Cytokines and growth factors in aqueous humor of the eye

The aqueous humor that bathes the inner ocular structures (corneal endothelium, iris, crystalline lens, trabecular meshwork, and retina) contains various cytokines and growth factors. TGF, especially TGF2, is the predominant cytokine. Physiologically, TGF is mainly produced in the ciliary epithelium and lens epithelium as a latent, inactive, form consisting of mature TGF, the latency-associated peptide (LAP) (small latent form), and the latent-TGF-binding protein (LTBP).^{17, 18, 19, 20, 21, 22, 23, 24} Heterogeneous expression patterns of each TGF isoform in the crystalline lens have been reported in humans and animals.²⁵ During the clinical course of various ocular diseases, the concentration of TGF2 in the aqueous humor changes. For example, in an eye with proliferative vitreoretinopathy (PVR), a disorder of post-retinal detachment and retinal fibrosis, the concentration of TGF2 in the vitreous humor increases in association with the progression of retinal fibrosis. The concentration of total and active TGF2 is also higher in patients with diabetic retinopathy and open-angle glaucoma than in normal subjects. In diabetic retinopathy, chronic obstruction of retinal microvessels induces upregulation of VEGF and chemotaxis of macrophages, a potent source of TGFs. VEGF and TGF cooperate to induce both retinal neovascularization and fibrosis around these new vessels, which may potentially cause retinal detachment or bleeding. Increased TGF2 levels induce matrix expression and deposition in trabecular meshwork cells, leading to obstruction of the aqueous drainage route and an increase of intraocular pressure in a glaucomatous eye. In each of these examples, TGF plays a role in disease pathogenesis. In eyes with pseudoexforiation syndrome, a kind of glaucoma with deposition of exforiative material on the lens, iris, or trabecular meshwork, the level of TGF1 increases, but the exact role of TGF1 in the pathogenesis of this disease is unknown.

TGF signal transduction

Upon TGF binding to its receptor, signaling occurs through a pair of transmembrane receptor serine–threonine kinases and the downstream mediator Smad proteins. Receptor-activated Smad proteins, Smad2 and Smad3, are phosphorylated directly by the TGF receptor type I kinase. They then partner with the common mediator, Smad4, and translocate to the nucleus where they play a prominent role in the activation of TGF-dependent gene targets. Smad6/7 are known to be inhibitory Smads, which block phosphorylation of Smads2/3.^{26, 27} However, the roles of Smad2 and Smad3 differ because the lack of Smad2 is lethal for mice at the embryonic stage, whereas those lacking Smad3 survive.²⁸

The bone morphogenetic proteins (BMPs), which are members of the TGF superfamily, bind to their own receptors and phosphorylate Smads 1, 5, and 8 which then bind to Smad4 for translocation to the nucleus. Additionally, in some cell types, TGF can potentially activate different arms of the mitogen-activated kinase (MAPK) pathway, including stress kinases (ie, c-Jun-N-terminal kinase, JNK), p38MAPK pathway, RhoA-related signals, phosphatase2A,²⁹ or PI3-kinase/AKT.^{30, 31, 32}

Although Smad2/3 signaling is relatively specific to the binding of ligands of the TGF/activin family to cell surface receptors, investigators are discovering that MAPKs (p42/p44 Erk1/2, JNK, or p38MAPK) phosphorylate specific sites in the middle linker region of Smad2/3, sites that are distinct from the C-terminus that is phosphorylated by the TGF II receptor (Figure 2).^{33, 34, 35, 36} Thus, ligands capable of activating MAPKs potentially modulate Smad signaling induced by TGF. It has recently been demonstrated that p38MAPK activate phosphorylation of Smad3 in the middle linker region, which enhances Smad3/4 complex formation and nuclear translocation,³⁷ consistent with our finding of diminished Smad3/4 reporter gene activity in the presence of a p38MAPK inhibitor. Such phosphorylation by MAPKs in the Smad3 linker region is reportedly required for the full activation of Smad signaling.^{38, 39} For example, inhibition of p38MAPK by the specific inhibitor SB202190 interferes with stimulatory effects of exogenous TGF2 on migration of cells and on production of ECM components, such as collagen type I and fibronectin, while having no effects on the basal activity. Moreover, p38 MAPK may affect these end points not only by direct phosphorylation of the Smad proteins in the middle linker region⁴⁰ but also by activation of cooperating transcription factors. For example, TGF-activated kinase (TAK1) has been shown to be an upstream activator of MKK6 and activation of this pathway results in phosphorylation of activating transcription factor 2 (ATF2) and enhancement of complex formation between Smad4 and ATF2.^{41, 42} However, another report shows that Smads and p38MAPK independently regulate collagen I 1 mRNA in hepatic stellate cells,⁴³ demonstrating the complexity of regulation of gene expression by TGF. Furthermore, TGF/Smad signaling is susceptible to modualtion by other cotranscription factors such as c-Ski and SnoN.

Figure 2.

Smad2/3 can be activated by non-TGF growth factors at the middle linker region.

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Embryogenesis and developmental disorders

Classical gene targeting techniques have provided important information about the role of each TGF isoform in eye morphogenesis, although the mice also have various systemic abnormalities. Although a mouse embryo that lacks TGF1 or TGF3 does not have any ocular abnormalities, a mouse embryo lacking TGF2 has multiple defects in ocular structures, that is, thin cornea with a loss of the corneal endothelium and anterior chamber, immature retina, and persistent vitreous vessels.^{44, 45, 46, 47, 48} These findings may coincide with the fact that TGF2 predominates in eye aqueous humor. Overexpression of TGF1 by using -crystalline promoter in TGF2-null mice rescues the abnormalities in ocular development caused by the deletion of TGF2.⁴⁹

