Background
The skin is vulnerable to every form of external radiation therapy administered to target internal organs [
1]. Skin reactions following irradiation have characteristics of the delay in the onset of clinical changes. Acute skin reaction related to radiation therapy usually manifest within 1–4 weeks of radiation start. Even with modern radiotherapy techniques, ~ 85% of patients will experience moderate to severe acute skin reactions in exposed areas [
2]. When severe, such exposures culminate in epidermal desquamation and potential ulceration or necrosis [
3‐
5], thereby curtailing treatments and perhaps undermining cancer control or prognosis. At present, conventional postirradiation skin management [
6] includes preventative measures, prompt conservative therapy, and even skin flaps in extreme cases. Still, conventional methods are less than ideal [
7]. The key to managing irradiated skin is its regenerative potential.
Skin injury triggers immediate stress responses in epidermal keratinocytes, which then begin to proliferate and migrate to wounded areas, giving rise to a layer of hyperproliferative epithelium [
8‐
11]. Epithelial tissues express differing keratin pairs, depending upon cell type. The K5/K14 pair is expressed by basal epidermis, wherein epidermal stem cells and transient amplifying (TA) cells reside [
12]. It has been shown that K14 cells adjacent to wounds are intimately involved in epithelial regenerative processes, producing daughter cells that gravitate to sites of injury and assist in skin repair [
12,
13]. However, cellular radiosensitivity is influenced by both phase of cellular proliferation and degree of differentiation, less differentiated or actively proliferating cells being more radiosensitive than highly differentiated or non-proliferating cells [
14,
15]. Thus, impaired keratinocyte proliferation and differentiation in the aftermath of irradiation impedes skin regeneration and healing.
Platelet-rich plasma (PRP) has been incorporated into a wide variety of surgical procedures and clinical treatments. Especially in chronic wounds, PRP has shown promising experimental and clinical results [
16]. The regenerative potential of PRP is generally attributed to supraphysiologic concentrations of various growth factors released by activated platelets [
17,
18]. The essential roles of these growth factors in tissue regeneration and wound healing have been confirmed by many studies [
19‐
21], particularly via PI3K/AKT/NFkappaB signaling pathways [
22]. According to a recent publication, the PI3K/AKT pathway is canonical pathway to promote the keratinocyte proliferation [
12]. Nevertheless, the ability of PRP to regulate postirradiation keratinocyte activity and its capacity to promote regeneration in irradiated skin is not fully understood. In the present study, we investigated the influence of radiation to the keratinocyte capacity and whether PRP enhances the regeneration efficacy of irradiated keratinocytes in vitro and in vivo in a mouse model of radiation-induced skin injury.
Materials and methods
PRP preparation
Umbilical cord blood (UCB) donated from ALLCORD (Seoul Metropolitan Government Public Cord Blood Bank) was used to prepare PRP, as detailed in a previously published protocol [
18]. The plasma was first collected by centrifugation (250×
g, 10 min). Platelets in collected plasma were then pelleted through a second centrifugation (1000×
g, 10 min). Thereafter, PRP was activated by a bead mill homogenizer (Precellys 24; OMNI International, Kennesaw, GA, USA). The supernatants were collected by centrifugation (12,000×
g, 20 min) and filtered (0.2-μm size) to furnish the activated PRP releasate.
Cell culture
Human keratinocyte HaCaT cells and 293T cells were grown in Dulbecco’s Modified Eagle’s Media (DMEM; Gibco, Gaithersburg, MD, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% antibiotic–antimycotic (Gibco) at 37 °C in a humidified atmosphere of 5% CO2. The cells were then irradiated at indicated dose using a 137Cs γ-ray source (Atomic Energy of Canada, Chalk River, ON, Canada) at a dose rate of 3.81 Gy/min. HaCaT cells were replaced to 0.5% FBS containing medium and added PRP or LY294002 (Sigma-Aldrich) for 24 h.
Scratch assay
Cells seeded in 12-well plates were incubated for 24 h until confluent monolayers formed. Using a pipette tip to wound or scratch monolayers, each was then rinsed twice with PBS and incubated in fresh media for eventual fixation (4% formaldehyde) and staining (crystal violet). Under an inverted phase-contrast microscope, images were captured and measurements taken.
