Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1ß protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Multiomics-Identified Intervention to Restore Ethanol-Induced Dysregulated Proteostasis and Secondary Sarcopenia in Alcoholic Liver Disease.

BACKGROUND/AIMS: Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. METHODS: Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. RESULTS: Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are ß-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by ß-hydroxymethyl-butyrate treatment. CONCLUSION: An innovative multiomics approach followed by experimental validation showed that ß-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

Prenatal Exposure to Respiratory Syncytial Virus Alters Postnatal Immunity and Airway Smooth Muscle Contractility during Early-Life Reinfections.

Maternal viral infections can have pathological effects on the developing fetus which last long after birth. Recently, maternal-fetal transmission of respiratory syncytial virus (RSV) was shown to cause postnatal airway hyperreactivity (AHR) during primary early-life reinfection; however, the influence of prenatal exposure to RSV on offspring airway immunity and smooth muscle contractility during recurrent postnatal reinfections remains unknown. Therefore, we sought to determine whether maternal RSV infection impairs specific aspects of cell-mediated offspring immunity during early-life reinfections and the mechanisms leading to AHR. Red fluorescent protein-expressing recombinant RSV (rrRSV) was inoculated into pregnant rat dams at midterm, followed by primary and secondary postnatal rrRSV inoculations of their offspring at early-life time points. Pups and weanlings were tested for specific lower airway leukocyte populations by flow cytometry; serum cytokine/chemokine concentrations by multiplex ELISA and neurotrophins concentrations by standard ELISA; and ex vivo lower airway smooth muscle (ASM) contraction by physiological tissue bath. Pups born to RSV-infected mothers displayed elevated total CD3+ T cells largely lacking CD4+ and CD8+ surface expression after both primary and secondary postnatal rrRSV infection. Cytokine/chemokine analyses revealed reduced IFN-γ, IL-2, IL-12, IL-17A, IL-18, and TNF-α, as well as elevated nerve growth factor (NGF) expression. Prenatal exposure to RSV also increased ASM reactivity and contractility during early-life rrRSV infection compared to non-exposed controls. We conclude that maternal RSV infection can predispose offspring to postnatal lower airways dysfunction by altering immunity development, NGF signaling, and ASM contraction during early-life RSV reinfections.

Small group learning: effect on item analysis and accuracy of self-assessment of medical students.

BACKGROUND: Small group sessions are regarded as a more active and student-centered approach to learning. Item analysis provides objective evidence of whether such sessions improve comprehension and make the topic easier for students, in addition to assessing the relative benefit of the sessions to good versus poor performers. Self-assessment makes students aware of their deficiencies. Small group sessions can also help students develop the ability to self-assess. This study was carried out to assess the effect of small group sessions on item analysis and students' self-assessment. METHODS: A total of 21 female and 29 male first year medical students participated in a small group session on topics covered by didactic lectures two weeks earlier. It was preceded and followed by two multiple choice question (MCQ) tests, in which students were asked to self-assess their likely score. The MCQs used were item analyzed in a previous group and were chosen of matching difficulty and discriminatory indices for the pre- and post-tests. RESULTS: The small group session improved the marks of both genders equally, but female performance was better. The session made the items easier; increasing the difficulty index significantly but there was no significant alteration in the discriminatory index. There was overestimation in the self-assessment of both genders, but male overestimation was greater. The session improved the self-assessment of students in terms of expected marks and expectation of passing. DISCUSSION: Small group session improved the ability of students to self-assess their knowledge and increased the difficulty index of items reflecting students' better performance.

Resolvin E1 analog RX-10045 0.1% reduces corneal stromal haze in rabbits when applied topically after PRK.