Lens epithelial cells are of ectodermal origin. During embryonic development, surface ectoderm invaginates into the optic cup and the vesicle is separated from surface ectoderm on embryonic day 11.5 in the mouse. At this time, the cells also start to express vimentin, an intermediate cytoskeletal protein of mesenchymal cell types, but also retain their epithelial character by expressing the epithelial surface marker, cadherins. Then, the cells located in the posterior part of the lens vesicle start to express various crystalline proteins to form a transparent lens. Members of the FGF and BMP family are potent inducers of lens fiber differentiation. They are expressed in various ocular tissues, that is, retina, ciliary body, and lens cell themselves. The retina produces FGF and insulin-like growth factor (IGF) family members as potential fiber cell differentiation factors. The nuclei of elongating lens fiber cells are positive for phospho-Smad1, an indicator of signaling through BMP receptors.⁵⁰ These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor. In mature lens fibers, the nuclei are degraded by apoptosis and this apoptotic process is also modulated by Smad signals.⁵⁰

In addition to BMPs, TGFs are also involved in lens fiber differentiation.⁵⁰ Overexpression of dominant-negative forms of either type I or type II TGF receptors in the lens fibers of transgenic mice using mouse A-crystallin promoter results in the development of pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus.

Although all three TGF isoforms are expressed in cornea, the lack of any one of them does not affect embyonic morphogenesis/differentiation of corneal epithelium as examined by the expression pattern of cornea-specific cytokeratin, keratin 12. Additionally, loss of Smad3, a prominent TGF-signaling molecule, does not produce ocular abnormalities, indicating that multiple signaling pathways are involved in ocular tissue morphogenesis.

TGF signal transduction and tissue fibrosis

TGF generally enhances gene expression related to tissue fibrosis in vivo and in vitro in mesenchymal cells in the eye. Details of differences between Smad2 and Smad3 were recently investigated using a gene expression array made of embryonic fibroblasts obtained from embryos lacking either Smad2 or Smad3.^{51, 52} Expression of -smooth muscle actin (SMA), important in fibroblast-myofibroblast conversion, is mediated by Smad2.^{53, 54, 55} However, expression of Snail, the master transcription factor involved in the earlier step of the epithelial–mesenchymal transition (EMT), as an important step in the process of tissue fibrosis in the eye, is controlled by Smad3.⁵⁶ The expression of the majority of the extracellular matrix components and enzymes involved in matrix reorganization/maturation depends on Smad3, whereas expression of matrix metalloproteinase-2 is Smad2 dependent. In Smad3-null mice, re-epithelialization is accelerated and fibrosis is reduced during tissue repair in skin.⁵⁷ However, blocking TGF type II receptor function by dominant negative expression in collagen I-expressing fibroblasts in a transgenic mouse results instead in a paradoxical systemic tissue fibrosis in association with an uncontrolled Smad signaling activation.⁵⁸ The mechanism of this phenomenon could be explained the fact that Smad3 is phosphorylated by various MAPK at their middle linker region, which might promote nuclear translocation of Smad and might stimulate fibrosis-related gene expression.

Wound healing reaction in the lens and post-cataract surgery complications

The crystalline lens is a unique tissue, consisting of epithelial cells, lens fibers, and the anuclear lens content inside a specialized basement membrane, the lens capsule. Following cataract surgery or lens capsular injury, cuboidal lens epithelial cells undergo EMT and transdifferentiate to myofibroblasts, expressing SMA on the residual lens capsular tissue (Figure 3).^{59, 60, 61, 62, 63, 64, 65, 66} Tissue fibrosis in association with EMT is also a key step in the process of fibrotic diseases in other tissues and organs, characterized by the presence of fibrous tissue accumulation and contraction by SMA-expressing myofibroblasts.^{67, 68} In this process, an epithelial cell changes its morphology and its transcriptional program to those characteristic of a mesenchymal cell type. Lens epithelium-derived myofibroblasts become capable of expressing components of the fibrous ECM and matrix-degrading enzymes. Structural and histological organization of postoperative capsular opacification is quite similar to that seen in healing tissue or tissue granulation formed around an implanted foreign body. Clinically, this fibrotic reaction in postoperative lens epithelium results in opacification and contraction of the residual lens capsule. Optical transparency is reduced, and implanted artifical intraocular lens (IOL) move off center, both of which decrease the patients' vision.

Figure 3.

Summary of biological reaction against an implanted IOL. A foreign body reaction is mediated by macrophages and foreign body giant cells generated through a fusion of many macrophages. A wound healing reaction occurs in lens epithelium. The equatorial region of the capsular bag is occupied by regenerated lenticular fibers of Sommerring's ring. Lens epithelial cells on the posterior capsule exhibit an elongated, fibroblast-like shape or Elschnig's pearl formation.

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TGF and Smad signaling in lens epithelium EMT

In the quiescent normal lens epithelial cell, Smads2/3 are detectable in the cytoplasm, indicating that the cells either are not or are minimally affected by endogenous TGF through this signaling pathway, as they are not detectable in the nucleus. However, TGF signaling is rapidly activated following wounding.⁶⁹ Following cataract surgery, Smad2/3 translocates to the nucleus prior to the appearance of SMA-positive cells (heralding the ocurrence of EMT). Similarly, in mice, an injury in the anterior capsule induces Smads3/4 nuclear translocation within 12 h, being followed by expression of Snail and subsequent EMT in the lens epithelium. This Smad nuclear translocation was abolished by local administration of anti-TGF2-neutralizing antibody. TGF is the growth factor involved in EMT of lens epithelial cells in vivo, as has been shown for other epithelial cell types in vitro. For example, overexpression of TGF1 in lens cells by transgenic techniques induces cataractous changes in the lens epithelial cells in association with EMT and accumulation of fibrous/collagenous extracellular matrix.^{70, 71}

Loss of Smad3 attenuates injury-induced EMT in the lens or renal epithelium. However, the suppression of EMT in lens epithelium seems to be dependent on the level of TGF stimuli. Our unpublished data show that severe intraocular inflammation caused by corneal exposure to alkali is associated with EMT in lens epithelium even in Smad3-null mice, although the extent of EMT is much lower. Similarly, overexpression of active TGF1 in lens epithelium using adenoviral gene introduction or transgenic technology induces EMT in lens epithelium in mice and this EMT (although to a lesser extent) is also seen in the absence of Smad3 (West-Mays J, personal communication, 2005). These findings suggest that upon uncontrolled stimulation by TGF, lens epithelium is capable of undergoing EMT, possibly via signaling via other as yet unidentified molecules. Alternatively, Smad2 might bypass the loss of Smad3. Nevertheless, the involvement of Smad3 signal in lens epithelium EMT raises the possibility of therapies for EMT-related fibrotic diseases gene transfer of Smad7 or other molecules which are capable of blocking Smad signaling, that is, BMP-7, Id2 or Id3, as discussed below.⁷²

Other signaling cascades are also required for TGF-induced EMT.^{66, 73} Our unpublished data show that specific inhibitors of PI3-kinase or Rho kinase also suppress TGF2-induced EMT of lens epithelium in organ culture.