RNA silencing and lentivirus replication
To generate K14 knockdown cells, a lentivirus-based plasmid kit (TR311838) encoding a 4-gene set (#1–#4) of unique 29mer shRNA sequences specific for the human K14 gene was purchased, along with non-effective control shRNA vectors (OriGene, Rockville, MD, USA). Lentivirus replication entailed transient co-transfection of HEK 293T cells with packaging vectors, using polyethylenimine (PEI) method. After 5 h, the medium was replaced with fresh; and 48 h later, the virus-laden medium was collected, cleared by centrifugation, and passed through 0.45-μm syringe filter.
HaCaT cells were infected with either K14 shRNA lentivirus (Lenti-K14 shRNA #1–#4) or negative control lentivirus (C) in the presence of 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO, USA) for 16 h, and the medium was refreshed. After 3 days the infection, the efficiency of infection was measured by western blot analysis.
Animal selection and care
Totally 72 male SKH-1 mice (7 weeks old, 30 ± 3 g) were obtained from the Orient Bio (Seongnam, South Korea). The mice were held under controlled conditions, including constant temperature, allowing free access to regular chow and 3-stage filtered water. After 1 week of acclimatization, mice were randomly divided into three groups. The Animal Investigation Committee of the Korea Institute of Radiological and Medical Sciences approved all animal experimentation.
Irradiation and PRP treatment protocol
Animals were anesthetized by intraperitoneal injection of alfaxalone (Alfaxan, 75 mg/kg; Careside, Gyeonggi-do, Korea) and xylazine (Rompun, 10 mg/kg; Bayer Korea, Seoul, South Korea) for irradiation (X-RAD 320; Softex Korea, Gyeonggi-do, South Korea). The dorsal skin was gently stretched to required dimensions (2 cm × 2 cm) and taped, falling within the field of irradiation. Other bodily areas were protected by lead shielding (6 mm). A single dose of 40 Gy was delivered at 260 kV, 10 mA, using a rate of 2 Gy/min. Dosage and rate of delivery were strictly monitored (UNIDOS E Universal Dosemeter; PTW-Freiburg, Freiburg, Germany). Afterwards, topical PRP was applied twice weekly for 2 weeks by intradermal injection, for a total of 100 ul of PRP per injury site in treated mice.
Transepidermal water loss in hairless mouse skin
Transepidermal water loss (TEWL) was quantified mechanically (Tewameter TM 300; Courage + Khazaka Electronic GmbH, Cologne, Germany) according to device protocol. Measurements were obtained under controlled conditions, including constant relative humidity and room temperature.
Histologic examination of hairless mouse skin
All animals were sacrificed and the dorsal skin promptly excised. The excised patches (2 cm × 2 cm) were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 μm, and stained [hematoxylin and eosin (H&E) and Sirius Red] for microscopic examination. Immunofluorescent stains were also performed to assess expression levels of K10, K14, Ki-67, and involucrin during wound healing. The following antibodies were purchased from commercial sources: anti-K10 (Abcam, Cambridge, UK), anti-K14 (Abcam), anti-Ki-67 (Acris Antibodies, Herford, Germany), and involucrin (Santa Cruz Biotechnology, Dallas, TX, USA). Quantitative assessment of immunoreactivity was enabled by proprietary image analysis software (i-Solution; IMT Inc,
https://www.IMT-Solution.com).
Whole-mount images of hairless mouse epidermis
Samples of promptly excised dorsal skin taken from sacrificed animals were incubated in 5 mM EDTA/PBS overnight at 4 °C, with an additional 2 h at 37 °C. Epidermis was smoothly peeled away using a pair of fine forceps and immediately placed in fresh PBS for Oil Red O staining of sebaceous glands.
Western blot analysis
Cells or tissue specimens were homogenized in RIPA lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA), separating lysates by electrophoresis in 12% sodium dodecyl sulfate-polyacrylamide gel. The various proteins were then electrophoretically transferred onto polyvinylidene fluoride membranes for blocking (5% skim milk, 1 h) and overnight incubation (4 °C) with the following primary antibodies: K1 (Abcam), K10 (Abcam), K14 (Abcam), Ki-67 (Acris Antibodies), p-AKT (Cell Signaling Technology, Danvers, MA, USA), AKT (Cell Signaling Technology), involucrin, and β-actin (Santa Cruz Biotechnology). Immunoreactive bands were developed using a horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology) and visualized as directed by light emission (Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate; PerkinElmer, Waltham, MA, USA). Quantitative assessment of immunoreactivity was software-enabled (IMT Inc).
