Critical Role of CD55 in Controlling Wound Healing

Abstract

How reparative processes are coordinated following injury is incompletely understood. In recent studies, we showed that autocrine C3a and C5a receptor (C3ar1 and C5ar1) G protein-coupled receptor signaling plays an obligate role in vascular endothelial growth factor receptor 2 growth signaling in vascular endothelial cells. We documented the same interconnection for platelet-derived growth factor receptor growth signaling in smooth muscle cells, epidermal growth factor receptor growth signaling in epidermal cells, and fibroblast growth factor receptor signaling in fibroblasts, indicative of a generalized cell growth regulatory mechanism. In this study, we examined one physiological consequence of this signaling circuit. We found that disabling CD55 (also known as decay accelerating factor), which lifts restraint on autocrine C3ar1/C5ar1 signaling, concomitantly augments the growth of each cell type. The mechanism is heightened C3ar1/C5ar1 signaling resulting from the loss of CD55's restraint jointly potentiating growth factor production by each cell type. Examination of the effect of lifted CD55 restraint in four types of injury (burn, corneal denudation, ear lobe puncture, and reengraftment of autologous skin) showed that disabled CD55 function robustly accelerated healing in all cases, whereas disabled C3ar1/C5ar1 signaling universally retarded it. In wild-type mice with burns or injured corneas, applying a mouse anti-mouse CD55 blocking Ab (against CD55's active site) to wounds accelerated the healing rate by 40-70%. To our knowledge, these results provide new insights into mechanisms that underlie wound repair and open up a new tool for accelerating healing.

Figure 1:. Recovery from burn wound and corneal denudation.

Panel A. An insulated 8 mm diameter steel rod was heated to 120 °C. A uniform full-thickness (8 mm) burn wound was made in CD55^−/−, WT, and C3ar1^−/−C5ar1^−/− mice (6 each group) by placing the rod on the shaved area for 20 s. The decrease in the size of the burn wound area was compared over a 9 d period. Results for male and female mice were comparable. The results were highly reproducible. Panel B. A uniform 0.5 cm deep (5–6 cell deep) circular 1.5 mm diameter layer of anesthetized corneal epithelium from CD55^−/−, WT, and C3ar1^−/−C5ar1^−/− mice (6 each group) was removed with a 1.5 mm trephine employing an Algerbrush II with a 0.5 mm Burr. After recovery from anesthesia, corneas were stained with fluorescein to quantitate wound size. Wound healing was compared at 4, 12, and 24 h. Results for males and females were similar. n=3

Figure 2:. Recovery from ear puncture and autologous skin transplant.

Panel A. Equal size ear lobe punctures were made in CD55^−/−, WT, and C3ar1^−/−C5ar1^−/− mice (6 each) with a 5 mm punch. Wound closure was measured over 29 d. Percentage decrease in size is shown on d 7, 21, and 29. Comparable results were observed in male and female mice. Studies done in triplicate. Panel B: Following shaving and depilation of backs, a 2 × 2 cm of skin was removed. Immediately thereafter a transplant from the donor mouse (cut to the same size) was secured in place. Mice were euthanized 14 d later and frozen sections of the skin transplant were stained with rat anti-mouse CD31 mAb followed by Alexa Fluor 594 labeled goat anti-rat CD31 (red). Rat IgG2a was included as a control. Nuclei were stained with Hoechst (blue). Representative images of 12 mice are shown at 20x magnification. Panel C. Blood vessel areas in the transplants in panel B for the three genotypes were quantified by NIS-Elements. Total areas (red) in each image were measured (n=5).

Figure 3:. Ability of CD55 blockade to accelerate wound healing.

Panel A. A uniform full-thickness burn wound was made in WT mice as in Figure 2 panel A. Wounds were covered for 24 h with bandages (3M) presoaked in mouse anti-mouse CD55 CCP23 anti-plasma (6 mice) or presoaked with pre-immunization plasma (6 mice). Burn wound size was quantitated as in Fig 2 panel A. The experiment was repeated 3 times with consistent results (p <0.05). Results for males and females were comparable. Panel B. A uniform circular layer of corneal epithelium was removed from WT mice (6 mice) as in Fig 2 panel B. The mice were treated with mouse anti-mouse CD55-CCPs23 antiserum or pre-immunization serum as control. After recovery from anesthesia, corneas were stained with fluorescein and wound healing measured at 4, 12, and 24 h. The experiment was repeated 3 times with consistent results (p <0.005).

Figure 4:. Up-regulatory effect of CD55 blockade on GF production and activation of mitotic signaling intermediates.

Panel A. NIH-3T3 fibroblasts and bEND.3 ECs express CD55 which control autocrine C3ar1/C5ar1 signaling (3, 4). Each cell type was incubated in 0.05% serum with threshold amounts (10 ng/ml) of GF in the absence or presence of anti-CD55 Ab for 2 and 6 h and growth factor mRNA expression was assessed by qPCR (n=3). Panel B. The growth of each cell type in the presence of threshold amounts of GF without or with anti-CD55 or control was measured kinetically at 24 and 48 h (n=3). Panel C. Cells were activated with GF + anti-CD55 Ab or GF + control, as in Panel B except cells were extracted at 10 min and extracts were assessed for p-ERK and p-AKT by immunoblot. Equal amounts of extract as assessed by protein concentration were added to each lane and blots were probed for phosphorylated signaling intermediates and β-actin.

Figure 5:. Schematic of the downstream effects of C3ar1/C5ar1 signaling in the presence and absence of CD55 restraint:

Panel A: In the absence of CD55 control, the generation of C3a and C5a activation fragments from cell endogenous C3 and C5 proteins is heightened and C3ar1/C5ar1 signaling potentiated. CD55 functions on the cell surface and in endosomes. As a result PI-3Kɣ activation is increased. It introduces three phosphorylations into PTEN which inactivate its phosphatase function. As a result, PIP₃ is stabilized and p-AKT production and its downstream signaling to mTOR (not shown) are potentiated. Concurrently, autophosphorylation of the RTK is augmented and its generation of p-Src and p-ERK increased. Panel B: In the presence of CD55 control, the restrained the generation of C3a and C5a activation fragments represses autocrine C3ar1/C5ar1 signaling. As a result, PI-3Kɣ production is repressed and phosphorylations of PTEN that inactivate its function are not introduced. As a result, the active PTEN dephosphorylates PIP₃ to PIP₂ and the generation of p-AKT and its downstream signaling to mTOR (not shown) is restrained. Concurrently, autophosphorylation of the RTK is reduced and its generation of p-Src and p-ERK repressed.