High fluence light emitting diode-generated red light modulates characteristics associated with skin fibrosis

Abstract

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.

Keywords: LED light; Skin fibrosis; low level light therapy; photobiomodulation; phototherapy.

Figure 1

High Fluence LED-RL (633 nm) decreases human dermal fibroblast cell count and proliferation in a dose-dependent manner without increasing apoptosis. (a) DHFs irradiated with 320 and 640 J/cm2 LED-RL were incubated for 24 or 48 hours and then counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. 320 and 640 J/cm2 LED-RL decreased relative cell count to 97.2 ± 4.7% (p=0.616) and 88.7 ± 3.3% (p = 0.043) compared to matched controls, respectively. DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 48 hours demonstrated decreased relative cell count to 81.0 ± 2.9% (p < 0.001) and 70.6 ± 4.5% (p < 0.001) compared to matched controls, respectively. (b) DHFs irradiated with 320 and 640 J/cm2 LED-RL every 12 hours for a total of 4 total doses were collected 48 hours following the first LED-RL dose and counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. Multiple doses of 320 and 640 J/cm2 LED-RL significantly decreased relative cell count to 75.8 ± 3.6% (p = 0.006) and 55.0 ± 10.3% (p < 0.001) compared to matched controls, respectively. (c) DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 18 hours with 4 μCi of tritiated thymidine (3H-Thy). The relative 3H-Thy incorporation of treated dishes was compared to matched control dishes as a surrogate of proliferation rate. 320 and 640 J/cm2 LED-RL decreased relative 3H-Thy incorporation to 84.2 ± 3.8% (p=0.003) and 33.4 ± 5.3% (p < 0.001) compared to matched controls, respectively. (d) Representative Annexin V vs. 7-AAD flow cytometry plots 0 hours, 2 hours, and 4 hours post-irradiation. (e) HF-LED-RL (633 nm) does not induce significant increases in total apoptosis levels at 0, 2, or 4 hours post-irradiation in HDFs. Bars represent average percent AV positive cells in each treatment group. For purposes of comparison to a positive control, we used temperature (50°C) to induce apoptosis levels of 98.3%. Error bars represent standard error of the mean. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 1

High Fluence LED-RL (633 nm) decreases human dermal fibroblast cell count and proliferation in a dose-dependent manner without increasing apoptosis. (a) DHFs irradiated with 320 and 640 J/cm2 LED-RL were incubated for 24 or 48 hours and then counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. 320 and 640 J/cm2 LED-RL decreased relative cell count to 97.2 ± 4.7% (p=0.616) and 88.7 ± 3.3% (p = 0.043) compared to matched controls, respectively. DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 48 hours demonstrated decreased relative cell count to 81.0 ± 2.9% (p < 0.001) and 70.6 ± 4.5% (p < 0.001) compared to matched controls, respectively. (b) DHFs irradiated with 320 and 640 J/cm2 LED-RL every 12 hours for a total of 4 total doses were collected 48 hours following the first LED-RL dose and counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. Multiple doses of 320 and 640 J/cm2 LED-RL significantly decreased relative cell count to 75.8 ± 3.6% (p = 0.006) and 55.0 ± 10.3% (p < 0.001) compared to matched controls, respectively. (c) DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 18 hours with 4 μCi of tritiated thymidine (3H-Thy). The relative 3H-Thy incorporation of treated dishes was compared to matched control dishes as a surrogate of proliferation rate. 320 and 640 J/cm2 LED-RL decreased relative 3H-Thy incorporation to 84.2 ± 3.8% (p=0.003) and 33.4 ± 5.3% (p < 0.001) compared to matched controls, respectively. (d) Representative Annexin V vs. 7-AAD flow cytometry plots 0 hours, 2 hours, and 4 hours post-irradiation. (e) HF-LED-RL (633 nm) does not induce significant increases in total apoptosis levels at 0, 2, or 4 hours post-irradiation in HDFs. Bars represent average percent AV positive cells in each treatment group. For purposes of comparison to a positive control, we used temperature (50°C) to induce apoptosis levels of 98.3%. Error bars represent standard error of the mean. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 1

