Skin fibrosis is a chronic debilitating feature of several skin diseases that lead to characteristic increases in dermal fibroblast proliferation and collagen deposition through upregulation in components of the transforming growth factor beta (TGF-B)/SMAD pathway. In contrast to ultraviolet phototherapy, high-fluence light-emitting diode-generated red light (HF-LED-RL, 633 ± 15 nm) is a safe, economic and non-invasive therapy with in vitro evidence that supports modulation of the key cellular characteristics involved in the pathogenesis of skin fibrosis. Limited data exists pertaining to the effects of HF-LED-RL on human skin fibroblast microRNA (miRNA). Herein, we explored the effects of HF-LED-RL on fibroblast miRNA levels using RNA-seq and miRNA expression analysis. Using RNA-seq analysis we found that HF-LED-RL at 320 and 640 J/cm² increased transcription of key miRNA that are involved in skin fibrosis including miRNA-29, miRNA-196a and Let-7a, and decreased transcription of miRNA-21, miRNA-23b and miRNA-31. These microRNA findings provide insight into the molecular underpinnings of HF-LED-RL and highlight potential therapeutic targets of interest for the treatment of skin fibrosis. Additional research on the specific molecular mechanisms underlying HF-LED-RL effects on fibroblasts may provide further mechanistic insight into this therapy and may reveal additional future therapeutic targets for skin fibrosis.

Skin fibrosis, often referred to as skin scarring, is a significant international health problem with limited treatment options. The hallmarks of skin fibrosis are increased fibroblast proliferation, collagen production, and migration speed. Recently published clinical observations indicate that visible red light may improve skin fibrosis. In this study we hypothesize that high-fluence light-emitting diode-generated red light (HF-LED-RL) modulates the key cellular features of skin fibrosis by decreasing cellular proliferation, collagen production, and migration speed of human skin fibroblasts. Herein, we demonstrate that HF-LED-RL increases reactive oxygen species (ROS) generation for up to 4 hours, inhibits fibroblast proliferation without increasing apoptosis, inhibits collagen production, and inhibits migration speed through modulation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. We demonstrate that HF-LED-RL is capable of inhibiting the unifying cellular processes involved in skin fibrosis including fibroblast proliferation, collagen production, and migration speed. These findings suggest that HF-LED-RL may represent a new approach to treat skin fibrosis. LED advantages include low cost, portability, and ease of use. Further characterizing the photobiomodulatory effects of HF-LED-RL on fibroblasts and investigating the anti-fibrotic effects of HF-LED-RL in human subjects may provide new insight into the utility of this therapeutic approach for skin fibrosis.

Background

Skin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL) is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease.

Objective

The goal of our study was to investigate the how reactive oxygen species (ROS) free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed.

Methods

High fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR). For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2) on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2.

Results

High fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158.4% relative to matched controls, respectively. These LED-RL associated increases in ROS were prevented by pretreating cells with 0.0001% or 0.001% resveratrol. Next, we quantified the effect of hydrogen peroxide (H2O2)-associated ROS on fibroblast migration speed, and found that while H2O2-associated ROS significantly decreased relative fibroblast migration speed, pretreatment with 0.0001% or 0.001% resveratrol significantly prevented the decreases in migration speed. Furthermore, we found that LED-RL at 480, 640 and 800 J/cm2 decreased fibroblast migration speed to 83.0%, 74.4%, and 68.6% relative to matched controls, respectively. We hypothesized that these decreases in fibroblast migration speed were due to associated increases in ROS generation. Pretreatment with 0.0001% and 0.001% resveratrol prevented the LED-RL associated decreases in migration speed.

Conclusion

High fluence LED-RL increases ROS and is associated with decreased fibroblast migration speed. We provide mechanistic support that the decreased migration speed associated with high fluence LED-RL is mediated by ROS, by demonstrating that resveratrol prevents high fluence LED-RL associated migration speed change. These data lend support to an increasing scientific body of evidence that high fluence LED-RL has anti-fibrotic properties. We hypothesize that our findings may result in a greater understanding of the fundamental mechanisms underlying visible light interaction with skin and we anticipate clinicians and other researchers may utilize these pathways for patient benefit.

Fibrosis occurs when collagen deposition and fibroblast proliferation replace healthy tissue. Red light (RL) may improve skin fibrosis via photobiomodulation, the process by which photosensitive chromophores in cells absorb visible or near-infrared light and undergo photophysical reactions. Our previous research demonstrated that high fluence RL reduces fibroblast proliferation, collagen deposition, and migration. Despite the identification of several cellular mechanisms underpinning RL phototherapy, little is known about the transcriptional changes that lead to anti-fibrotic cellular responses. Herein, RNA sequencing was performed on human dermal fibroblasts treated with RL phototherapy. Pathway enrichment and transcription factor analysis revealed regulation of extracellular matrices, proliferation, and cellular responses to oxygen-containing compounds following RL phototherapy. Specifically, RL phototherapy increased the expression of MMP1, which codes for matrix metalloproteinase-1 (MMP-1) and is responsible for remodeling extracellular collagen. Differential regulation of MMP1 was confirmed with RT-qPCR and ELISA. Additionally, RL upregulated PRSS35, which has not been previously associated with skin activity, but has known anti-fibrotic functions. Our results suggest that RL may benefit patients by altering fibrotic gene expression.

Therapeutic applications of light emitting diode-red light (LED-RL) are expanding, yet data on its clinical effects are lacking. Our goal was to evaluate the safety of high fluence LED-RL (≥160 J/cm² ). In two phase I, single-blind, dose escalation, randomized controlled trials, healthy subjects received LED-RL or mock irradiation to the forearm thrice weekly for 3 weeks at fluences of 160-640 J/cm² for all skin types (STARS 1, n = 60) and at 480-640 J/cm² for non-Hispanic Caucasians (STARS 2, n = 55). The primary outcome was the incidence of adverse events (AEs). The maximum tolerated dose was the highest fluence that did not elicit predefined AEs. Dose-limiting AEs, including blistering and prolonged erythema, occurred at 480 J/cm² in STARS 1 (n = 1) and 640 J/cm² in STARS 2 (n = 2). AEs of transient erythema and hyperpigmentation were mild. No serious AEs occurred. We determined that LED-RL is safe up to 320 J/cm² for skin of color and 480 J/cm² for non-Hispanic Caucasian individuals. LED-RL may exert differential cutaneous effects depending on race and ethnicity, with darker skin being more photosensitive. These findings may guide future studies to evaluate the efficacy of LED-RL for the treatment of various diseases.