PURPOSE: To perform a masked study to determine whether resolvin E1 (RvE1), a lipid-derived immunomodulator, could regulate the development of corneal haze and opacity-related myofibroblasts after opacity-generating high correction photorefractive keratectomy (PRK) in rabbits. METHODS: Three groups of eight rabbits each were included in the study. Nine diopter (D) PRK for myopia was performed in each test cornea, and the eyes were treated with 30 µl of topical solution every 4 h (six times a day) for 5 days starting immediately after PRK. Group 1 was treated with 0.1% RX-10045, a prodrug of an RvE1 analog; group 2 was treated with 0.01% RX-10045; and group 3 was treated with vehicle control solution. At 1 month after PRK, haze was graded at the slit-lamp by a masked observer. Immunohistochemistry for α-smooth muscle actin (SMA) was performed on the central cornea of each test eye to determine the anterior stromal myofibroblast density. RESULTS: Corneal opacity was significantly lower in the 0.1% RX-10045 group, but not the 0.01% RX-10045 group, compared to the vehicle control group (p=0.029), at 1 month after -9.0D PRK. At 1 month after -9.0D PRK, SMA+ myofibroblast densities in the anterior stroma were not statistically significantly different among the three groups, although a trend toward lower myofibroblast generation was noted in the 0.1% RX-10045 group. CONCLUSIONS: Topical 0.1% RX-10045, a prodrug of an RvE1 analog, reduces corneal opacity after haze-generating PRK in rabbits. Further studies are needed to determine the precise points at which RvE1 decreases corneal opacity after injury.

Mouse strain variation in SMA(+) myofibroblast development after corneal injury.

The purpose of present study was to investigate differences in myofibroblast development after haze-generating injury in different commonly used strains of mice. The inbred mouse strains used in this study were Balb/c, C57BL/6, C3H/HeJ and DBA/1J. All mice had uniform irregular phototherapeutic keratectomy with an excimer laser according to a previously published method to generate stromal haze. DBA/1J mice generated significantly greater density of alpha smooth muscle actin (SMA)-positive myofibroblasts in the anterior stroma compared to Balb/c (p < 0.05), C57BL/6 (p < 0.05) and C3H/HeJ (p < 0.01) mice. The C3H/HeJ strain had significantly lower density of SMA-positive myofibroblasts compared to other three mouse strains. These results indicate that mouse strain must be considered in designing experiments and interpreting the results of experiments in which corneal haze and myofibroblast generation is studied in mice. Further investigation of genetics underlying mouse strain variation could provide insight into the corneal wound healing and haze generation processes.

Transmission electron microscopy analysis of epithelial basement membrane repair in rabbit corneas with haze.

PURPOSE: To assess the ultrastructure of the epithelial basement membrane using transmission electron microscopy (TEM) in rabbit corneas with and without subepithelial stroma opacity (haze). METHODS: Two groups of eight rabbits each were included in this study. Photorefractive keratectomy (PRK) was performed using an excimer laser. The first group had -4.5-diopter (-4.5D) PRK and the second group had -9.0D PRK. Contralateral eyes were unwounded controls. Rabbits were sacrificed at 4 weeks after surgery. Immunohistochemical analysis was performed to detect the myofibroblast marker α-smooth muscle actin (SMA). TEM was performed to analyze the ultrastructure of the epithelial basement membrane and stroma. RESULTS: At 4 weeks after PRK, α-SMA+ myofibroblasts were present at high density in the subepithelial stroma of rabbit eyes that had -9.0D PRK, along with prominent disorganized extracellular matrix, whereas few myofibroblasts and little disorganized extracellular matrix were noted in eyes that had -4.5D PRK. The epithelial basement membrane was irregular and discontinuous and lacking typical morphology in all corneas at 1 month after -9D PRK compared to corneas at 1 month in the -4.5D PRK group. CONCLUSIONS: The epithelial basement membrane acts as a critical modulator of corneal wound healing. Structural and functional defects in the epithelial basement membrane correlate to both stromal myofibroblast development from precursor cells and continued myofibroblast viability, likely through the modulation of epithelial-stromal interactions mediated by cytokines. Prolonged stromal haze in the cornea is associated with abnormal regeneration of the epithelial basement membrane.

Biological and biomechanical responses to traditional epithelium-off and transepithelial riboflavin-UVA CXL techniques in rabbits.