PVR and retinal pigment epithelium (RPE)

PVR is a disease caused by the formation of fibrotic tissue on the detached retina, which reduces the flexibility of the retina and may potentially make it difficult to reattach the retina. RPE cells are normally located in the cell layer external to the retina. Following retinal break and detachment, RPE cells disseminate in the subretinal space and vitreous humor through the retinal break(s), and then settle on the luminal retinal surface following development of rhegmatogenous retinal detachment.^{74, 75} RPE cells then undergo transformation to fibroblast-like cells, proliferate, and produce extracellular matrix components, participating in this fibrotic sequelae. Muller glia cells are also involved in the fibrotic reaction of the detached retina.

As in the EMT-related fibrosis in the lens, TGF can induce transformation of RPE cells to myofibroblast-like cells in vitro,^{76, 77, 78} suggesting that TGF is likely a key player in the development of PVR, although various other growth factors, including PDGF, HGF, and activin, are all reportedly involved in its pathogenesis.^{79, 80, 81, 82, 83, 84, 85} The concentration of TGF2 in the vitreous humor of the eye correlates with the severity of the PVR, underlying its importance.⁷⁹ Similar to other cell types, RPE-cell-EMT is also suppressed by the loss of Smad3 in vivo, resulting in the attenuation of development of PVR. Unlike gene introduction of active TGF1 in the lens epithelium, adenoviral TGF1 does not induce EMT in RPE in the absence of Smad3 (J West-Mays, personal communication, 2005).

Gene introduction to suppress EMT and subsequent tissue fibrosis in lens or retinal pigment epithelia

Suppression of EMT might be beneficial to prevent or treat the lens capusar fibrosis that leads to fibrotic-type cataracts, or post-cataract surgery capsular opacification, both of which potentially reduce vision. Based on the finding that loss of Smad3 attenuates injury-induced lens epithelial EMT, we tested adenoviral gene introduction of cDNAs for Smad7, BMP-7, Id2 or Id3, all of which antagonize TGF/Smad signal, in vivo to an injured mouse lens epithelium. While all of these genes attenuated injury-induced EMT of the lens epithelium,⁸⁶ Smad7 gave the greatest degree of inhibition of in vivo lens cell EMT.

Attenuation of injury-induced EMT of lens epithelial cells by Smad3 gene ablation or Smad7 gene introduction also suggests that such approaches might suppress EMT in RPE cells, and therefore prove beneficial in the treatment of PVR. Indeed, loss of Smad3 attenuates PVR in vivo,⁸⁷ and Smad7 gene introduction actually does suppress PVR in mice (S Saika, unpublished data, 2005).

Activation of Smads by phosphorylation at its middle linker region by MAPK is also a potential target of inhibition of TGF signaling. It has been reported that p38MAPK is involved in EMT in cultured cells. A p38MAPK inhibitor reduces reporter gene expression using an Smad-dependent promoter, indicating that p38MAPK signal may be involved in Smad-dependent gene expression. Chemical inhibition of p38MAPK attenuates migration and ECM production of the RPE cell line, ARPE-19.⁸⁸ In vivo adenoviral gene transfer of dominant-negative p38MAPK suppresses the fibrotic reaction by RPE cells in an experimental mouse PVR model.⁸⁸ Further study is needed to establish the clinical application of this treatment strategy.

Roles of TGF siganling in corneal wound healing

The cornea consists of a nonkeratinizing stratified epithelium, lying on Bowman's membrane, and a stroma consisting of collagenous lamellae and keratocytes (corneal fibroblasts) (Figure 4). Although the cornea lacks vasculature, the main components are quite similar to those of the skin; stratified epithelium and a collagenous matrix containing mesenchymal cells lying beneath it. TGF is expressed in corneal tissue.^{89, 90, 91, 92, 93, 94, 95, 96, 97}

Figure 4.

Light microscopic histology of the human cornea. The surface is covered with nonkeratinizing stratified epithelium. Keratocytes are the mesenchymal (fibroblast-like) cells in the stroma. Dense matrix membrane of Descemet's membrane locates beneath the epithelial basement membrane. Bar, 10 m.

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An epithelial defect in the cornea must be rapidly resurfaced to avoid microbial infection and further damage to the underlying stroma. Healing of an epithelial defect is achieved by migration of epithelial cells, followed by an enhancement of cell proliferation for re-establishment of the epithelium stratification. Although there is some difference in the expression pattern of TGF isoforms in the cornea, it is believed that TGF is upregulated upon wounding. In mouse cornea, intracellular TGF1 is detected in corneal epithelium, but extracellular, secreted TGF1 is not observed in an uninjured healthy cornea; but TGF2 and TGF3 are both detected in uninjured epithelium. Following an injury, extracellular TGF1, TGF2, and TGF3 are all detected in subepithelial stromal tissue. In vitro cell or organ culture reveals that endogenous TGF enhances cell migration of corneal epithelium. Such migrating epithelium lacks proliferative activity presumably due to inhibition of cell proliferation by TGFl.

