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We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-β1 (TGFβ1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8-12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFβ1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFβ1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFβ1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFβ1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.

We examined whether the loss of transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing ion channel, or TRPA1 antagonist treatment affects the severity inflammation and scarring during tissue wound healing in a mouse cornea injury model. In addition, the effects of the absence of TRPA1 on transforming growth factor β1 (TGF-β1)-signaling activation were studied in cell culture. The lack of TRPA1 in cultured ocular fibroblasts attenuated expression of TGF-β1, interleukin-6, and α-smooth muscle actin, a myofibroblast the marker, but suppressed the activation of Smad3, p38 MAPK, ERK, and JNK. Stroma of the healing corneas of TRPA1(-/-) knockout (KO) mice appeared more transparent compared with those of wild-type mice post-alkali burn. Eye globe diameters were measured from photographs. An examination of the corneal surface and eye globes suggested the loss of TRPA1 suppressed post-alkali burn inflammation and fibrosis/scarring, which was confirmed by histology, immunohistochemistry, and gene expression analysis. Reciprocal bone marrow transplantation between mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO mouse wound healing with reduced inflammation and fibrosis. Systemic TRPA1 antagonists reproduced the KO phenotype of healing. In conclusion, a loss or blocking of TRPA1 in mice reduces inflammation and fibrosis/scarring in the corneal stroma during wound healing following an alkali burn. The responsible mechanism may include the inhibition of TGF-β1-signaling cascades in fibroblasts by attenuated TRPA1 signaling. Inflammatory cells are considered to have a minimum involvement in the exhibition of the KO phenotype after injury.

We investigated the effects of loss of tenascin C on the healing of the stroma using incision-injured mice corneas. Tenascin C was upregulated in the stroma following incision injury to the cornea. Wild-type (WT) and tenascin C-null (knockout (KO)) mice on a C57BL/6 background were used. Cell culture experiments were also conducted to determine the effects of the lack of tenascin C on fibrogenic gene expression in ocular fibroblasts. Histology, immunohistochemistry and real-time reverse transcription PCR were employed to evaluate the healing process in the stroma. The difference in the incidence of wound closure was statistically analyzed in hematoxylin and eosin-stained samples between WT and KO mice in addition to qualitative observation. Healing of incision injury in corneal stroma was delayed, with less appearance of myofibroblasts, less invasion of macrophages and reduction in expression of collagen Iα1, fibronectin and transforming growth factor β1 (TGFβ1) in KO mice compared with WT mice. In vitro experiments showed that the loss of tenascin C counteracted TGFβ1 acceleration of mRNA expression of TGFβ1, and of collagen Iα1 and of myofibroblast conversion in ocular fibroblasts. These results indicate that tenascin C modulates wound healing-related fibrogenic gene expression in ocular fibroblasts and is required for primary healing of the corneal stroma.

Transforming growth factor-beta (TGF-beta) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-beta1 in the salivary gland, we created a transgenic mouse (beta1(glo)) that conditionally overexpresses active TGF-beta1 upon genomic recombination by Cre recombinase. beta1(glo) mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (beta1(glo)/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those beta1(glo)/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-beta signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-beta1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-beta in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögren's syndrome, or with radiation therapy given to head-and-neck cancer patients.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

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)-beta2-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-beta2 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-beta2-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.

An alkali burn in the cornea is a common serious clinical problem often leading to permanent visual impairment. Since transforming growth factor-beta (TGF-beta) is involved in the response to corneal injury, we evaluated the therapeutic effects of adenoviral gene transfer of mouse bone morphogenic protein-7 (BMP-7), which has antagonistic effects on TGF-beta in tissue fibrosis. Burned cornea did not express endogenous BMP-7 mRNA and protein. Resurfacing of the burned cornea by invading conjunctival epithelium was accelerated by adenoviral introduction of BMP-7. Exogenous BMP-7 expression also suppressed myofibroblast generation, appearance of monocytes/macrophages and expression of MCP-1, TGF-betas, and collagen I alpha2 chain in the affected stroma. Ectopic BMP-7 did not suppress stromal neovascularization throughout the interval studied and also did not reduce VEGF mRNA expression at Day 10. Ectopic BMP-7 in burned corneal tissue resulted in activation of Smad1/5/8 signaling and partial suppression of the phospho-Smad2 signal. These data suggest that overexpression of BMP-7 is an effective strategy for treatment of ocular alkali burns.

We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFbeta/activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 microl 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 alpha-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 TGFbeta1 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.

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-beta (TGF-beta) 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-beta 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 alpha-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-beta 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.