PURPOSE: To compare the biological effects of riboflavin-ultraviolet A (UVA) corneal cross-linking (CXL) performed with a traditional epithelium-off method to several transepithelial methods in a rabbit model. Preliminary experiments on biomechanical rigidity were also performed. METHODS: Four treatment groups were included: (1) standard epithelium-off, (2) tetracaine transepithelial, (3) benzal-konium chloride-ethylenediaminetetraacetic acid (BKC-EDTA) transepithelial, and (4) femtosecond laser-assisted transepithelial riboflavin-UVA CXL. Six eyes from each treatment group and the untreated control group were analyzed at 24 hours and 2 months after treatment in wound healing studies. The TUNEL assay was performed to detect the extent of stromal cell death. Optical density was measured with a Scheimpflug analyzer. The corneal stiffening effect was quantitated in three eyes from each group using optical coherence elastography performed 2 months after treatments. RESULTS: Twenty-four hours after CXL, stromal cell death extended full corneal thickness with both standard epithelium-off CXL and femtosecond laser-assisted CXL, but only approximately one-third stromal depth after BKC-EDTA transepithelial CXL. Negligible stromal cell death was detected with tetracaine transepithelial CXL. Cell death results were statistically different between the BKC-EDTA transepithelial CXL and standard epithelium-off CXL groups (P < .0001). Significant corneal opacity differences were noted. Standard epithelium-off CXL had the greatest density and tetracaine transepithelial CXL had the least density compared to the control group after treatment. As measured with optical coherence elastography, a trend toward greater mean stiffening was observed with BKC-EDTA transepithelial CXL than with epithelium-off CXL, femtosecond laser-assisted CXL, or tetracaine transepithelial CXL, but the result did not reach statistical significance. All of the CXL treatment groups exhibited significantly smaller variance of stiffness compared to the control group. CONCLUSION: In the rabbit model, BKC-EDTA transepithelial CXL produced less stromal cell death and less risk of endothelial cell damage than standard epithelium-off CXL or femtosecond laser-assisted CXL. Additional study is needed to determine whether biomechanical stiffness is significantly different between the epithelium-off CXL and transepithelial CXL groups.

Use of formative assessment as an educational tool.

BACKGROUND: Though formative assessments are popular in medical education, but data to establish their educational benefits are lacking. This study was conducted to determine whether participation and performance of MBBS students in regular formative assessments are associated with positive outcomes and has measurable effects on learning. METHODS: One hundred and fifty MBBS students of semester II attending Biochemistry classes were studied by dividing into two groups till the completion of a topic. End-of-topic summative assessment marks were analysed with respect to the effect of participation and performance in formative assessments. RESULTS: Participation in formative assessments had a statistically significant positive relationship with summative assessment marks. Mean difference in formative and summative assessment marks for group that participated in formative assessments is 1.6 (95% CI = 0.9-2.4, p < 0.001). The mean difference in summative assessment marks for two groups is 3.4 (95% CI = 2.3-4.6, p < 0.001). The mean difference in marks obtained by solving case studies given in Summative Assessment for two groups is 1.2 (95% CI = 0.7-1.6, p < 0.001). CONCLUSIONS: Formative assessment not only assesses students' achievements but it also enables students to recognise the areas in which they are having difficulty and to concentrate their future efforts on those areas. Adequate frequency of formative assessment with immediate feedback is beneficial as it stimulates meaningful and multifaceted learning. The results of this study encourage the use of formative assessment as an educational tool in all MBBS subjects for they have significant positive effects on learning.

Short-term cell death and inflammation after intracorneal inlay implantation in rabbits.

PURPOSE: To investigate the cell death and inflammatory response to insertion of the KAMRA inlay (AcuFocus Inc) for presbyopia. METHODS: Twenty-four rabbits were included in the study. Each rabbit had pockets generated in both corneas with a femtosecond laser. One eye of each rabbit had an inlay inserted into the pocket and the opposite control eye had the pocket dissected. Eight rabbits were studied at 24 hours, 48 hours, or 6 weeks after surgery. Tissue sections were analyzed with TUNEL assay to detect cell death and immunohistochemistry for CD11b to detect monocytes as a marker of inflammation. RESULTS: The inlay group had significantly more stromal cell death than the control group at 48 hours after surgery (P=.038). At 24 hours and 6 weeks after surgery, no significant difference was noted in stromal cell death between the inlay and control groups. Significantly more CD11b+ cells were noted in the stroma in the inlay group compared to the control group at 24 and 48 hours after surgery (P=.025 and P=.001, respectively). However, at 6 weeks after surgery, no significant difference in CD11b+ cells was observed between the control and inlay groups (P=.05). CONCLUSIONS: Although an early increase in stromal cell death and inflammation occurred in eyes that underwent femtosecond laser pocket creation and KAMRA inlay insertion compared to a control group with the pocket only, no significant difference was noted between the inlay and control groups in stromal cell death or inflammation at 6 weeks after surgery.

Corneal wound healing after ultraviolet-A/riboflavin collagen cross-linking: a rabbit study.

