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) GPCR signaling plays an obligate role in vascular endothelial cell receptor-2 (VEGFR2) growth signaling in vascular endothelial cells. We documented the same interconnection for platelet derived growth factor (PDGFR) growth signaling in smooth muscle cells, epidermal growth factor receptor (EGFR) growth signaling in epidermal cells, and fibroblast growth factor FGFR signaling in fibroblasts, indicative of a generalized cell growth regulatory mechanism. Herein, we examined one physiological consequence of this signaling circuit. We found that disabling CD55 [aka decay accelerating factor (DAF)], which lifts restraint on autocrine C3ar1/C5ar1 signaling, concomitantly augments the growth of each cell type. The mechanism is that heightened C3ar1/C5ar1 signaling resulting from the loss of CD55’s restraint jointly potentiates growth factor (GF) production by each cell type. Examination of the effect of lifted CD55 restraint in four types of injury: 1) burn, 2) corneal denudation, 3) ear lobe puncture, and 4) re-engraftment of autologous skin, showed that disabled CD55 function robustly accelerated healing in all cases, whereas disabled C3ar1/C5ar1 signaling universally retarded it. In WT mice with burns or injured corneas, applying a mouse anti-mouse CD55 blocking antibody (against CD55’s active site) to wounds accelerated the healing rate by 40–70%. The results provide new insights into mechanisms that underlie wound repair and open-up a new tool for accelerating healing.

INTRODUCTION

Many processes participate in wound repair. Central among them is potentiated growth factor (GF) signaling. Vascular endothelial cell growth factor receptor (VEGFR2) and platelet derived growth factor receptor (PDGFR) signaling initiate vascular tube formation. Fibroblast growth factor receptor (FGFR) signaling evokes stromal cell ingress into the wound while epidermal growth factor receptor (EGFR) signaling induces striated muscle ingrowth and epithelial surface reconstitution. In addition to augmenting growth, the receptor tyrosine kinase (RTK) signaling promotes integrin dependent cell-cell adhesion at repair sites (1, 2) and pro-mitotic cytokine production. IL-6 ligation of IL-6 receptor-gp130 (IL-6R-gp130) induces the nuclear translocation of p-STAT3 which together with associated transcription factors (TFs) triggers the production of growth relevant genes that enable wound closure. How GF signaling is regulated during wound repair is incompletely understood.

RTKs have classically been regarded as conferring their growth signaling in and of themselves. Contrary to this concept, our recent studies showed that VEGFR2, PDGFR, and EGFR each interconnect in their respective cell types with C3a and C5a receptors (C3ar1/C5ar1) G protein coupled receptors (GPCRs) and function together with these GPCRs in eliciting cell growth (3, 4). The two GPCRs operate in an autocrine fashion in each cell type as a result of their activating ligands C3a and C5a being generated from endogenously synthesized C3 and C5 proteins. This extrahepatic synthesis and nontraditional function of complement proteins is in line with the growing appreciation (5, 6) that complement proteins are produced by most cell types and participate in many cellular functions, e.g. adaptive immune responses (7–12). In this vein, our recent studies (3, 4).showed that joint C3ar1 and C5ar1 signaling is essential for VEGF-A, PDGF, EGF-2, and FGF2 induction of vascular endothelial cell (EC), smooth muscle cell (SMC), epithelial cell (EPC) and fibroblast growth. Studies in vivo validated this requisite role of autocrine C3ar1/C5ar1 signaling in physiological cell growth (3, 4) The mechanism is that C3ar1 and C5ar1 in each cell type are physically associated with the respective RTK together with IL-6 receptor-glycoprotein 130 (IL-6R-gp130) in a signalosome, and combinatorial signaling of all four partners is required for the production of pro-mitotic cellular intermediates and the cognate GF by each cell (3, 4). The combinatorial signaling is an essential participant in the transition of each cell type through the cell cycle (3, 4).