Unlike epidermis, loss of Smad3 does not affect re-epithelialization following corneal debridement in mice (S Saika, unpublished data, 2002). Nevertheless, this finding does not exclude the possibility that TGF has an important function in modulation of corneal epithelial healing. Migrating epithelium upregulates phosphorylation of p38MAPK as early as 1 h postinjury. Organ-culture experiments using a TGF-neutralizing antibody and the specific p38MAPK inhibitors, SB202190 and SB203580, revealed that TGF/p38MAPK signal is required for epithelial cell migration and cessation of cell proliferation in migrating cells.⁹⁸ p38MAPK is known to modulate signaling cascades toward cell death or extracellular matrix expression, depending on cell types and kinds of stimuli.^{99, 100, 101} Involvement of p38MAPK in cell migration has been observed in various cell types including corneal epithelium and cancer cell lines. However, it has not been fully elucidated whether or not p38MAPK's involvement in the cell migration is mediated by the phosphorylation of the linker regions of Smads2/3 or by its direct effect.

Stromal healing is initiated by inflammatory cells, that is, macrophages that activate mesenchymal cells via their expression of cytokines, including TGF. The activated mesenchymal cells (keratocytes) express matrix components and various growth factors and contract the scarring stroma. VEGF, expressed in both invading macrophages and myofibroblasts, induces stromal neovascularization that also potentially causes corneal opacification.

Gene therapy for the treatment of corneal inflammation and fibrosis by targeting TGF signaling

In corneas affected by chemical or thermal burn, or Stevens–Johnson's syndrome, various cytokines and growth factors, including interleukins, EGF, KGF, HGF, TNF , TGF, VEGF, and macrophage/monocyte chemoattractant protein-1 (MCP-1), are believed to orchestrate cellular interactions and behaviors. The resulting pathological outcomes include scarring, conjunctivalization of the corneal surface and neovascularization. TGF is chemotactic to macrophages and also activates stromal fibroblasts (keratocytes), leading to the generation of myofibroblasts and induction of other cytokines such as VEGF and MCP-1 which also have chemotactic activity. A key outcome is EMT-related tissue fibrosis. It is then reasonable to posit that an excessive wound healing reaction from inflammation and fibrobalst acticvation can also be a target of TGF-inhibition therapy. Blocking the activity of TGF by systemic expression of soluble TGF receptor by adenoviral gene transfer results both in the suppression of liver fibrosis and acceleration of tissue repair in injured corneas in rats.^{102, 103} Although these studies clearly demonstrate that endogenous TGF is critical in the corneal tissue destruction after alkali exposure, blocking TGF activity at the receptor level might potentially perturb healing of the corneal epithelial component by interfering with the p38MAPK activity that is required for epithelial cell migration. Smad3-null cutaneous repair is associated with hyperproliferation of epidermal keratinocytes and decreased chemotaxis of macrophages, resulting in acceleration of epithelial resurfacing and less scarring. This information prompted us to test whether blocking TGF activity at the level of Smad signaling level might yield a more favorable result, since other TGF signaling cascades would remain intact. Using a mouse corneal alkali burn model, we have shown that loss of Smad3 suppresses tissue destruction of the healing cornea in association with a reduction of macrophage infiltration, inhibition of myofibroblast generation, and suppression of growth factor expression.¹⁰⁴

Neovascularization also potentially impairs vision. Adenoviral gene transfer of mouse Smad7 cDNA has been used in the treatment of tissue fibrosis in several disease models, that is, bleomycin-induced pulmonary fibrosis, drug-induced liver fibrosis, or kidney fibrosis by unilateral ureteral obstruction.^{105, 106, 107} Our results show that in mice Smad7 gene introduction by topical application suppresses scarring and neovascularization of the burned cornea, restoring its transparency.¹⁰⁴ Smad7 also suppressed generation of myofibroblasts, macrophage invasion, and the expression of wound healing-related cytokines. The effects were more marked than those seen in Smad3-null mice, probably because Smad7 also suppresses phosphorylated RelA of the NF-B pathway, which leads to suppression of inflammation cascades.^{104, 108} Signals derived from bone morphogenic protein-7 (BMP-7) are known to antagonize TGF/Smad signal via Smad1/5/8 signal and induction of Id2 and Id3. We have shown that gene introduction of BMP-7 also has a therapeutic effect on an alkali burn in mice, although its efficacy is less than that of Smad7.¹⁰⁹

Unlike in an alkali-burned cornea, Stevens–Johnson's syndrome is an inflammatory ocular surface disease caused by an autoimmune mechanism. Nevertheless, the main component of the disease consists of inflammation and scarring that are similar to those seen in a burned eye. Thus, there is a possibility that interference of TGF signaling might have a therapeutic effect on this disorder.

In conclusion, it is apparent that further understanding of the roles of TGF in physiological and pathological processes of the eye is needed to develop new strategies in the treatment of ocular diseases; and Smad signaling is an important target for development of treatments of fibrosis-related diseases in the eye.