PURPOSE: To investigate corneal wound healing following ultraviolet-A (UVA)/riboflavin corneal collagen cross-linking (CXL) in rabbit corneas. METHODS: Thirty-six rabbits were enrolled in the study. Animals were divided into three treatment groups and corneas were analyzed at 24 hours and 4 weeks postoperatively. Thus, each group had 6 rabbits at each time point. Treatment groups were: 1) standard UVA+riboflavin CXL, 2) UVA alone, and 3) riboflavin alone. One eye of each rabbit served as an untreated control eye. TUNEL assay was performed to detect stromal cell apoptosis. Immunocytochemistry was performed to detect the inflammatory marker CD11b expressed in monocytes and the alpha-smooth muscle actin (SMA) marker expressed in myofibroblasts. RESULTS: At 24 hours, corneas from the UVA+riboflavin CXL group had significantly more apoptosis than the UVA alone and riboflavin alone groups. Eyes from all three groups had significantly more inflammatory cell influx into the cornea than unwounded controls. Four weeks after the procedure, many corneas in the UVA+riboflavin CXL group had mild haze, but very few SMA-positive myofibroblasts could be detected in the central cornea. CONCLUSIONS: Riboflavin+UVA CXL triggers more anterior keratocyte apoptosis than corneal scrape with UVA alone or riboflavin alone. Inflammation monitored by the monocyte marker CD11b was present, but not statistically different among the three groups. Very little myofibroblast generation could be detected after UVA+riboflavin CXL, indicating that the mild stromal haze associated with this procedure is normally related to transient corneal fibroblast generation rather than more persistent haze due to generation of myofibroblasts.

Corneal myofibroblast generation from bone marrow-derived cells.

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP-, SMA-GFP+ and SMA-GFP- cells, as well as the number of DAPI+ cell nuclei, per 400x field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9x more SMA+GFP+ than SMA+GFP- myofibroblasts. This difference was significant (p < 0.01). There were significantly more (p < 0.01) SMA-GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP- cells, although SMA-GFP- cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60-95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

Effect of femtosecond laser energy level on corneal stromal cell death and inflammation.

PURPOSE: To analyze the effects of variations in femtosecond laser energy level on corneal stromal cell death and inflammatory cell influx following flap creation in a rabbit model. METHODS: Eighteen rabbits were stratified in three different groups according to level of energy applied for flap creation (six animals per group). Three different energy levels were chosen for both the lamellar and side cut: 2.7 microJ (high energy), 1.6 microJ (intermediate energy), and 0.5 microJ (low energy) with a 60 kHz, model II, femtosecond laser (IntraLase). The opposite eye of each rabbit served as a control. At the 24-hour time point after surgery, all rabbits were euthanized and the corneoscleral rims were analyzed for the levels of cell death and inflammatory cell influx with the terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunocytochemistry for monocyte marker CD11b, respectively. RESULTS: The high energy group (31.9+/-7.1 [standard error of mean (SEM) 2.9]) had significantly more TUNEL-positive cells in the central flap compared to the intermediate (22.2+/-1.9 [SEM 0.8], P=.004), low (17.9+/-4.0 [SEM 1.6], P< or =.001), and control eye (0.06+/-0.02 [SEM 0.009], P< or =.001) groups. The intermediate and low energy groups also had significantly more TUNEL-positive cells than the control groups (P< or =.001). The difference between the intermediate and low energy levels was not significant (P=.56). The mean for CD11b-positive cells/400x field at the flap edge was 26.1+/-29.3 (SEM 11.9), 5.8+/-4.1 (SEM 1.6), 1.6+/-4.1 (SEM 1.6), and 0.005+/-0.01 (SEM 0.005) for high energy, intermediate energy, low energy, and control groups, respectively. Only the intermediate energy group showed statistically more inflammatory cells than control eyes (P=.015), most likely due to variability between eyes. CONCLUSIONS: Higher energy levels trigger greater cell death when the femtosecond laser is used to create corneal flaps. Greater corneal inflammatory cell infiltration is observed with higher femtosecond laser energy levels.

Expression of PDGF receptor-alpha in corneal myofibroblasts in situ.