Our earlier studies of immune cell activation (9, 10) showed that the intensity of C3ar1/C5ar1 signaling and the consequent strength of cellular proliferation is controlled by the cell associated regulator CD55 [aka decay accelerating factor (DAF)]. When its restraint is lifted, cellular C3a/C5a production is increased and autocrine C3ar1/C5ar1 signaling is amplified (diagrammed in Figure 6). In contrast, when CD55 expression is upregulated, C3a/C5a production is inhibited and C3ar1/C5ar1 signaling is repressed. While our studies of RTK signaling showed that GF production that drives RTK signaling is interconnected with autocrine C3ar1/C5ar1 signaling, they didn’t definitively investigate the role of CD55.

In the studies in this report, we first exploited CD55^−/− and C3ar1^−/−C5ar1^−/− mice (each congenic on C57BL/6) and compared the rates of wound healing in these mice to that of wild type (WT) mice in four independent wound models. With the use of a mouse anti-mouse CD55 Ab directed against CD55’s active site that confers it convertase inhibitory activity (13, 14), we then tested whether blockade of CD55 in WT mice would accelerate wound healing, a procedure that could be translated clinically. We then characterized signaling processes interconnecting CD55’s control of C3ar1/C5ar1 signaling with GF production and cell growth.

METHODS

Mice and reagents

WT C57BL/6 mice were purchased from Jackson Labs. CD55^−/−- and C3ar1^−/−C5ar1^−/− were bred 12 generations on the C57BL/6 background as described previously (8, 15). Male and female mice aged 7–12 wk were used for all models. Mice were housed in a specific pathogen–free facility and were confirmed to be negative for common murine viral pathogens by routine sera analysis. Experiments were conducted employing established guidelines for animal care, and all protocols were approved by the Case IACUC institutional animal care committee.

Burn model

The backs of 8–10-week-old mice were shaved and depilated one day prior to experiments and the mice given normal food and access to water. Immediately prior to the burn injury, mice were anesthetized with ketamine (8–10 mg/100 g), xylazine (0.5–1.0 mg/100 g), and acepromazine (0.3 mg/100 g) intraperitoneally (i.p.) and the depilated skin cleaned with 70% ethanol. A uniform full-thickness burn wound was produced by placing an insulated 8 mm diameter steel rod heated to 120 °C on the shaved skin for 20 seconds. The pressure applied to the rod was the weight of the rod itself. Buprenorphine (0.3 mg/ml) and 0.9% saline were given i.p. for analgesia and fluid resuscitation was given immediately after injury. Animals were observed daily and sacrificed if signs of distress were apparent. The wound area was photographed at 0, 3, 6, and 9 d following the procedure and the change in size was analyzed using MetaMorph employing the Region Measurement tool and Photoshop.. Mice were sacrificed 14 d after the injury by CO₂ asphyxiation. Burn wound tissues were harvested and fixed in 4% paraformaldehyde for 24 h and stained with H&E.

Corneal Epithelialization

Mice were placed in an induction chamber connected to an oxygen source and isoflurane vaporizer oxygen flow was initiated at 0.9 liters/min and the isoflurane vaporizer adjusted to 1–2%. As soon as the mouse became unresponsive and had shallow breathing, it was transferred to an anesthesia platform with a nose cone and placed on a heating pad. One drop of 1% proparacaine hydrochloride was applied to the right eye prior to the procedure. After marking the cornea with a 1.5 mm trephine, a uniform circular 5–6 cell epithelial layer of corneal cells was removed using an Algerbrush II with 0.5 mm Burr (The Alger company, Inc, TX, USA). Fluorescein solution was applied to the cornea and the size of staining used as a measure of the unhealed wound.

Ear wound model

Seven-12 wk old male and female mice of each genotype were anesthetized. The ear was punctured using a 5 mm biopsy punch. The size of the wound was measured on days (d) 7, 21, and 29 after the procedure.