References

1. Martin P. Wound healing—aiming for perfect skin regeneration. Science 1997;276:75–81. | Article | PubMed | ISI | ChemPort |
2. Baum CL, Arpey CJ. Normal cutaneous wound healing: clinical correlation with cellular and molecular events. Dermatol Surg 2005;31:674–686. | PubMed | ISI |
3. Klenkler B, Sheardown H. Growth factors in the anterior segment: role in tissue maintenance, wound healing and ocular pathology. Exp Eye Res 2004;79:677–688. | Article | PubMed | ISI | ChemPort |
4. Grose R, Werner S. Wound-healing studies in transgenic and knockout mice. Mol Biotechnol 2004;28:147–166. | Article | PubMed | ISI | ChemPort |
5. Efron PA, Moldawer LL. Cytokines and wound healing: the role of cytokine and anticytokine therapy in the repair response. J Burn Care Rehabil 2004;25:149–160. | PubMed | ISI |
6. Flanders KC. Smad3 as a mediator of the fibrotic response. Int J Exp Pathol 2004;85:47–64. | Article | PubMed | ISI | ChemPort |
7. Kim IY, Kim MM, Kim SJ. Transforming growth factor-beta: biology and clinical relevance. J Biochem Mol Biol 2005;38:1–8. | PubMed | ISI | ChemPort |
8. Rockey DC. Antifibrotic therapy in chronic liver disease. Clin Gastroenterol Hepatol 2005;3:95–107. | Article | PubMed | ISI | ChemPort |
9. Saika S. TGF- signal transduction in corneal wound healing as a therapeutic target. Cornea 2004;23(Suppl):S25–S30. | PubMed |
10. Schiller M, Javelaud D, Mauviel A. TGF--induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing. J Dermatol Sci 2004;35:83–92. | Article | PubMed | ISI | ChemPort |
11. Leask A, Abraham DJ. TGF- signaling and the fibrotic response. FASEB J 2004;18:816–827. | Article | PubMed | ChemPort |
12. Moustakas A, Pardali K, Gaal A, et al. Mechanisms of TGF- signaling in regulation of cell growth and differentiation. Immunol Lett 2002;82:85–91. | Article | PubMed | ISI | ChemPort |
13. Roberts AB, Russo A, Felici A, et al. Smad3: a key player in pathogenetic mechanisms dependent on TGF-. Ann NY Acad Sci 2003;995:1–10. | PubMed | ChemPort |
14. ten Dijke P, Goumans MJ, Itoh F, et al. Regulation of cell proliferation by Smad proteins. J Cell Physiol 2002;191:1–16. | Article | PubMed | ISI | ChemPort |
15. Van Obberghen-Schilling E, Roche NS, Flanders KC, et al. Transforming growth factor 1 positively regulates its own expression in normal and transformed cells. J Biol Chem 1988;263:7741–7746. | PubMed | ChemPort |
16. Holmes A, Abraham DJ, Sa S, et al. CTGF and SMADs, maintenance of scleroderma phenotype is independent of SMAD signaling. J Biol Chem 2001;276:10594–10601. | Article | PubMed | ISI | ChemPort |
17. Jampel HD, Roche N, Stark WJ, et al. Transforming growth factor- in human aqueous humor. Curr Eye Res 1990;9:963–969. | PubMed | ISI | ChemPort |
18. Tripathi RC, Li J, Chan WF, et al. Aqueous humor in glaucomatous eyes contains an increased level of TGF-2. Exp Eye Res 1994;59:723–727. | Article | PubMed | ISI | ChemPort |
19. Kokawa N, Sotozono C, Nishida K, et al. High total TGF-2 levels in normal human tears. Curr Eye Res 1996;15:341–343. | PubMed | ISI | ChemPort |
20. Hu DN, McCormick SA, Lin AY, et al. TGF-2 inhibits growth of uveal melanocytes at physiological concentrations. Exp Eye Res 1998;67:143–150. | Article | PubMed | ISI | ChemPort |
21. Wallentin N, Wickstrom K, Lundberg C. Effect of cataract surgery on aqueous TGF- and lens epithelial cell proliferation. Invest Ophthalmol Vis Sci 1998;39:1410–1418. | PubMed | ISI | ChemPort |
22. Chen KH, Harris DL, Joyce NC. TGF-2 in aqueous humor suppresses S-phase entry in cultured corneal endothelial cells. Invest Ophthalmol Vis Sci 1999;40:2513–2519. | PubMed | ISI | ChemPort |
23. Connor Jr TB, Roberts AB, Sporn MB, et al. Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye. J Clin Invest 1989;83:1661–1666. | PubMed | ISI | ChemPort |
24. Picht G, Welge-Luessen U, Grehn F, et al. Transforming growth factor 2 levels in the aqueous humor in different types of glaucoma and the relation to filtering bleb development. Graefes Arch Clin Exp Ophthalmol 2001;239:199–207. | PubMed | ISI | ChemPort |
25. Saika S, Miyamoto T, Kawashima Y, et al. Immunolocalization of TGF-1, -2, and -3, and TGF- receptors in human lens capsules with lens implants. Graefes Arch Clin Exp Ophthalmol 2000;238:283–293. | Article | PubMed | ISI | ChemPort |
26. Shi Y, Massague J. Mechanisms of TGF- signaling from cell membrane to the nucleus. Cell 2003;113:685–700. | Article | PubMed | ISI | ChemPort |
27. ten Dijke P, Goumans MJ, Itoh F, et al. Regulation of cell proliferation by Smad proteins. J Cell Physiol 2002;191:1–16. | Article | PubMed | ISI | ChemPort |
28. Yang X, Letterio JJ, Lechleider RJ, et al. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-. EMBO J 1999;18:1280–1291. | Article | PubMed | ISI | ChemPort |
29. Petritsch C, Beug H, Balmain A, et al. TGF- inhibits p70 S6 kinase via protein phosphatase 2A to induce G(1) arrest. Genes Dev 2000;14:3093–3101. | Article | PubMed | ISI | ChemPort |
30. Gotzmann J, Huber H, Thallinger C, et al. Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-1 and Ha-Ras: steps towards invasiveness. J Cell Sci 2002;115:1189–1202. | PubMed | ISI | ChemPort |
31. Peron P, Rahmani M, Zagar Y, et al. Potentiation of Smad transactivation by Jun proteins during a combined treatment with epidermal growth factor and transforming growth factor- in rat hepatocytes. Role of phosphatidylinositol 3-kinase-induced AP-1 activation. J Biol Chem 2001;276:10524–10531. | Article | PubMed | ISI | ChemPort |
