Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury

Abstract

The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFβ requisite for myofibroblast survival.

Fibrocytes, Wound Healing, and Corneal Fibrosis

“…It is widely accepted that fibrocytes arise from bone marrow cells. Lassance et al, 18 demonstrate, using green fluorescent protein (GFP) chimeric mice, that cells derived from bone marrow give rise to fibrocytes that enter the cornea after injury. Similarly, in an experiment using sex-mismatched bone marrow chimeric mice (female), Mori et al 17 found the Y chromosome (from transplanted male bone marrow) in the nuclei of the majority of fibrocytes.…”

“…They conclude that fibrocytes migrate from bone marrow as predifferentiated cells-reinforcing that bone marrow is the primary location of fibrocytes differentiation. 14 Monocyte differentiation into fibrocytes has been found to take approximately 10 days in vitro, whereas fibrocytes can be found by 1 day after epithelial-stromal injury to the cornea, 18 suggesting that either local differentiation of fibrocytes from monocytes takes a shorter amount of time or they are present in the blood ready to respond to injuries by moving into tissues and Downloaded from iovs.arvojournals.org on 07/06/2020…”

“…The immunohistochemical study of fibrocytes in the cornea is challenging since an important marker for detection of fibrocytes is collagen type I, 6 and the normal corneal stroma itself contains large amounts of collagen type I. 18 Fibrocytes themselves typically produce low amounts of collagen. 71 Thus Lassance et al 18 find intense stromal staining for collagen type I in corneas-which interferes with the tracking of collagen type I expressed by fibrocytes.…”

“…18 Fibrocytes themselves typically produce low amounts of collagen. 71 Thus Lassance et al 18 find intense stromal staining for collagen type I in corneas-which interferes with the tracking of collagen type I expressed by fibrocytes. De Roo et al 83 report they can identify fibrocytes in the cornea by immunohistochemistry for the collagen type I marker.…”

“…6,7 More recently, fibrocytes have been defined as spindleshaped cells with oval nuclei, that express markers of hematopoietic progenitor cells, leukocytes and mesenchymal cells, 8 and that contribute to several functions involved in the healing response and angiogenesis 9,10 that can progress to pathological fibrosis in organs such as the lung, 11 liver, 12 kidney, 13,14 heart, 15 blood vessel, 16 skin, 17 and eyes. 18 These bone marrow-derived cells have recently become a focus of corneal wound healing in the cornea and likely contribute to fibrotic processes in the retina, vitreous, and other structures in the eye.…”