Syngeneic skin transplant model

Adult recipient mice were shaved and depilated 3 d prior to transplantation. Mice were anesthetized. A 2 × 2-cm midline area of their backs were delineated, shaved and depilated. The dorsal area was prepared for wounding using a Betadine scrub and was wiped with 70% alcohol. All instruments used were either sterile disposable or sterilized prior to use. The skin (~1.0–1.5 cm²) transplant from the same mouse or donor syngeneic mouse (cut to the same size) was secured in place using either sutures or Band-Aid, the graft was protected from injury by bandaging the area following transplant. Clinical observations and assessments were made daily throughout the experiment and recorded. Photographs of the grafts were taken at least twice for all animals. Mice were monitored daily and those showing any signs of morbidity (site infection, poor healing (redness and itching not disappearing in 1–3 d) losing more than 15% of their body mass, and/or loss of ambulation or eating) were euthanized. All skin samples were divided, and half processed for histology, and half used for CD31 analyses of ECs. Tissue destined for histology/ISH was fixed in 10% neutral buffered formalin for 16 h, then transferred to 70% ethyl alcohol and paraffin-embedded. The fixation duration was recorded for each sample.

CD55 Blockade on Corneal Wound Healing

For studies of CD55 blockade, corneas of WT mice were injured as above and treated immediately after wounding and at 2 and 6 h thereafter with a 20 uL drop of mouse anti-mouse CD55 CCP23 Ab (1:50 diluted anti-CD55 plasma). An identical group of WT mice was treated with pre-immunization plasma at same time intervals,. Procedures were performed under a Leica stereo microscope with a fixed magnification and brightness in order to maintain consistency. Pictures were taken at 0, 4, 12 and 24 h using a green fluorescence filter on the microscope and the size decrease of the de-epithelialized area was analyzed using MetaMorph again employing the region measurement tool. Another group of mice was not treated and photographed to assess their natural course of healing.

Preparation of mouse anti-mouse CD55 CCP23 Ab

Twelve C57BL/6 CD55^−/− mice were immunized with recombinant mouse CD55 protein (60 ug/mouse) emulsified with complete Freund’s adjuvant (CFA) or with PBS as control. Blood was collected from the tail vein prior to immunization and weekly thereafter. Plasmas were applied to an ELISA to assess levels of anti-CD55 CCPs23 Ab.

Immunohistochemistry

Frozen skin sections of the graft bed were allowed to air dry for 30 m at room temperature. They were fixed in cold acetone for 10 m, and after rinsing with PBS, blocked with 4% normal bovine serum for 1 h at room temperature. Rat anti-mouse CD31 mAb (15.6 μg/ml) from BD Biosciences was applied at 1:100 dilution and sections were incubated at 4°C overnight after which they were washed with PBS three times. Sections were then incubated with Alexa Fluor 594 goat anti-rat IgG2a mAb (2 mg/ml) for one h. ProLong Gold Antifade Mounting Media (Life Technologies) was applied on the sections and they were cover-slipped.

qPCR

Murine bEnd.3 and NIH-3T3 cells were seeded at 1.25 × 10⁵ cells per well in 6-well plates in complete DMEM (10% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL Penicillin/Streptomycin) and allowed to adhere overnight. Reduced serum DMEM (0.05% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL Penicillin/Streptomycin) was then substituted fo the complete media and the plates were again incubated overnight. Cultures were then treated with 10 ng/mL mVEGF or mPDGF ± anti-CD55 Ab (1:1000 dilution) and incubated for 2 and 6 h. Cells were harvested and RNA isolated using RNeasy Plus Mini Kit (Qiagen)l. cDNA was synthesized using High-Capacity RNA-cDNA Reverse Transcription Kit (Applied Biosystems) according to manufacturer’s protocol. cDNA was diluted 1:10 with nuclease-free H₂O and 8 uL of diluted cDNA were mixed with 2 uL of 10 uM primers and 10 uL of LightCycler 480 SYBR Green I Master Mix (Roche Life Sciences). Samples were assayed in triplicate on a LightCycler 96. Data are given as fold increases of RNA expression relative to basal levels and standardized to RNA expression of murine Beta-Actin (mACTB).

