H. Kaur

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields alpha-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p<0.01). Intermediate filaments--vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery.

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.

Corneal myofibroblast viability: Opposing effects of IL-1 and TGF β1

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<or=0.001). Although there was also lower corneal haze at the slit lamp at one month after surgery, the difference in haze after PDGF-B blockade was not statistically significant compared to either control group. Stromal PDGF-B blockade during the early postoperative period following PRK decreases stromal alphaSMA+ myofibroblast generation. PDGF is an important modulator of myofibroblast development in the cornea.

Stromal interleukin-1 expression in the cornea after haze-associated injury

The purpose of this study was to determine whether myofibroblasts or other cells in the stroma in the cornea produce interleukin (IL)-1alpha or IL-1beta that could modulate myofibroblast viability in corneas with haze after photorefractive keratectomy (PRK). Twenty-four female rabbits had haze-generating PRK for 9 diopters of myopia and were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were removed, frozen in OCT at -80 degrees C, and analyzed by immunocytochemistry using primary antibodies to IL-1alpha, IL-1beta and alpha smooth muscle actin (SMA). Double immunostaining was performed for the co-localization of SMA with IL-1alpha or IL-1beta. Central dense haze and peripheral slight haze regions of each cornea were analyzed. SMA+ cells that expressed IL-1alpha protein were detected in both regions of the corneas at most time points following PRK. However, in the haze region at the 1, 3 and 4 week time points, significantly more (p<0.01) SMA+ cells did not express IL-1alpha. Also, in the haze region at all three time points, significantly more (p<0.01) SMA- cells than SMA+ cells expressed interleukin-1alpha protein. IL-1beta expression patterns in SMA+ and SMA- stromal cells was similar to that of IL-1alpha after PRK. Previous studies have demonstrated that IL-1alpha or IL-1beta triggers myofibroblast apoptosis in vitro, depending on the available concentration of apoptosis-suppressive TGFbeta. This study demonstrates that SMA- cells such as corneal fibroblasts, keratocytes, or inflammatory cells may produce IL-1alpha and/or IL-1beta that could act in paracrine fashion to regulate myofibroblast apoptosis--especially in the region where there is haze in the cornea after PRK was performed and SMA+ myofibroblasts are present at higher density. However, some SMA+ myofibroblasts themselves produce IL-1alpha and/or IL-1beta, suggesting that myofibroblast viability could also be regulated via autocrine mechanisms.

Expression of PDGF Receptor-α 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.