with TNF-alpha, SN50, or an inhibitor of c-Jun N-terminal kinase (JNK) and examined cell proliferation in healing corneal epithelium in TNF-alpha-/- mice treated with SN50. An acceleration of epithelial cell proliferation by SN50 treatment was found to depend on TNF-alpha/JNK signaling. In conclusion, topical application of SN50 is effective in treating corneal alkali burns in mice.

Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial–mesenchymal transition of lens epithelium in mice

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFβ/activin signaling, on injury-induced epithelial–mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 μl of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and α-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFβ1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue.

TGFβ-Smad signalling in postoperative human lens epithelial cells

Aims: To localise Smads3/4 proteins in lens epithelial cells (LECs) of fresh and postoperative human specimens. Smads3/4 are involved in signal transduction between transforming growth factor β (TGFβ) cell surface receptors and gene promoters. Nuclear localisation of Smads indicates achievement of endogenous TGFβ signalling in cells. Methods: Three circular sections of the anterior capsule, one lens, and 17 capsules undergoing postoperative healing were studied. Immunohistochemistry was performed for Smads3/4 in paraffin sections of the specimens. The effect of exogenous TGFβ2 on Smad3 subcellular localisation was examined in explant cultures of extracted human anterior lens epithelium. Results: The cytoplasm, but not the nuclei, of LECs of uninjured lenses was immunoreactive for Smads3/4. In contrast, nuclear immunoreactivity for Smads3/4 was detected in LECs during capsular healing. Nuclei positive for Smads3/4 were observed in monolayered LECs adjacent to the regenerated lens fibres of Sommerring’s ring. Interestingly, the nuclei of LECs that were somewhat elongated, and appeared to be differentiating into fibre-like cells, were negative for Smads3/4. Fibroblast-like, spindle-shaped lens cells with nuclear immunoreactivity for nuclear Smads3/4 were occasionally observed in the extracellular matrix accumulated in capsular opacification. Exogenous TGFβ induced nuclear translocation of Smad3 in LECs of anterior capsule specimens in explant culture. Conclusions: This is consistent with TGFβ induced Smad signalling being involved in regulating the behaviour of LECs during wound healing after cataract surgery.

Inhibition of p38MAP kinase suppresses fibrotic reaction of retinal pigment epithelial cells

Proliferative vitreoretinopathy (PVR) is one of the major causes of the failure of retinal detachment surgery. Its pathogenesis includes a fibrotic reaction by the retinal pigment epithelium and other retina-derived non-neural cells, leading to fixation of the detached retina. We examined the role of p38 mitogen-activated protein kinase (MAPK) in transforming growth factor (TGF)-β2-dependent enhancement of the fibrogenic reaction in a human retinal pigment epithelial cell line, ARPE-19, and also evaluated the therapeutic efficacy of inhibiting p38MAPK by adenoviral gene transfer of dominant-negative (DN) p38MAPK in a mouse model of PVR. Exogenous TGF-β2 activates p38MAPK in ARPE-19 cells. It also suppresses cell proliferation, but this was unaffected by addition of the p38MAPK inhibitor, SB202190. SB202190 interfered with TGF-β2-dependent cell migration and production of collagen type I and fibronectin, but had no effect on basal levels of these activities. While SB202190 did not affect phosphorylation of the C-terminus of Smads2/3, it did suppress the transcriptional activity of Smads3/4 as indicated by a reporter gene, CAGA12-Luc. Gene transfer of DN-p38MAPK attenuated the post-retinal detachment fibrotic reaction of the retinal pigment epithelium in vivo in mice, supporting its effectiveness in preventing/treating PVR.

Lens epithelial cell outgrowth and matrix formation on intraocular lenses in rabbit eyes

Purpose: To evaluate the presence and distribution of lens epithelial cells (LECs) and extracellular matrix on intraocular lenses (IOLs) implanted in the capsular bag in rabbit eyes. Setting: Department of Ophthalmology, Wakayama Medical College, Wakayama, Japan. Methods: Five adult albino rabbits had phacoemulsification and IOL implantation in both eyes. Two or 11 months later, the animals were killed by intravenous pentobarbital. The IOLs were removed and observed under scanning and transmission electron microscopy. Results: In addition to the macrophages and giant cells on the IOLs, all eyes had a monolayer of flattened cells growing out from the residual anterior lens capsule and a fibrous meshwork of extracellular matrix. Unlike those of a macrophagic origin, these cells had no central cytoplasmic elevation of nuclei and few cell surface microvilli and were considered to be proliferating LECs. Conclusion: Lens epithelial cells are involved in the eye’s cellular reaction to IOLs and in the formation of extracellular matrix on IOLs. Further study of LEC behavior on IOLs should be done to improve IOL biocompatibility.