Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye

Human Sclera Maintains Common Characteristics with Cartilage throughout Evolution

BACKGROUND: The sclera maintains and protects the eye ball, which receives visual inputs. Although the sclera does not contribute significantly to visual perception, scleral diseases such as refractory scleritis, scleral perforation and pathological myopia are considered incurable or difficult to cure. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. METHODOLOGY/PRINCIPAL FINDINGS: We have demonstrated microarray data of cultured human infant scleral cells. Hierarchical clustering was performed to group scleral cells and other mesenchymal cells into subcategories. Hierarchical clustering analysis showed similarity between scleral cells and auricular cartilage-derived cells. Cultured micromasses of scleral cells exposed to TGF-betas and BMP2 produced an abundant matrix. The expression of cartilage-associated genes, such as Indian hedge hog, type X collagen, and MMP13, was up-regulated within 3 weeks in vitro. These results suggest that human 'sclera'-derived cells can be considered chondrocytes when cultured ex vivo. CONCLUSIONS/SIGNIFICANCE: Our present study shows a chondrogenic potential of human sclera. Interestingly, the sclera of certain vertebrates, such as birds and fish, is composed of hyaline cartilage. Although the human sclera is not a cartilaginous tissue, the human sclera maintains chondrogenic potential throughout evolution. In addition, our findings directly explain an enigma that the sclera and the joint cartilage are common targets of inflammatory cells in rheumatic arthritis. The present global gene expression database will contribute to the clarification of the pathogenesis of developmental diseases such as high myopia

Choroidal-scleral cell interplay and the regulation of scleral biomechanics

Worldwide prevalence and severity of myopia have increased dramatically nowadays. High myopia and its irreversible associated eye elongation increase the risk of sight-threatening conditions. However, the exact mechanisms that drive myopia progression are still unknown. Myopia almost exclusively occurs in childhood, suggesting the adult sclera is functionally different from the young sclera. During myopia progression, the sclera becomes thinner and more elastic, and the composition of its scleral extracellular matrix changes. Similarly, the choroid is thinner and accommodation-induced choroidal secreted factors are linked to tissue biomechanics that may regulate scleral remodelling and eye elongation. Thus, we hypothesize that signals from the choroid are crucial to the regulation of scleral biomechanics. In addition, light exposure and subsequent dopamine release may regulate scleral remodelling and eye elongation, and myopia development and progression could be induced by near work. We used human primary fibroblasts isolated from the sclera and choroid of donor eyes from different ages and antero-posterior positions to test our hypothesis. We found that paediatric scleral fibroblastsembedded in the 3D collagen gels had greater contractility than adult ones, particularly those from the anterior part of the sclera. Scleral fibroblasts’ ability to contract collagen gels was enhanced following stimulation with the choroid-conditioned medium, and this promotion was not due to an increase in proliferation or change in a-SMA expression. Furthermore, the ability of choroid conditioned medium to stimulate scleral fibroblasts was completely abolished when choroid cells were treated with dopamine. This suggests that normal scleral development is regulated by a balance between positive biochemical signals from the choroid, and negative signals resulting from a direct effect of retina-derived dopamine on the choroid cells. These findings may help improve clinical practice to control myopia development and progression in the future

The Lymphogenic and Hemangiogenic factors affecting The Human Sclera

Our project “The Lymphogenic and Hemangiogenic factors affecting the Human Sclera” is a novel study focused on the vascular and cellular changes of human sclera in healthy status and under pathological conditions Our research work based on observations and clinical experience, along with a literature review based on a PubMed search. We found that the vascular privilege of healthy human scleral characterized by tight vascular network showed by a wide spectrum of anti-angiogenic and anti-lymphangiogenic factors that is highly expressed, while pro-angiogenic factors expression is reduced. Where the healthy human scleral layers are devoid of lymphatic vessels which contribute to the ocular immune privilege of the inner eye. Under pathological conditions vascular and cellular privileges of the sclera are compromised where the fibrocytes change into active fibroblasts causing remodelling and proliferation of the scleral matrix. Also, cellular activation and secondary ingrowth of intraocular lymphatic vessels, associated LYVE-1+ macrophages invaded the sclera and intraocular structures affecting healing out-comes. immunologic defense against intruding microorganism, and autoimmune reactions against intraocular antigens

All-trans retinoic acid regulates the expression of the extracellular matrix protein fibulin-1 in the guinea pig sclera and human scleral fibroblasts

Purpose: Fibulin-1 (FBLN1) mRNA is expressed in human sclera and is an important adhesion modulatory protein that can affect cell-matrix interactions and tissue remodeling. Scleral remodeling is influenced by all-trans retinoic acid (RA). Our purpose was to confirm the presence of fibulin-1 protein in guinea pig sclera and investigate the effect of RA on the expression of fibulin-1 in guinea pig sclera in vivo and in cultured human scleral fibroblasts (HSFs). Methods: Confocal fluorescence microscopy was used to study fibulin-1 and aggrecan expression and localization in sclera from control guinea pigs and in animals given RA by daily gavage from 4 to 8 days of age. The effects of RA (from 10⁻⁹ to 10⁻⁵ M) on fibulin-1 expression in HSFs were observed by immunohistochemistry and assayed by real-time PCR and western blot analysis. Results: Fibulin-1 protein expression was detected by confocal fluorescence microscopy in guinea pig sclera and in cultured HSFs. Upregulation of fibulin-1 in scleral tissue was observed after feeding with RA. In vitro, the level of Fbln1 mRNA was increased after treatment of HSFs with RA (at concentrations of 10⁻⁸ to 10⁻⁶ M; p<0.001), with a maximum effect at 10⁻⁷ M. Fibulin-1 protein levels were significantly increased after treatment of HSFs with 10⁻⁷ M of RA for 24 or 48 h (p<0.05). Conclusions: Fibulin-1 protein was expressed in guinea pig sclera and cultured HSFs. Expression was regulated by RA, a molecule known to be involved in the regulation of eye growth. Further studies on the role of fibulin-1 in the regulation of eye growth, including during the development of myopia, are therefore warranted

Expression of muscarinic receptor subtypes in tree shrew ocular tissues and their regulation during the development of myopia

Muscarinic receptors are known to regulate several important physiologic processes in the eye. Antagonists to these receptors such as atropine and pirenzepine are effective at stopping the excessive ocular growth that results in myopia. However, their site of action is unknown. This study details ocular muscarinic subtype expression within a well documented model of eye growth and investigates their expression during early stages of myopia induction. Total RNA was isolated from tree shrew corneal, iris/ciliary body, retinal, choroidal, and scleral tissue samples and was reverse transcribed. Using tree shrew-specific primers to the five muscarinic acetylcholine receptor subtypes (CHRM1-CHRM5), products were amplified using polymerase chain reaction (PCR) and their identity confirmed using automated sequencing. The expression of the receptor proteins (M1-M5) were also explored in the retina, choroid, and sclera using immunohistochemistry. Myopia was induced in the tree shrew for one or five days using monocular deprivation of pattern vision, and the expression of the receptor subtypes was assessed in the retina, choroid, and sclera using real-time PCR. All five muscarinic receptor subtypes were expressed in the iris/ciliary body, retina, choroid, and sclera while gene products corresponding to CHRM1, CHRM3, CHRM4, and CHRM5 were present in the corneal samples. The gene expression data were confirmed by immunohistochemistry with the M1-M5 proteins detected in the retina, choroid, and sclera. After one or five days of myopia development, muscarinic receptor gene expression remained unaltered in the retinal, choroidal, and scleral tissue samples. This study provides a comprehensive profile of muscarinic receptor gene and protein expression in tree shrew ocular tissues with all receptor subtypes found in tissues implicated in the control of eye growth. Despite the efficacy of muscarinic antagonists at inhibiting myopia development, the genes of the muscarinic receptor subtypes are neither regulated early in myopia (before measurable axial elongation) nor after significant structural change