Western Blots

Murine NIH-3T3 cells were grown to 70% confluency in 10cm TC-Treated plates in complete DMEM (10% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL Penicillin/Streptomycin), after which media complete media was substituted for reduced serum DMEM (0.05% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL Penicillin/Streptomycin) and cells were treated with 10 ng/mL mPDGF ± anti-CD55 Ab. After 5 minutes, cells were lysed in 1x Cell Lysis Buffer (10x) (Cell Signaling Technology, #9803) supplemented with one Complete Mini protease inhibitor tablet (Roche, #11836153001) and 1mM sodium orthovanadate (to inhibit phosphatase activity) for 10 minutes on ice. Lysates were sonicated 3x for 2 minutes and centrifuged for 10 minutes at 12,000 x g. Lysates were then subjected to reducing SDS-PAGE and Western Blotting, and phosphorylation of ERK and AKT was assessed via Optical Density relative to unstimulated samples and Beta-Actin protein levels. Antibodies for p-ERK, p-AKT, and Beta-Actin were obtained from Cell Signaling Technology (#4370T, #4060T, #4970T).

Growth Curves

Murine bEnd.3 ECs and NIH-3T3 cells were seeded at 3 × 10⁴ cells per well in 6-well plates in complete DMEM (10% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL Penicillin/Streptomycin). The cells were allowed to adhere overnight, after which reduced serum DMEM (0.05% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/m) was substituted as above and the plates incubated overnight again. The cells were treated with 10 ng/mL mVEGF-A or mPDGF ± anti-CD55 Ab (1:1000 diluted anti-CD55 or control normal rabbit sera). Cell counts were performed with trypan blue exclusion at 0, 24, and 48 h after treatment.

Murine mIMCD-3 myocyte cells were seeded at 3 × 10⁴ cells per well in 6-well plates in complete DMEM:F12 (10% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL) and re-equilibrated in reduced serum DMEM:F12 (0.05% Fetal Bovine Serum, 2mM L-Glutamine, 100 U/mL). Cells were treated with 10 ng/mL mEGF ± anti-CD55 Ab or normal rabbit Serum (1:1000 dilution) and. cell counts were performed in an identical fashion.

Human aortic endothelial cells (HAEC) were seeded in EBM-2 supplemented with EGM-2 Endothelial SingleQuots Kit (Lonza Bioscience), re-equilibrated in EBM-2 media alone without growth supplements and cells were treated with 10 ng/mL hVEGF-A ± anti-CD55 Ab or normal rabbit serum (1:1000 dilution) and counted in the same fashion.

Statistics

Statistical significance for all experimental data was determined by Student’s t-test (unpaired, two-tailed) with Microsoft Excel or GraphPad Prism 5.

RESULTS

In initial studies, we examined the effect of lifted CD55 restraint on C3ar1/C5ar1 signaling in vivo in different murine models of wound repair.

Burn recovery.

In a first model, we examined recovery from burn injury. Round 0.8 cm burns were made by placing a heated (120 ^o) metal rod on shaved skin on the back of mice of each genotype. All mice were given continued pain medication and displayed no sign of prolonged pain. The burn wound diameter decreased ~150% faster in CD55^−/− mice compared to WT mice (Figure 1A). This contrasted with a >200% lesser decrease in C3ar1^−/−C5ar1^−/− mice compared to WTs (n = 11).

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

Re-epithelialization of the corneas

In a second model, we compared healing of injured corneas. We removed a uniform 1.5 mm circular 5–6 cell layer of corneal epithelium by marking the site with a 1.5 mm trephine and employing an Algerbrush II with a 0.5 mm Burr. The anesthetized mice experienced no pain. After recovery from anesthesia, we stained the wounded corneas with fluorescein to detect wounded tissue. We then measured the change in wound size at 4, 12, and 24 h after injury by re-staining the cornea with fluorescein. The corneas of CD55^−/− mice re-epithelialized 200% faster than WTs, whereas those of C3ar1^−/−C5ar1^−/− mice re-epithelialized 150% slower than WTs (Figure 1B).