32. Bhowmick NA, Ghiassi M, Bakin A, et al. Transforming growth factor-1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell 2001;12:27–36. | PubMed | ISI | ChemPort |
33. Vadlamudi R, Adam L, Talukder A, et al. Serine phosphorylation of paxillin by heregulin-1: role of p38 mitogen activated protein kinase. Oncogene 1999;18:7253–7264. | Article | PubMed | ISI | ChemPort |
34. Mori S, Matsuzaki K, Yoshida K, et al. TGF- and HGF transmit the signals through JNK-dependent Smad2/3 phosphorylation at the linker regions. Oncogene 2000;23:7416–7429. | Article |
35. Tahashi Y, Matsuzaki K, Date M, et al. Differential regulation of TGF- signal in hepatic stellate cells between acute and chronic rat liver injury. Hepatology 2002;35:49–61. | Article | PubMed | ISI | ChemPort |
36. Yoshida K, Matsuzaki K, Mori S, et al. Transforming growth factor- and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury. Am J Pathol 2005;166:1029–1039. | PubMed | ISI | ChemPort |
37. Furukawa F, Matsuzaki K, Mori S, et al. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts. Hepatology 2003;38:879–889. | Article | PubMed | ISI | ChemPort |
38. Geller SF, Lewis GP, Fisher SK. FGFR1 signaling and AP-1 expression after retinal detachment: reactive Muller and RPE cells. Invest Ophthalmol Vis Sci 2001;42:1363–1369. | PubMed | ISI | ChemPort |
39. Yu L, Hebert MC, Zhang Y. TGF- receptor-activated p38 MAP kinase mediates Smad-independent TGF- responses. EMBO J 2002;21:3749–3759. | Article | PubMed | ISI | ChemPort |
40. Saklatvala J, Dean J, Finch A. Protein kinase cascades in intracellular signalling by interleukin-1 and tumour necrosis factor. Biochem Soc Symp 1999;64:63–77. | PubMed | ChemPort |
41. Yosimichi G, Nakanishi T, Nishida T, et al. CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular-signal regulated kinase (ERK). Eur J Biochem 2001;268:6058–6065. | Article | PubMed | ISI | ChemPort |
42. Kim JY, Choi JA, Kim TH, et al. Involvement of p38 mitogen-activated protein kinase in the cell growth inhibition by sodium arsenite. J Cell Physiol 2002;190:29–37. | Article | PubMed | ISI | ChemPort |
43. Mori Y, Chen SJ, Varga J. Modulation of endogenous Smad expression in normal skin fibroblasts by transforming growth factor-beta. Exp Cell Res 2000;258:374–383. | Article | PubMed | ISI | ChemPort |
44. Shull MM, Doetschman T. Transforming growth factor-beta 1 in reproduction and development. Mol Reprod Dev 1994;39:239–246. | PubMed | ISI | ChemPort |
45. Kaartinen V, Voncken JW, Shuler C, et al. Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects ofepithelial–mesenchymal interaction. Nat Genet 1995;11:415–421. | Article | PubMed | ISI | ChemPort |
46. Proetzel G, Pawlowski SA, Wiles MV, et al. Transforming growth factor-beta 3 is required for secondary palate fusion. Nat Genet 1995;11:409–414. | Article | PubMed | ISI | ChemPort |
47. Sanford LP, Ormsby I, Gittenberger-de Groot AC, et al. TGF2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development 1997;124:2659–2670. | PubMed | ISI | ChemPort |
48. Saika S, Saika S, Liu CY, et al. TGF2 in corneal morphogenesis during mouse embryonic development. Dev Biol 2001;240:419–432. | Article | PubMed | ISI | ChemPort |
49. Zhao S, Overbeek PA. Elevated TGF signaling inhibits ocular vascular development. Dev Biol 2001;237:45–53. | Article | PubMed | ISI | ChemPort |
50. Beebe D, Garcia C, Wang X, et al. Contributions by members of the TGFbeta superfamily to lens development. Int J Dev Biol 2004;48:845–856. | Article | PubMed | ISI | ChemPort |
51. Yang YC, Piek E, Zavadil J, et al. Hierarchical model of gene regulation by transforming growth factor . Proc Natl Acad Sci USA 2003;100:10269–10274. | Article | PubMed | ChemPort |
52. Piek E, Ju WJ, Heyer J, et al. Functional characterization of transforming growth factor beta signaling in Smad2- and Smad3-deficient fibroblasts. J Biol Chem 2001;276:19945–19953. | Article | PubMed | ISI | ChemPort |
53. Flanders KC, Major CD, Arabshahi A, et al. Interference with transforming growth factor-/Smad3 signaling results in accelerated healing of wounds in previously irradiated skin. Am J Pathol 2003;163:2247–2257. | PubMed | ISI | ChemPort |
54. Evans RA, Tian YC, Steadman R, et al. TGF-1-mediated fibroblast–myofibroblast terminal differentiation—the role of Smad proteins. Exp Cell Res 2003;282:90–100. | Article | PubMed | ISI | ChemPort |
55. Roberts AB, Russo A, Felici A, et al. Smad3: a key player in pathogenetic mechanisms dependent on TGF-. Ann NY Acad Sci 2003;995:1–10. | PubMed | ChemPort |
56. Saika S, Kono-Saika S, Ohnishi Y, et al. Smad3 signaling is required for epithelial–mesenchymal transition of lens epithelium after injury. Am J Pathol 2004;16:651–663.
57. Ashcroft GS, Yang X, Glick AB, et al. Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol 1999;1:260–266. | Article | PubMed | ISI | ChemPort |
58. Denton CP, Zheng B, Evans LA, et al. Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGF) receptor leads to paradoxical activation of TGF signaling pathways with fibrosis in transgenic mice. J Biol Chem 2003;278:25109–25119. | Article | PubMed | ISI | ChemPort |
59. Apple DJ, Solomon KD, Tetz MR, et al. Posterior capsule opacification. Surv Ophthalmol 1992;37:73–116. | Article | PubMed | ISI | ChemPort |
60. Saika S, Kawashima Y, Miyamoto T, et al. Immunolocalization of prolyl 4-hydroxylase subunits, a-smooth muscle actin, and extracellular matrix components in human lens capsules with lens implants. Exp Eye Res 1998;66:283–294. | Article | PubMed | ISI | ChemPort |