High Fluence LED-RL (633 nm) decreases human dermal fibroblast cell count and proliferation in a dose-dependent manner without increasing apoptosis. (a) DHFs irradiated with 320 and 640 J/cm2 LED-RL were incubated for 24 or 48 hours and then counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. 320 and 640 J/cm2 LED-RL decreased relative cell count to 97.2 ± 4.7% (p=0.616) and 88.7 ± 3.3% (p = 0.043) compared to matched controls, respectively. DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 48 hours demonstrated decreased relative cell count to 81.0 ± 2.9% (p < 0.001) and 70.6 ± 4.5% (p < 0.001) compared to matched controls, respectively. (b) DHFs irradiated with 320 and 640 J/cm2 LED-RL every 12 hours for a total of 4 total doses were collected 48 hours following the first LED-RL dose and counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. Multiple doses of 320 and 640 J/cm2 LED-RL significantly decreased relative cell count to 75.8 ± 3.6% (p = 0.006) and 55.0 ± 10.3% (p < 0.001) compared to matched controls, respectively. (c) DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 18 hours with 4 μCi of tritiated thymidine (3H-Thy). The relative 3H-Thy incorporation of treated dishes was compared to matched control dishes as a surrogate of proliferation rate. 320 and 640 J/cm2 LED-RL decreased relative 3H-Thy incorporation to 84.2 ± 3.8% (p=0.003) and 33.4 ± 5.3% (p < 0.001) compared to matched controls, respectively. (d) Representative Annexin V vs. 7-AAD flow cytometry plots 0 hours, 2 hours, and 4 hours post-irradiation. (e) HF-LED-RL (633 nm) does not induce significant increases in total apoptosis levels at 0, 2, or 4 hours post-irradiation in HDFs. Bars represent average percent AV positive cells in each treatment group. For purposes of comparison to a positive control, we used temperature (50°C) to induce apoptosis levels of 98.3%. Error bars represent standard error of the mean. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 1

High Fluence LED-RL (633 nm) decreases human dermal fibroblast cell count and proliferation in a dose-dependent manner without increasing apoptosis. (a) DHFs irradiated with 320 and 640 J/cm2 LED-RL were incubated for 24 or 48 hours and then counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. 320 and 640 J/cm2 LED-RL decreased relative cell count to 97.2 ± 4.7% (p=0.616) and 88.7 ± 3.3% (p = 0.043) compared to matched controls, respectively. DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 48 hours demonstrated decreased relative cell count to 81.0 ± 2.9% (p < 0.001) and 70.6 ± 4.5% (p < 0.001) compared to matched controls, respectively. (b) DHFs irradiated with 320 and 640 J/cm2 LED-RL every 12 hours for a total of 4 total doses were collected 48 hours following the first LED-RL dose and counted using the trypan blue exclusion assay. Cells counts of groups treated with HF-LED-RL were compared to matched control dishes to calculate relative cell count, a surrogate of cell proliferation. Multiple doses of 320 and 640 J/cm2 LED-RL significantly decreased relative cell count to 75.8 ± 3.6% (p = 0.006) and 55.0 ± 10.3% (p < 0.001) compared to matched controls, respectively. (c) DHFs irradiated with 320 and 640 J/cm2 LED-RL and then incubated for 18 hours with 4 μCi of tritiated thymidine (3H-Thy). The relative 3H-Thy incorporation of treated dishes was compared to matched control dishes as a surrogate of proliferation rate. 320 and 640 J/cm2 LED-RL decreased relative 3H-Thy incorporation to 84.2 ± 3.8% (p=0.003) and 33.4 ± 5.3% (p < 0.001) compared to matched controls, respectively. (d) Representative Annexin V vs. 7-AAD flow cytometry plots 0 hours, 2 hours, and 4 hours post-irradiation. (e) HF-LED-RL (633 nm) does not induce significant increases in total apoptosis levels at 0, 2, or 4 hours post-irradiation in HDFs. Bars represent average percent AV positive cells in each treatment group. For purposes of comparison to a positive control, we used temperature (50°C) to induce apoptosis levels of 98.3%. Error bars represent standard error of the mean. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 2