Ear wound healing

We next assessed wound closure following ear lobe puncture, a model widely used by others (16). We made equivalent 5 mm punctures in the ear lobes of WT, CD55^−/−, and C3ar1^−/−C5ar1^−/− mice and made daily measurements of the area of the puncture remaining open. In C3ar1^−/−C5ar1^−/− mice, the 5-mm wound declined slowly at d 29 post puncture reaching 44.2% of the original size, whereas it declined much more rapidly to 13.9% of the original size in CD55^−/− mice, a >200% greater decrease compared to C3ar1^−/−C5ar1^−/− mice. These results contrasted with a 31.1% decrease by WT mice (Figure 2A).

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).

Skin transplant engraftment

We lastly studied re-engraftment of excised autologous skin. Two cm by 2 cm skin grafts excised from the left flanks of mice of the three genotypes were immediately re implanted at the same site and covered with gauze. Analysis of the grafts 14 d post-engraftment showed that the transplanted skin in CD55^−/− mice re-granulated 90% faster than in WTs. Conversely, the skin in C3ar1^−/−C5ar1^−/− mice showed ~70% delayed re-granulation compared to the WTs, with less hair regrowth. Histological quantitation of blood vessels at the junction of the transplant showed 50% higher blood vessel density in CD55^−/− mice than in WT mice. This contrasted with ~400% lower blood vessel density in C3ar1^−/−C5ar1^−/− mice (Figure 2B and 2C).

Acceleration of wound healing in WT mice by Ab blockade of CD55

In all four models, deficiency of CD55 (which potentiates C3ar1/C5ar1 dependent RTK signaling) markedly accelerated wound healing. Conversely, deficiency of C3ar1/C5ar1 [which disables autocrine C3ar1/C5ar1 signaling (3, 4)] uniformly slowed healing. These consistent differences prompted the question of whether Ab blockade of CD55 would accelerate wound healing in WT mice, a treatment which could be translated clinically. To test this, we developed a mouse anti-CD55 Ab against the second and third of CD55’s four 60 amino acid long complement control protein repeats (CCPs) which contain its active site (13, 14). We generated the anti-CCPs23 Ab by immunizing our CD55^−/− mice selectively devoid of CCP23 (15) with full length recombinant mouse CD55 protein (17) (see Methods). The elicited Abs were specific for CD55 CCPs23 as other regions of the CD55 immunogen were homologous with recipient CD55. ELISAs of 11 cultured splenic cell clones from the immunized CD55^−/− mice identified the anti-CD55 CCPs23 Ab plasma possessing the highest anti-CD55 activity (Figure S1). Functional studies showed that the anti-CD55 Abs abolished the function of both mouse and human CD55 (not shown). We used that anti-CD55 plasma together with pre-immunization control plasma in WT mice subjected to 1) burn wound or to 2) corneal injury to test whether Ab blockade of CD55 function would accelerate healing similarly to CD55 deficiency.

To assess the effect of the anti-CD55 CCP23 Ab in the burn model, we covered burn wounds in WT mice with bandages (3M) presoaked in the mouse anti-mouse CD55 CCP23 plasma. An identical set of mice received bandages presoaked in pre-immune plasma as a control. Daily comparison of wound sizes showed that bandages presoaked in the anti-CD55 CCP23 plasma accelerated burn repair 150–250% faster than those presoaked in nonimmune plasma (Figure 3A).

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).

To determine if the anti-CCP23 Ab blockade of CD55 would similarly accelerate corneal wound healing, we produced circular epithelial layer lesions in WT mice as done in the above studies of the knockouts. We added 20 μl of 1:50 diluted anti-CD55 plasma immediately following injury (time 0) and at 2 and 6 h thereafter to the tears of one set of mice and the same amount of pre-immune plasma to the tears of a set of identically injured mice. We then stained eyes with fluorescein at 0, 4, 12, and 24 h as in the above studies comparing the anti-CD55 CCP23 and control treated mice. The corneas of the mice treated with anti-mouse CD55 CCP23 plasma healed 350% faster than those of mice treated with pre-immune plasma (Figure 3B). Side by side comparisons of the effect of the anti-CD55 blockade to that of the CD55 deficiency (Fig 1B vs Fig 3B) showed the rate of healing in the anti-CD55 CCPs23 treated mice approximated that in CD55^−/− mice.