61. Saika S, Miyamoto T, Tanaka S, et al. Response of lens epithelial cells to injury: role of lumican in epithelial–mesenchymal transition. Invest Ophthalmol Vis Sci 2003;44:2094–2102. | Article | PubMed | ISI |
62. Saika S. Relationship between posterior capsule opacification and intraocular lens biocompatibility. Prog Retin Eye Res 2004;23:283–305. | Article | PubMed | ISI | ChemPort |
63. Hay ED. An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 1995;154:8–20. | PubMed | ChemPort |
64. Hay ED, Zuk A. Transformations between epithelium and mesenchymae: normal, pathological, and experimentally induced. Am J Kidney Dis 1995;26:678–690. | PubMed | ISI | ChemPort |
65. Kang P, Svoboda KK. PI-3 kinase activity is required for epithelial–mesenchymal transformation during palate fusion. Dev Dyn 2002;225:316–321. | Article | PubMed | ISI | ChemPort |
66. Masszi A, Di Ciano C, Sirokmany G, et al. Central role for Rho in TGF-1-induced alpha-smooth muscle actin expression during epithelial–mesenchymal transition. Am J Physiol Renal Physiol 2003;284:F911–F924. | PubMed | ISI | ChemPort |
67. Sato M, Muragaki Y, Saika S, et al. Targeted disruption of TGF-1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin Invest 2003;112:1486–1494. | Article | PubMed | ISI | ChemPort |
68. Tomasek J, Gabbiani G, Hinz B, et al. Myofibroblasts and mechanoregulation of connective tissue remodeling. Nat Rev Mol Cell Biol 2002;3:349–463. | Article | PubMed | ISI | ChemPort |
69. Saika S, Okada Y, Miyamoto T, et al. Smad translocation and growth suppression in lens epithelial cells by endogenous TGF2 during wound repair. Exp Eye Res 2001;76:679–686. | Article |
70. Srinivasan Y, Lovicu FJ, Overbeek PA. Lens-specific expression of transforming growth factor 1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest 1998;101:625–634. | PubMed | ISI | ChemPort |
71. Lovicu FJ, McAvoy JW. FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling. Development 2001;128:5075–5084. | PubMed | ISI | ChemPort |
72. Saika S, Ikeda K, Yamanaka O, et al. Adenoviral gene transfer of BMP-7, Id2 or Id3 suppresses injury-induced epithelial–mesenchymal transition of lens epithelium in mice. Am J Physiol Cell Physiol 2005 (in press).
73. Kang Y, Massague J. Epithelial–mesenchymal transitions: twist in development and metastasis. Cell 2004;118:277–279. | Article | PubMed | ISI | ChemPort |
74. Pastor JC, de la Rua ER, Martin F. Proliferative vitreoretinopathy: risk factors and pathobiology. Prog Retin Eye Res 2002;21:127–144. | Article | PubMed | ISI |
75. Bochaton-Piallat ML, Kapetanios AD, Donati G, et al. TGF-1, TGF- receptor II and ED-A fibronectin expression in myofibroblast of vitreoretinopathy. Invest Ophthalmol Vis Sci 2000;41:2336–2342. | PubMed | ISI | ChemPort |
76. Casaroli-Marano RP, Pagan R, Vilaro S. Epithelial–mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1999;40:2062–2072. | PubMed | ISI | ChemPort |
77. Grisanti S, Guidry C. Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype. Invest Ophthalmol Vis Sci 1995;36:391–405. | PubMed | ISI | ChemPort |
78. Lee SC, Kwon OW, Seong GJ, et al. Epitheliomesenchymal transdifferentiation of cultured RPE cells. Ophthalmic Res 2001;33:80–86. | Article | PubMed | ISI | ChemPort |
79. Roberts AB, Sporn MB. The transforming growth factors-. In: Sporn MB, Roberts AB (eds). Handbook of Experimental Pharmacology. Peptide Growth Factors and Their Receptors. Springer-Verlag: New York, 1990, pp 419–472.
80. Carrington L, McLeod D, Boulton M. IL-10 and antibodies to TGF-2 and PDGF inhibit RPE-mediated retinal contraction. Invest Ophthalmol Vis Sci 2000;41:1210–1216. | PubMed | ISI | ChemPort |
81. Cassidy L, Barry P, Shaw C, et al. Platelet derived growth factor and fibroblast growth factor basic levels in the vitreous of patients with vitreoretinal disorders. Br J Ophthalmol 1998;82:181–185. | PubMed | ISI | ChemPort |
82. Choudhury P, Chen W, Hunt RC. Production of platelet-derived growth factor by interleukin-1 and transforming growth factor--stimulated retinal pigment epithelial cells leads to contraction of collagen gels. Invest Ophthalmol Vis Sci 1997;38:824–833. | PubMed | ISI | ChemPort |
83. Hinton DR, He S, Jin ML, et al. Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy. Eye 2002;16:422–428. | Article | PubMed | ISI | ChemPort |
84. Jaffe GJ, Harrison CE, Lui GM, et al. Activin expression by cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 1994;35:2924–2931. | PubMed | ISI | ChemPort |
85. Taylor LM, Khachigian LM. Induction of platelet-derived growth factor B-chain expression by transforming growth factor- involves transactivation by Smads. J Biol Chem 2000;275:16709–16716. | Article | PubMed | ISI | ChemPort |
86. Saika S, Ikeda K, Yamanaka O, et al. Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial–mesenchymal transition of lens epithelium in mice. Lab Invest 2004;84:1259–1270. | Article | PubMed | ISI | ChemPort |
87. Saika S, Kono-Saika S, Tanaka T, et al. Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice. Lab Invest 2004;84:1245–1258. | Article | PubMed | ISI | ChemPort |
88. Saika S, Yamanaka O, Ikeda K, et al. Inhibition of p38MAP kinase suppresses fibrotic reaction of retinal pigment epithelial cells. Lab Invest 2005;85:838–850. | Article | PubMed | ISI | ChemPort |
89. Imanishi J, Kamiyama K, Iguchi I, et al. Growth factors: importance in wound healing and maintenance of transparency of the cornea. Prog Retin Eye Res 2000;19:113–120. | Article | PubMed | ISI | ChemPort |
