Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

Human optical axial length and defocus

Purpose: To investigate the short term influence of imposed monocular defocus upon human optical axial length (the distance from anterior cornea to retinal pigment epithelium) and ocular biometrics. Methods: Twenty-eight young adult subjects (14 myopes and 14 emmetropes) had eye biometrics measured before and then 30 and 60 minutes after exposure to monocular (right eye) defocus. Four different monocular defocus conditions were tested, each on a separate day: control (no defocus), myopic (+3 D defocus), hyperopic (-3 D defocus) and diffuse (0.2 density Bangerter filter) defocus. The fellow eye was optimally corrected (no defocus). Results: Imposed defocus caused small but significant changes in optical axial length (p<0.0001). A significant increase in optical axial length (mean change +8 ± 14 μm, p=0.03) occurred following hyperopic defocus, and a significant reduction in optical axial length (mean change -13 ± 14 μm, p=0.0001) was found following myopic defocus. A small increase in optical axial length was observed following diffuse defocus (mean change +6 ± 13 μm, p=0.053). Choroidal thickness also exhibited some significant changes with certain defocus conditions. No significant difference was found between myopes and emmetropes in the changes in optical axial length or choroidal thickness with defocus. Conclusions: Significant changes in optical axial length occur in human subjects following 60 minutes of monocular defocus. The bi-directional optical axial length changes observed in response to defocus implies the human visual system is capable of detecting the presence and sign of defocus and altering optical axial length to move the retina towards the image plane.

Confocal microscopy of the bulbar conjunctiva in contact lens wear

Purpose: The aim of this study was to investigate the capabilities of laser scanning confocal microscopy (LSCM) for undertaking qualitative and quantitative investigations of the response of the bulbar conjunctiva to contact lens wear. Methods: LSCM was used to observe and measure morphological characteristics of the bulbar conjunctiva of 11 asymptomatic soft contact lens wearers and 11 healthy volunteer subjects (controls). Results: The appearance of the bulbar conjunctiva is consistent with known histology of this tissue based on light and electron microscopy. The thickness of the bulbar conjunctival epithelium of lens wearers (30.9 ± 1.1 μm) was less than that of controls (32.9 ± 1.1 μm) (P < 0.0001). Superficial and basal bulbar conjunctival epithelial cell densities in contact lens wearers were 91% and 79% higher, respectively, than that in controls (P < 0.0001). No difference was observed in goblet and Langerhans cell density between lens wearers and controls. Conjunctival microcysts were observed in greater numbers, and were larger in size, in lens wearers compared with controls. Conclusions: The effects of contact lens wear on the human bulbar conjunctiva can be investigated effectively at a cellular level using LSCM. The observations in this study suggest that contact lens wear can induce changes in the bulbar conjunctiva such as epithelial thinning and accelerated formation and enlargement of microcysts, increased epithelial cell density, but has no impact on goblet or Langerhans cell density.

Estrogen receptor-Beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNF-alpha mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor–β (ERβ) in BPH and PCa. ERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFα knock-out mice fail to respond to ERβ agonist, demonstrating the requirement for TNFα signaling. In human tissues, ERβ agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133+ enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERβ causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERβ agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERβ agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

Transport of IgG across the blood-luminal barrier of the male reproductive tract of the rat and the effect of estradiol administration on reabsorption of fluid and IgG by the epididymal ducts

In rats immunized systemically with tetanus toxoid the concentration of specific anti-tetanus-toxoid-specific IgG in fluid from the rete testis and cauda epididymidis were respectively 0.6% and 1.4% the concentration in blood serum. The extratesticular duct system reabsorbed 97% of the IgG and 99% of the fluid leaving the rete, but estradiol administration affected the site of reabsorption. In untreated rats, the ductuli efferentes reabsorbed 94% of the IgG and 96% of the fluid leaving the rete, whereas estradiol-treated rats reabsorbed 83% of the IgG and 86% of the fluid, and the ductus epididymidis fully compensated for these different effects of estradiol on the ductuli efferentes. The concentrations of IgG in secretions of the seminal vesicles and prostate gland were lower (0.1% and 0.3% respectively of the titers in blood serum) than in fluids from the extratesticular ducts, and were not affected by the administration of estradiol. RT-PCR showed that Fcgrt (neonatal Fc receptor, also known as FcRn) is expressed in the reproductive ducts, where IgG is probably transported across epithelium, being particularly strong in the ductuli efferentes (where most IgG was reabsorbed) and distal caput epididymidis. It is concluded that IgG enters the rete testis and is concentrated only 2.5-fold along the extratesticular duct system, unlike spermatozoa, which are concentrated 95-fold. Further, the ductus epididymidis can recognize and compensate for changes in function of the ductuli efferentes.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

Background: The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. Methods: RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. Results: All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. Conclusions: EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target. © 2005 Lee et al., licensee BioMed Central Ltd.