These findings in the corneal surface and burn healing models taken together with the four injury models with the knockouts and the GF studies show that CD55 control of C3ar1/C5ar1 signaling jointly regulates the mitotic activity of multiple RTKs in vivo, and argue that anti-CD55 blockade could be efficacious in accelerating tissue regeneration.

Anti-CD55 blockade induces endogenous GF production that augments the proliferation of tissue cells.

To validate how lifted CD55^−/− restraint on C3ar1/C5ar1 signaling influences VEGFR2, PDGFR, and EGFR signaling in ECs, SMCs and EPCs, we measured GF production, cell growth, and RTK signaling intermediates following treatment with the respective GFs in the absence and presence of anti-CD55 blocking Ab. For analyses of GF production, we incubated 1) bEnd.3 ECs with threshold amounts of VEGF-A and 2) NIH-3T3 cells with threshold amounts of PDGF or FGF-1 (not shown), in the absence and presence of anti-CD55 Ab and measured mRNA levels of the respective GFs by qPCR. Abolishing CD55 restraint upregulated GF production by each cell type (Fig 4A left and right). To determine if the increased GF mRNA production correlated with increased cell growth, we measured cell numbers at 24 and 48 h following incubation of 1) bEnd.3 ECs with threshold amounts of VEGF-A, and 2) NIH-3T3 fibroblasts with threshold amounts PDGF without or with added anti-CD55 Ab. CD55 blockade accelerated cell growth of both cell types at both time points (Fig 4B left and center-left). To establish if the results apply to human cells, we incubated human aortic endothelial cells (HAEC) with threshold amounts of human VEGF-A ± anti-human CD55 Ab and measured HAEC growth. The HAECs showed a comparable growth response to the anti-CD55 Ab documenting human cell relevance. (Fig 4B center-right). Treatment of miMCD-3 myocytes with EGF + anti CD55 over 48 h gave the same results showing that growth enhancing effect also applied to striated muscle cells (Fig 4B right). In line with anti-CD55 blockade promoting cell growth by augmenting the intensity of RTK signaling (3, 4), it increased the phosphorylation of both AKT and ERK by at ~2-fold (Fig 4C).

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.

DISCUSSION

In this study, we examined in vivo wound healing following four different types of injury. Our experiments showed that disabling CD55 function uniformly accelerated healing in each wound setting, whereas disabling C3ar1/C5ar1 function uniformly inhibited it. The central process underlying this result is that autocrine C3ar1/C5ar1 signaling is a requisite process in VEGFR2, PDGFR, and EGFR as well as other RTK (3, 4) mitotic signaling and that lifting CD55’s restraint on this GPCR signaling concomitantly potentiates growth signaling in each cell type and promotes cell cycle transition of each (3, 4). Our past work showed that C3ar1/C5ar1 and each RTK are physically associated and the joint C3ar1/C5ar1-RTK signaling is essential for the downstream growth inductive signaling of each RTK and the induction of endogenous GF production by each cell type. The studies herein show that in the absence of CD55’s restraint, RTK signaling is amplified and mitotic activity is potentiated. Conversely, abrogated C3ar1/C5ar1 signaling represses RTK mitotic activity. Of relevance therapeutically, we found that Ab blockade of CD55 in WT mice was as effective as CD55 genetic deficiency. The uniformly accelerated wound repair in the burn and corneal epithelial murine models argues for studies of CD55 blockade in human injury models. Since topical application of the Ab was effective in both the murine injury models, the anti-CD55 blockade could be adaptable to several types of human wounds. Humanized anti-human CD55 Abs are available and in preparation. The 40% to >200% accelerating effect of the anti-CD55 blockade on the healing in the mouse models argues that the treatment, should be effective.

Our connection of C3ar1/C5ar1 signaling with GF mitotic signaling was informed by our prior studies of adaptive T cell activation (8, 9, 12). Those studies (18–21) found that when antigen presenting dendritic cells (DCs) engage cognate T cells, the DCs as well as the T cells endogenously generate C3a and C5a. The immune cell produced C3a/C5a establish autocrine signaling loops with upregulated C3ar1/C5ar1 on both partners that transduce synergistic signals into each that are integral to T effector cell (Teff) proliferation (8), differentiation (8, 9) and viability (8, 22). Studies with CD55^−/− mice or mice devoid of the C3/C5 sources (C3^−/−C5^−/− mice) of C3a/C5a or their receptors (C3ar1^−/−C5ar1^−/− mice) uncovered the dependence of the C3ar1/C5ar1 signaling strength on CD55 expression levels and validated CD55’s regulatory effect on T cell differentiation in vivo (8, 9, 23, 24). Subsequent work (ourselves and others) showed that the T cell activation process operates intracellularly in T cells (20, 25) and is essential intracellularly in DCs as well as in B2 cells (26) to produce Abs.

CD55 was originally characterized as a shield that protects self-cells from autologous complement attack (27). Findings that autocrine C3ar1/C5ar1 signaling operates in many if not most cell types and that CD55 control of this signaling regulates multiple cell functions revealed that CD55 is a broadly functioning cellular regulator (3, 4). It acts both on the cell surface and intracellularly. It tonically regulates the generation of C5a/C3a and consequently regulates the signaling level of C3ar1/C5ar1 that are tonically associated with RTKs. Following GF occupation of the RTK, C3a/C5a generation increases (4), the C3ar1/C5ar/ RTK signaling complex activates downstream intracellular intermediates (3, 4), and transits intracellularly to endosomes (4). The Gβɣ subunits of the C3ar1/C5ar1 G proteins activate phosphoinositide-3 kinase ɣ (PI-3Kɣ) (8) and Gα subunits repress adenylyl cyclase (12) in concert with activating phospholipase C (PLC) (28).

Mechanistic studies of the role of C3ar1/C5ar1 co-signaling in RTK signaling (3) traced its effects to repressing tonically activated PTEN, SOCS1/3 and PHLPP that together tonically inhibit cell growth (3). These phosphatases dominantly repress inner leaflet phosphatidylinositol 3,4,5 trisphosphate (PIP₃) assembly and phosphorylation of AKT and STAT3, and thereby homeostatically restrain VEGFR2, PDGFR, and EGFR induction of cell growth. Engagement of C3ar1/C5ar1 signaling overcomes these dominantly restraining processes by its activation of PI-3Kɣ which confers inactivating phosphorylations in PTEN (that prevent its dephosphorylation of PIP₃). The C3ar1/C5ar1 induced activation of PI-3kɣ thereby synergizes with conventional RTK-associated PI-3Kα in promoting PIP₃ assembly (3)_. The PI-3Kɣ signaling also reverses suppression of p-STAT3 and p-AKT by SOCS1/3 and PHLPP respectively (3). The physical association of C5ar1/C5ar1 with RTKs leads to their transactivation of the RTK and consequent participation of C5ar1/C5ar1 in RTK autophosphorylation (4). Because of the multiple linkages by others of different RTKs, and multiple signaling intermediates and cytokines e.g., ERK, PLC and IL-6 (29–36), with healing, the existence of a unified process that could account for the joint activation of many of them has not been envisioned. Identification of CD55 control of C5ar1/C5ar1 signaling as constituting one such process and its blockade as robustly promoting healing thus should be broadly relevant to accelerating tissue regeneration (37).