HDFs treated with 320 and 640 J/cm2 LED-RL demonstrate significantly increased ROS levels compared to matched controls for up to 4 hours post-irradiation. (a) Representative flow cytometry curves. Red curves represent mean fluorescent intensity of HDFs treated with HF-LED-RL, blue curves represent matched controls. (b) At 0, 1, 2, and 4 hours following irradiation, ROS levels were significantly elevated compared to matched controls. Peak ROS levels were observed 1 hour following 320 and 640 J/cm2 LED-RL and found to be 127% (p=0.0026) and 145% (p=0.0018) relative to matched controls, respectively. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 2

HDFs treated with 320 and 640 J/cm2 LED-RL demonstrate significantly increased ROS levels compared to matched controls for up to 4 hours post-irradiation. (a) Representative flow cytometry curves. Red curves represent mean fluorescent intensity of HDFs treated with HF-LED-RL, blue curves represent matched controls. (b) At 0, 1, 2, and 4 hours following irradiation, ROS levels were significantly elevated compared to matched controls. Peak ROS levels were observed 1 hour following 320 and 640 J/cm2 LED-RL and found to be 127% (p=0.0026) and 145% (p=0.0018) relative to matched controls, respectively. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 3

High Fluence LED-RL inhibits human dermal fibroblast collagen production.HDFs were irradiated, fixed, and stained with picrosirius red (a collagen-specific dye). Picrosirius red was then eluted and measured using spectrophotometry. HF-LED-RL irradiated groups demonstrated a dose-dependent decrease in collagen levels with 320 J/cm2 = 83.2 ± 2.0% of matched control (p=0.002), 640 J/cm2 = 54.1 ± 6.3% of matched control (p < 0.001). (b) HDFs treated with HF-LED-RL and stained with picrosirius red demonstrated a decrease in staining intensity compared with matched controls when viewed with light microscopy at 40x magnification. (A, top left panel) Control for 320 J/cm2 LED-RL. (B, top right panel) Treatment group irradiated with 320 J/cm2 LED-RL. (C, bottom left panel) Control for 640 J/cm2 LED-RL. (D, bottom right panel) Treatment group irradiated with 640 J/cm2 LED-RL. (c) Western blot staining of HDFs treated with HF-LED-RL demonstrated no significant decrease in procol1A1 levels immediately following irradiation. A representative western blot is shown. After normalizing to GAPDH, we found 320 J/cm2 LED-RL resulted in 91% procol1A1 and 640 J/cm2 LED-RL resulted in 97% procol1A1, relative to matched controls, respectively. (d) Western blot staining of HDFs treated with HF-LED-RL demonstrated a decrease in procol1A1 levels at 2 hours following irradiation. A representative western blot is shown. After normalizing to GAPDH, we found 320 J/cm2 LED-RL resulted in 55% procol1A1 and 640 J/cm2 LED-RL resulted in 51% procol1A1, relative to matched controls, respectively. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 3

High Fluence LED-RL inhibits human dermal fibroblast collagen production.HDFs were irradiated, fixed, and stained with picrosirius red (a collagen-specific dye). Picrosirius red was then eluted and measured using spectrophotometry. HF-LED-RL irradiated groups demonstrated a dose-dependent decrease in collagen levels with 320 J/cm2 = 83.2 ± 2.0% of matched control (p=0.002), 640 J/cm2 = 54.1 ± 6.3% of matched control (p < 0.001). (b) HDFs treated with HF-LED-RL and stained with picrosirius red demonstrated a decrease in staining intensity compared with matched controls when viewed with light microscopy at 40x magnification. (A, top left panel) Control for 320 J/cm2 LED-RL. (B, top right panel) Treatment group irradiated with 320 J/cm2 LED-RL. (C, bottom left panel) Control for 640 J/cm2 LED-RL. (D, bottom right panel) Treatment group irradiated with 640 J/cm2 LED-RL. (c) