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

Abstract

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.

Fig. 1

Immunocytochemistry for vimentin and α-smooth muscle actin in the unwounded rabbit corneal stroma (A) and at 1 week (B), 2 weeks (C), 3 weeks (D), and 4 weeks (E) after -9 diopter photorefractive keratectomy. Six corneas were studied at each time point and the panels show representative results. Cells nuclei are stained blue with DAPI, SMA-positive cells are stained green and vimentin-positive cells are stained red. Representative positive-stained cells are indicated with arrows in some panels. Note that in the unwounded cornea there are vimentin+ cells in the stroma beneath the epithelial basement membrane. These vimentin+ cells increase in number and have increased levels of vimentin at 1, 2, 3, and 4 weeks after PRK (arrows B, C, D, and E). SMA is not detectible until 2 weeks after PRK (arrows). In the double-stained column of C, D, and E, it can be seen that many (arrows E DAPI/vimentin/SMA), but not all, cells in the anterior stroma are positive for both vimentin and SMA. Magnification 400×. The bar in DAPI staining E shows 50 μm. With cryopreservation of rabbit corneas it is common during sectioning to have artifactual separation of the epithelium from the underlying stroma. White vertical bars in the DAPI image for each week delineate this separation, if any, that is also present in the other images for that week. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Immunocytochemistry for α-smooth muscle actin and desmin in the unwounded rabbit corneal stroma (A) and at 1 week (B), 2 weeks (C), 3 weeks (D), and 4 weeks (E) after -9 diopter photorefractive keratectomy. Six corneas were studied at each time point and the panels show representative results. Cells nuclei are stained blue with DAPI, SMA-positive cells are stained green and desmin-positive cells are stained red. Representative positive-stained cells are indicated with arrows in some panels. Note that no SMA or desmin were detected in the unwounded corneas or at 1 week after PRK (A and B). At two weeks (C) and 3 weeks (D) after PRK, many SMA-positive cells could be detected beneath the epithelial basement membrane. Some of these cells, if not all, were also desmin+, although in some desmin was expressed at low levels, if at all. By four weeks after PRK (E) there appeared to be 100% concordance between SMA and desmin expression in the anterior stromal myofibroblast cells. Magnification 400×. The bar in DAPI staining E shows 50 μm. With cryopreservation of rabbit corneas it is common during sectioning to have artifactual separation of the epithelium from the underlying stroma. White vertical bars in the DAPI image for each week delineate this separation, if any, that is also present in the other images for that week.

Fig. 3

Box and whisker plots for vimentin (A), α-smooth muscle actin (B) and desmin (C) expression over time after 9 diopter PRK for myopia in rabbits. The line in the center of each box is the median and the top and bottom of the boxes are the 25th and 75th percentiles. The distance between the 25th and 75th percentiles (the length of the box) is the interquartile range. The top and bottom of the “whiskers” are at the “upper and lower adjacent values” which represent the points furthest from the median that fall within 1.5 times the interquartile range from the median. Note that vimentin was the only marker of the three that was detected in stromal cells (presumably keratocytes) in the unwounded cornea (data from the unwounded corneas are not shown in C for desmin, but none was detected, identical to week 1).