While our data are consistent with some past studies of the involvement of complement in wound healing, they differ from some others (38, 39). Two important distinctions between our studies and those of others are that 1) other studies focused on systemic complement, and 2) none examined CD55 in the context of regulating cell growth via cellular (as opposed to systemic) complement. Prior studies consistent with our findings found that topical addition of either purified C3 or C5 proteins individually or in combination to skin increases vascular permeability, fibroblast migration, and collagen deposition (40–43). Other studies consistent with ours found that augmented C3ar1 or C5ar1 signaling promotes hepatic regeneration following CCl₄ hepatic poisoning (44) and bone repair following fracture (45). The interpretation in both cases however was that the C3a/C5a ligands derived from systemic complement activation. The role of downregulated CD55 and endogenous C3a/C5a production elicited by the cells themselves in accelerating healing was not envisioned.

With respect to contrasting literature, it was reported that C3 deficiency favors wound healing (39), but the highly inflammatory model and the absence of blocking CD55 could have caused different results and the effect consequently may have related to neutrophil and macrophage infiltration and their attendant effects. CD55 was not assessed and may have been upregulated. In contrast to C3 deficiency, which would impair immunity, CD55 deficiency could be anti-infectious as it markedly heightens Teff (Th1/Th17 cell) generation and anti-bacterial function (8, 46). Alternatively, as our studies of immune cells found (12), C3 deficiency that would disable C3ar1 signaling in the presence of intact CD55 function in WT cells would promote Foxp3⁺ T regulatory cell (Treg) induction (12). Another possibility derives from a paper (21) that showed that C3 can be deficient systemically, but C3a function and consequent C3ar1 signaling could be retained intracellularly. Also seemingly contradictory but in fact consistent, C3ar1 or C5ar1 blockade was reported to arrest the progression of FGF induced fibrosis in a mouse model of pulmonary fibrosis (47). We have found that FGF function, like VEGF-A/PDGF/EGF function, is inhibited by C3ar1/C5ar1 blockade (3).

The results herein together with our findings (3) that FGF induced growth is similarly interconnected with autocrine C3ar1/C5ar1 signaling could relate to findings that applying FGF onto wounded rabbit ears shortened the healing time, an effect connected with FGF induction of angiogenesis via ERK and AKT activation (48). Our data also may account for some reported effects of cytokines on wound healing. Among these reports, TGF-β (associated in our studies (12) with repressed C3ar1/C5ar1 signaling) was connected with retarding wound healing (49). Conversely, IL-6 (associated in our studies with amplified C3ar1/C5ar1 signaling) has been connected with augmenting wound healing (50). These observations are in accordance with our findings in immune cells that autocrine TGF-β signaling by Tregs inhibits cell endogenous C3 and C5 production (12), whereas IL-6 production by Teff augments them (12). These findings would translate via the function of the RTK-C3ar1-C5ar1-IL-6R-gp130 signalosome to suppressed vs potentiated C3ar1/C5ar1 signaling (3, 4).

The findings herein that adding mouse anti-mouse CD55 CCPs23 Ab to murine burns and injured corneas accelerates healing of both argue that this treatment should be applicable to human wounds. In principle, it could be useful for poorly healing wounds in individuals in whom healing is impaired, e.g. diabetics, individuals with inflammatory arthritis, or aged persons. In line with this, our pilot studies in diabetic mice have shown more rapid closure of chronic ulcers (Hammelef, E, Bapputty R, Gubitosi-Klug R, and Medof ME unpublished).

A unique feature of autocrine C3ar1/C5ar1 signaling and its control by CD55 exploitable for tissue regeneration is that it concomitantly regulates multiple RTKs (3). In blood vessels it not only controls VEGF-A (and FGF-2) induction of EC growth, but also controls coordinate PDGF and EGF induction of SMC and EPC/muscle cell growth (3) Our studies of stable ECs in blood vessels have shown that it regulates thrombomodulin (TM) and nitric acid synthesis (eNOS) expression which participate in the health of the vasculature (An FQ, Medof ME, et al, under review). More studies will be needed to determine how it precisely integrates with other signaling systems in each cell type in human injuries.

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.

Key points:

Disabling CD55 globally accelerates the growth of multiple cell types.

Potentiated C3ar1/C5ar1 signaling integrates with the signaling of receptor RTKs.