Statistical analysis
All data were subjected to Student’s t-tests and expressed as standard error of the mean (SEM). Statistical significance was set at p < 0.05.
Discussion
Improving endogenous basal keratinocytes and their inherent functions is a key factor in skin repair, especially under suboptimal conditions. In the course of this study, we discovered that unlike normal controls, keratinocytes from radiogenic wounds were marked by shifts in keratin expression (K10 to K14 keratin); and in immunofluorescent analysis, K14 expression broadened to include suprabasal cells. These revelations were further confirmed in irradiated HaCaT cells. In addition, the proliferation and migration of such altered keratinocytes seemed to be impaired. K14 is typically found in the basal layer where it serves to control the proliferation of cells. Previous reports have shown that K14 is explicitly expressed in the mitotically active basal layer of stratified epithelia, wherein epidermal stem cells and TA cells reside [
26]. In another in vitro study, primary cultures developed from K14-null mice showed a reduced capacity for cellular proliferation [
27]. However, our results indicate that K14 in irradiated keratinocytes hampered the ability to participate in skin repair. Consequently, restoration of functional K14 in keratinocytes may be essential for adequate repair of irradiated skin.
Clinical applications intended to functionally activate endogenous basal cells are part of an advanced strategy adopted by regenerative medicine and tissue engineering for reduction of risk. Since the 1970s, PRP has been a familiar clinical commodity, recognized for its regenerative and healing properties [
28‐
31]. It has been shown to enhance the proliferation and differentiation of cells, primarily through a variety of growth factors secreted by platelets (i.e., PDGF-AB, TGF-β, and FGF) [
32‐
34]. In a previous study of ours, we have proven that our method of PRP activation yields a more concentrated release of growth factors for superior growth and migration of mesenchymal stromal cells (MSCs), compared with traditional methods of PRP activation
18. Thus, we launched this investigation of radiation-induced skin damage, hoping to improve the reparative process through activated PRP application.
To assess the regenerative effects of PRP on irradiated skin, we examined the wound healing capacity of irradiated HaCaT cells incubated with PRP. According to one in vitro study, PRP treatment promotes wound healing properties and activates AKT signaling in irradiated HaCaT cells. These particular effects are also blocked by AKT inhibitor treatment. Activation of PI3K/AKT signaling has been viewed as the chief mechanism driving proliferation and migration of K14 cells [
12]. As recent reports indicate, PRP may induce PI3K/AKT signaling, serving to enhance survival and regenerative functions of MSCs [
22,
35]. To further explore the relation between PRP on K14-positive cells, we used shRNA to create a K14-knockdown HaCaT cell line, which readily displayed inhibition of PRP-induced wound healing properties.
To validate the wound-healing effects of PRP on irradiated skin, we used a preclinical model of radiation-induced skin injury in mice. Through clinical observations, histopathologic analysis, and functional evaluation of the skin barrier, we have ascertained that local injection of PRP clearly promotes cutaneous wound healing. Microscopic examination of irradiated mouse skin injected with PRP revealed an lush mantle of granulation tissue, including new and thicker type III collagen and epithelial tongues composed of K14-positive cells, all materializing within 14 days. At day 28, structures resembling sebaceous glands were also observed in PRP-treated skin.
When proliferating keratinocytes in irradiated lesions were subsequently immunostained, focusing on Ki-67 expression and AKT signaling activation, Ki-67-positive cells were frequent in regenerative PRP-treated skin, compared with controls; and results were analogous for AKT signaling. Finally, the diminished expression of K10 we observed after irradiation was reversed through PRP treatment. K10 is expressed in the suprabasal layer of skin, and in K10-deficient mice, the permeability barrier function is diminished [
36]. Reduced K10 expression leads to changes in expression levels of cornified envelope proteins, which form a scaffold for permeability barrier purposes [
36,
37]. Expression of involucrin, a cornified envelope protein, increased in PRP-treated irradiated skin, and heightened TEWL values were reversed. Overall, these findings demonstrate that PRP enhances the proliferation of K14 cells and accelerates cutaneous barrier restoration in a mouse model of radiation-induced skin injury.
Conclusion
The present efforts indicate a shift from K1/10 to K14 status in irradiated keratinocytes and demonstrable impairment of wound healing. However, PRP acts to reverse radiation injury via AKT signaling, thus enhancing the wound-healing and barrier functions of skin. PRP may therefore be clinically beneficial in this sphere, helping to overcome poor cutaneous healing after radiation exposure.
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