Active regulation of the epidermal calcium profile

A distinct calcium profile is strongly implicated in regulating the multi-layered structure of the epidermis. However, the mechanisms that govern the regulation of this calcium profile are currently unclear. It clearly depends on the relatively impermeable barrier of the stratum corneum (passive regulation) but may also depend on calcium exchanges between keratinocytes and extracellular fluid (active regulation). Using a mathematical model that treats the viable sublayers of unwounded human and murine epidermis as porous media and assumes that their calcium profiles are passively regulated, we demonstrate that these profiles are also actively regulated. To obtain this result, we found that diffusion governs extracellular calcium motion in the viable epidermis and hence intracellular calcium is the main source of the epidermal calcium profile. Then, by comparison with experimental calcium profiles and combination with a hypothesised cell velocity distribution in the viable epidermis, we found that the net influx of calcium ions into keratinocytes from extracellular fluid may be constant and positive throughout the stratum basale and stratum spinosum, and that there is a net outflux of these ions in the stratum granulosum. Hence the calcium exchange between keratinocytes and extracellular fluid differs distinctly between the stratum granulosum and the underlying sublayers, and these differences actively regulate the epidermal calcium profile. Our results also indicate that plasma membrane dysfunction may be an early event during keratinocyte disintegration in the stratum granulosum.

A continuum model of the growth of engineered epidermal skin substitutes

We present a porous medium model of the growth and deterioration of the viable sublayers of an epidermal skin substitute. It consists of five species: cells, intracellular and extracellular calcium, tight junctions, and a hypothesised signal chemical emanating from the stratum corneum. The model is solved numerically in Matlab using a finite difference scheme. Steady state calcium distributions are predicted that agree well with the experimental data. Our model also demonstrates epidermal skin substitute deterioration if the calcium diffusion coefficient is reduced compared to reported values in the literature.

Clinical performance of a new silicone hydrogel contact lens over 6 months of extended wear

Purpose: Silicone hydrogel contact lenses (CLs) are becoming increasingly popular for daily wear (DW), extended wear (EW) and continuous wear (CW), due to their higher oxygen transmissibility compared to hydrogel CLs. The aim of this study was to investigate the clinical and subjective performance of asmofilcon A (Menicon Co., Ltd), a new surface treated silicone hydrogel CL, during 6-night EW over 6 months (M). Methods: A prospective, randomised, single-masked, monadic study was conducted. N=60 experienced DW soft CL wearers were randomly assigned to wear either asmofilcon A (test: Dk=129, water content (WC)=40%, Nanogloss surface treatment) or senofilcon A (control: Dk=103, WC=38%, PVP internal wetting agent, Vistakon, Johnson & Johnson Vision Care) CLs bilaterally for 6 M on an EW basis. A PHMB-preserved solution (Menicon Co., Ltd) was dispensed for CL care. Evaluations were conducted at CL delivery and after 1 week (W), 4 W, 3 M and 6 M of EW. At each visit, a range of objective and subjective clinical performance measures were assessed. Results: N=50 subjects (83%) successfully completed the study, with the majority of discontinuations due to loss to follow-up (n=3) or moving away/travel (n=5). N=2 subjects experienced adverse events; n=1 unilateral red eye with asmofilcon A and n=1 asymptomatic infiltrate with senofilcon A. There were no significant differences in high or low contrast distance visual acuity (HCDVA or LCDVA) between asmofilcon A and senofilcon A; however, LCDVA decreased significantly over time with both CL types (p<0.05). The two CL types did not vary significantly with respect to any of the objective and subjective measures assessed (p>0.05); CL fitting characteristics and CL surface measurements were very similar and mean bulbar and limbal redness measures were always less than grade 1.0. Superior palpebral conjunctival injection showed a statistically, but not clinically, significant increase over time with both CL types (p<0.05). Corneal staining did not vary significantly between asmofilcon A and senofilcon A (p>0.05), with low median gradings of less than 0.5 observed for all areas assessed. There were no solution-related staining reactions observed with either CL type. The asmofilcon A and senofilcon A CLs were both rated highly with respect to overall comfort, with medians of 14 or 15 hours of comfortable lens wearing time per day reported at each of the study visits (p>0.05). Conclusions: Over 6 months of EW, the asmofilcon A and senofilcon A CLs performed in a similar manner with respect to visual acuity, ocular health and CL performance measures. Some changes over time were observed with both CL types, including reduced LCDVA and increased superior palpebral injection, which warrant further investigation in longer-term EW studies. Asmofilcon A appeared to be equivalent in performance to senofilcon A.