The purpose of this study was to investigate the expression of platelet-derived growth factor receptor-alpha (PDGFR-alpha) in the myofibroblasts of corneas with stromal haze. Central corneal sections from rabbit eyes that had -9 diopter PRK were analyzed by immunocytochemistry (IHC) for the expression of PDGFR-alpha at 4 week after surgery. PDGFR-alpha was expressed immediately beneath the epithelial basement membrane in the anterior stroma. Double IHC studies revealed the expression of PDGFR-alpha in the anterior stroma co-localized with alpha-smooth muscle actin (SMA) marker for myofibroblasts. In vitro studies have suggested that PDGF is important in the development and viability of myofibroblasts after corneal injury. Expression of PDGFR-alpha in myofibroblasts supports these findings.

Corneal myofibroblast viability: opposing effects of IL-1 and TGF beta1.

The purpose of this study was to test the effect of corneal epithelial scrape on myofibroblasts associated with haze and elucidate the effect of interleukin-1 and transforming growth factor beta1 on corneal stromal myofibroblasts viability and death in vitro. Corneal epithelial scrape was performed in rabbit eyes with severe haze at one month after -9 diopter photorefractive keratectomy. Corneas were processed for immunocytochemistry for myofibroblast marker alpha-smooth muscle actin (alpha-SMA) and the TUNEL assay to detect apoptosis. Rabbit corneal fibroblasts were cultured with 2 ng/ml of transforming growth factor beta1 (TGF beta1) to induce myofibroblast differentiation confirmed by monitoring alpha-SMA expression. Fluorescence-based TUNEL assay was performed to analyze the apoptotic response of myofibroblasts to IL-1alpha or IL-1beta, in the presence or absence of TGF beta1. Dose response experiments were performed after withdrawal of TGF beta1 and exposure to 1, 5, or 10 ng/ml of IL-1alpha or IL-1beta for 1 h. Subsequent experiments were performed with myofibroblasts exposed to 5 ng/ml of IL-1alpha or IL-1beta in conjunction with 0, 1, 5, or 10 ng/ml of TGF beta1. Corneal epithelial scrape with a scalpel blade produced myofibroblast apoptosis. Exposure to TGF beta1 in vitro resulted in greater than 99% transformation of corneal fibroblasts to alpha-SMA+ myofibroblasts. There was a statistically significant dose-dependent increase in the percentage of TUNEL+ cells with either IL-1alpha or IL-1beta initiated at concentrations as low as 1 ng/ml. For example, after withdrawal of TGF beta1, the % TUNEL+ cells at 1 h after exposure to IL-1alpha increased significantly with increasing concentration (0 ng/ml, 2.4 +/- 0.8% [S.E.M.]; 1 ng/ml, 15.4 +/- 1.8%; 5 ng/ml, 47.4 +/- 3.9%; or 10 ng/ml, 70.3 +/- 3.2%). Similar results were obtained with IL-1beta. The differences between the means of apoptotic myofibroblasts for the different concentrations of cytokine for either IL-1alpha or IL-1beta were significantly different (ANOVA, p < 0.001). When myofibroblasts were exposed to 5 ng/ml of IL-1alpha or IL-1beta, the % TUNEL+ cells at 1 h were reduced in a significant dose-dependent manner when TGF beta1 at a concentration of 5 ng/ml or 10 ng/ml was present in the medium (ANOVA p < 0.01). IL-1alpha or IL-1beta triggers the death of myofibroblasts in vitro and TGF beta1 reduces the IL-1 effect on cell death. TGF beta1 and IL-1 have opposing effects on myofibroblast viability and likely interact to modulate haze generation after corneal injury.

Corneal stroma PDGF blockade and myofibroblast development.

Myofibroblast development and haze generation in the corneal stroma is mediated by cytokines, including transforming growth factor-beta (TGF-beta), and possibly other cytokines. This study examined the effects of stromal PDGF-beta blockade on the development of myofibroblasts in response to -9.0 diopter photorefractive keratectomy in the rabbit. Rabbits that had haze generating photorefractive keratectomy (PRK, for 9 diopters of myopia) in one eye were divided into three different groups: stromal application of plasmid pCMV.PDGFRB.23KDEL expressing a subunit of PDGF receptor b (domains 2-3, which bind PDGF-B), stromal application of empty plasmid pCMV, or stromal application of balanced salt solution (BSS). The plasmids (at a concentration 1000ng/microl) or BSS was applied to the exposed stroma immediately after surgery and every 24h for 4-5 days until the epithelium healed. The group treated with pCMV.PDGFRB.23KDEL showed lower alphaSMA+ myofibroblast density in the anterior stroma compared to either control group (P