90. Wilson SE, Chen L, Mohan RR, et al. Expression of HGF, KGF, EGF and receptor messenger RNAs following corneal epithelial wounding. Exp Eye Res 1999;68:377–397. | Article | PubMed | ISI | ChemPort |
91. Wilson SE, Liu JJ, Mohan RR. Stromal–epithelial interactions in the cornea. Prog Retin Eye Res 1999;18:293–309. | Article | PubMed | ISI | ChemPort |
92. Pelton RW, Saxena B, Jones M, et al. Immunohistochemical localization of TGF1, TGF2, and TGF3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development. J Cell Biol 1991;115:1091–1105. | Article | PubMed | ISI | ChemPort |
93. Li DQ, Tseng SC. Three patterns of cytokine expression potentially involved in epithelial–fibroblast interactions of human ocular surface. J Cell Physiol 1995;163:61–79. | Article | PubMed | ISI | ChemPort |
94. Wilson SE, Schultz GS, Chegini N, et al. Epidermal growth factor, transforming growth factor a, transforming growth factor , acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res 1994;59:63–71. | Article | PubMed | ISI | ChemPort |
95. Nishida K, Sotozono C, Adachi W, et al. Transforming growth factor-1, -2 and -3 mRNA expression in human cornea. Curr Eye Res 1995;14:235–241. | PubMed | ISI | ChemPort |
96. Joyce NC, Zieske JD. Transforming growth factor- receptor expression in human cornea. Invest Ophthalmol Vis Sci 1997;38:1922–1928. | PubMed | ISI | ChemPort |
97. Zieske JD, Hutcheon AEK, Guo X, et al. TGF- receptor types I and II are differentially expressed during corneal epithelial wound repair. Invest Ophthalmol Vis Sci 2001;42:1465–1471. | PubMed | ISI | ChemPort |
98. Saika S, Okada Y, Miyamoto T, et al. Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium. Invest Ophthalmol Vis Sci 2004;45:100–109. | Article | PubMed | ISI |
99. Dumon N, Bakin AV, Arteaga CL. Autocrine transforming growth factor- signaling mediates Smad-independent motility in human cancer cells. J Biol Chem 2003;278:3275–3285. | Article | PubMed | ISI | ChemPort |
100. Klekotka PA, Santoro SA, Zutter MM. Alpha 2 integrin subunit cytoplasmic domain-dependent cellular migration requires p38 MAPK. J Biol Chem 2001;276:9503–9511. | Article | PubMed | ISI | ChemPort |
101. Li W, Nadelman C, Henry G, et al. The p38-MAPK/SAPK pathway is required for human keratinocyte migration on dermal collagen. J Invest Dermatol 2001;117:1601–1611. | Article | PubMed | ISI | ChemPort |
102. Qi Z, Atsuchi N, Ooshima A, et al. Blockade of type transforming growth factor signaling prevents liver fibrosis and dysfunction in the rat. Proc Natl Acad Sci USA 1999;96:2345–2349. | Article | PubMed | ChemPort |
103. Sakamoto T, Ueno H, Sonoda K, et al. Blockade of TGF- by in vivo gene transfer of a soluble TGF- type II receptor in the muscle inhibits corneal opacification, edema and angiogenesis. Gene Therapy 2000;7:1915–1924. | Article | PubMed | ISI | ChemPort |
104. Saika S, Ikeda K, Yamanaka O, et al. Expression of Smad7 in mouse eyes accelerates healing of corneal tissue after exposure to alkali. Am J Pathol 2005;166:1405–1418. | PubMed | ISI | ChemPort |
105. Nakao A, Fujii M, Matsumura R, et al. Transient gene transfer and expression of Smad7 prevents bleomycin-induced lung fibrosis in mice. J Clin Invest 1999;104:5–11. | PubMed | ISI | ChemPort |
106. Dooley S, Hamzavi J, Breitkopf K, et al. Smad7 prevents activation of hepatic stellate cells and liver fibrosis in rats. Gastroenterology 2003;125:178–191. | Article | PubMed | ISI | ChemPort |
107. Hou CC, Wang W, Huang XR, et al. Ultrasound-microbubble-mediated gene transfer of inducible Smad7 blocks transforming growth factor-beta signaling and fibrosis in rat remnant kidney. Am J Pathol 2005;166:761–771. | PubMed | ISI | ChemPort |
108. Saika S, Miyamoto T, Yamanaka O, et al. Therapeutic effect of topical administration of SN50, an inhibitor of nuclear factor-B, in treatment of corneal alkali burns in mice. Am J Pathol 2005;166:1393–1403. | PubMed | ISI | ChemPort |
109. Saika S, Ikeda K, Yamanaka O, et al. Therapeutic effects of adenoviral gene transfer of bone morphogenic protein-7 on a corneal alkali injury model in mice. Lab Invest 2005;85:474–486. | Article | PubMed | ISI | ChemPort |

Acknowledgements

We thank the following doctors (listed in the alphabetical order) as well as the staff in my laboratory for their daily support in my research activity: Dr Kathleen C Flanders, who proofread the manuscript (Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute/National Institutes of Health), Dr Kazuo Ikeda (Department of Anatomy, Graduate School, Osaka City University School of Medicine), Professor Winston Whei-Yang Kao (Department of Ophthalmology, University of Cincinnati Medical Center), Dr Koichi Matsuzaki (Department of Internal Medicine, Kansai Medical University), Professor John McAvoy (Save Sight Institute), Professor Yasuteru Muragaki (Department of Pathology, Wakayama Meidcal University), Professor Yuji Nakajima (Department of Anatomy, Graduate School, Osaka City University School of Medicine), Professor Yoshitaka Ohnishi (Department of Ophthalmology, Wakayama Medical University), Professor Emeritus. Akira Ooshima (Department of Pathology, Wakayama Meidcal University), Professor Peter S Reinach (School of Optometry, State University of New York), and Dr Anita B Roberts (Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute/National Institutes of Health). This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan [13771038], a Grant from Uehara Memorial Foundation, a Research Grant on Priority Areas from Wakayama Medical University.