Solution methods for advection-diffusion-reaction equations on growing domains and subdomains, with application to modelling skin substitutes

Problems involving the solution of advection-diffusion-reaction equations on domains and subdomains whose growth affects and is affected by these equations, commonly arise in developmental biology. Here, a mathematical framework for these situations, together with methods for obtaining spatio-temporal solutions and steady states of models built from this framework, is presented. The framework and methods are applied to a recently published model of epidermal skin substitutes. Despite the use of Eulerian schemes, excellent agreement is obtained between the numerical spatio-temporal, numerical steady state, and analytical solutions of the model.

Expression and distribution of cell-surface proteoglycans in the normal Lewis rat molar periodontium

Cell-surface proteoglycans participate in several biological functions such as cell cell and cell-matrix interactions, cell adhesion, the binding to various growth factors as co-receptors and repair. To understand better the expression and distribution of cell-surface proteoglycans in the periodontal tissues, an immunohistochemical evaluation of the normal Lewis rat molar periodontium using panels of antibodies for syndecan-1, -2, -4, glypican and betaglycan was carried out. Our results demonstrated the expression and distribution of all proteoglycans in the suprabasal gingival epithelium, soft and hard connective tissues. Both cellular and matrix localization was evident within the various periodontal compartments. The presence of these cell-surface proteoglycans indicates the potential for roles in the process of tissue homeostasis, repair or regeneration in periodontium of which each function requires further study.

Differential expression and distribution of syndecan-1 and -2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell–cell and cell–matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell–cell and cell–matrix interactions, while syndecan-2 showed a predilection to associate with cell–matrix interactions during hard tissue formation.

GABAergic modification of myopic and hyperopic ocular tissue effects on scleral fibroblast growth

Purpose: Myopia is a common eye disorder affecting up to 90% of children in South East Asia and 30% of the population worldwide. Myopia of high severity is a leading cause of blindness around the world (4th to 5th most common). Changes and remodelling of the sclera i.e. increase cellular proliferation & increase protein synthesis within scleral cells (↑ scleral DNA) and thinning and lose of extracellular matrix of sclera (↓ scleral GAG synthesis) have been linked to myopic eye growth in animal models. Signals acting on the sclera are thought to originate in the retina, and are modulated by the retinal pigment epithelium (RPE) with limited evidence suggesting that the RPE can modify scleral cell growth in culture. However, the mechanism of retinal signal transmission and the role of posterior eye cup tissue, including the RPE, in mediating changes in scleral fibroblast growth during myopia development are unclear. Retinal transmitter systems are critically involved in pathways regulating eye growth, which ultimately lead to alterations in the sclera if eye size is to change. A dopaminergic agonist and muscarinic antagonists decrease the proliferation of scleral chondrocytes when co-cultured with chick’s retinal pigment epithelium (RPE). GABA receptors have recently been localised to chick sclera. We therefore hypothesised that posterior eye cup tissue from myopic eyes would stimulate and from hyperopic eyes would inhibit growth of scleral fibroblasts in vitro and that GABAergic agents could directly interact with scleral cells or indirectly modify the effects of myopic and hyperopic posterior eye cup tissue on scleral fibroblast growth. Method: Fibroblastic cells obtained from 8-day-old chick sclera were used to establish cell banks. Two major experiments were performed. Experiment 1: To determine if posterior eye cup tissues from myopic eye stimulates and hyperopic eye inhibits scleral cell proliferation, when co-cultured with scleral cells in vitro. This study comprised two linked experiments, i) monocular visual treatments of FDM (form-deprivation myopia), LIM (lens-induced myopia) and LIH (lens-induced hyperopia) with assessment of the effect of full punch eye cup tissue on DNA and GAG synthesis by cultured chick scleral fibroblasts, and ii) binocular visual treatments comprising LIM and LIH with assessment of the effect of individual layers of eye cup tissues (neural retina, RPE and choroid) on cultured chick scleral fibroblasts. Visual treatment was applied for 3 days. Experiment 2: To determine the direct interaction of GABA agents on scleral cell growth and to establish whether GABA agents modify the stimulatory/inhibitory effect of myopic and hyperopic posterior eye cup tissues on cultured scleral cell growth in vitro. Two linked experiments were performed. i) GABA agonists (muscimol and baclofen) and GABA antagonists (bicuculine (-), CGP46381 and TPMPA) were added to scleral cell culture medium to determine their direct effect on scleral cells. ii) GABAergic agents (agonists and antagonists) were administered to scleral fibroblasts co-cultured with posterior eye cup tissue (retina, RPE, retina/RPE, RPE/choroid). Ocular tissues were obtained from chick eyes wearing +15D (LIH) or -15D lenses (LIM) for 3 days. In both experiments, tissues were added to hanging cell culture insert (pore size 1.0ìm) placed over each well of 24 well plates while scleral cells were cultured in DMEM/F12, Glutamax (Gibco) plus 10% FBS and penicillin/streptomycin (50U/ml)) and fungizone (1.25ug/ml) (Gibco), at seeding density of 30,000 cells/well at the bottom of the well and allowed to grow for 3 days. Scleral cells proliferation rate throughout the study was evaluated by determining GAG and DNA content of scleral cells using Dimethylmethylene blue (DMMB) dye and Quant-iTTm Pico Green® dsDNA reagent respectively. Results and analysis: Based on DNA and GAG content, there was no significant difference in tissue effect of LIM and LIH eyes on scleral fibroblast growth (DNA: 8.4 ± 1.1μg versus 9.3 ± 2.3 μg, p=0.23; GAG: 10.13 ± 1.4 μg versus 12.67 ± 1.2 μg, F2,23=6.16, p=0.0005) when tissues were obtained from monocularly treated chick eyes (FDM or +15D lens or -15D lens over right eyes with left eyes untreated) and co-cultured as full punch. When chick eyes were treated binocularly with -15D lens (LIM) right eye and +15D lens (LIH) left eyes and tissue layers were separated, the retina from LIM eyes did not stimulate scleral cell proliferation compared to LIH eyes (DNA: 27.2 ± 6.7 μg versus 23.2 ± 1.5 μg, p=0.23; GAG: 28.1 ±3.7 μg versus 28.7 ± 4.2 μg, p=0.21). Similarly, the LIH and LIM choroid did not produce a differential effect based on DNA (LIM 46.9 ± 6.4 μg versus LIH 53.5 ± 4.7 μg, p=0.18), however the choroid from LIH eyes induced higher scleral GAG content than from LIM eyes (32.5 ± 6.7 μg versus 18.9 ± 1.2 μg, p=0.023). In contrast, the RPE from LIM eyes caused a significant increase in fibroblast proliferation whereas the RPE from LIH eyes was relatively inhibitory (72.4 ± 6.3 μg versus 27.9 ± 2.3 μg, F1, 6=69.99, p=0.0005). GAG data were opposite to DNA data e.g. the RPE from LIH eyes increased (33.7 ± 7.9 μg) while the RPE from LIM eyes decreased (28.2 ± 3.0 μg) scleral cell growth (F1, 6=13.99, p=0.010). Based on DNA content, GABA agents had a small direct effect on scleral cell growth; GABA agonists increased (21.4 ± 1.0% and 18.3 ± 1.0% with muscimol and baclofen, p=0.0021), whereas GABA antagonists decreased fibroblast proliferation (-23.7 ± 0.9% with bicuculine & CGP46381 and -28.1 ± 0.5% with TPMPA, p=0.0004). GABA agents also modified the effect of LIM and LIH tissues (p=0.0005).The increase in proliferation rate of scleral fibroblasts co-cultured with tissues (RPE, retina, RPE/retina and RPE/choroid) from LIM treated eyes was enhanced by GABA agonists (muscimol: 27.4 ± 1.2%, 35.8 ± 1.6%, 8.4 ± 0.3% and 11.9 ± 0.6%; baclofen: 27.0 ± 1.0%, 15.8 ± 1.5%, 16.8 ± 1.2% and 15.4 ± 0.4%, p=0.014) whereas GABA antagonists further reduced scleral fibroblasts growth (bicuculine: -52.5 ± 2.5%, -36.9 ± 1.4%, -37.5 ± 0.6% and -53.7 ± 0.9%; TPMPA: 57.3 ± 1.3%, -15.7 ± 1.2%, -33.5 ± 0.4% and -45.9 ± 1.5%; CGP46381: -51.9 ± 1.6%, -28.5 ± 1.5%, -25.4 ± 2.0% and -45.5 ± 1.9% respectively, p=0.0034). GAG data were opposite to DNA data throughout the experiment e.g. GABA agonists further inhibited while antagonists relatively enhanced scleral fibroblasts growth for both LIM and LIH tissue co-culture. The effect of GABA agents was relatively lower (p=0.0004) for tissue from LIH versus LIM eyes but was in a similar direction. There was a significant drug effect on all four tissue types e.g. RPE, retina, RPE/retina and RPE/choroid for both LIM and LIH tissue co-culture (F20,92=3.928, p=0.0005). However, the effect of GABA agents was greatest in co-culture with RPE tissue (F18,36=4.865, p=0.0005). Summary and Conclusion: 1) Retinal defocus signals are transferred to RPE and choroid which then exert their modifying effect on scleral GAG and DNA synthesis either through growth stimulating factors or directly interacting with scleral cells in process of scleral remodeling during LIM and LIH visual conditions. 2) GABAergic agents affect the proliferation of scleral fibroblasts both directly and when co-cultured with ocular tissues in vitro.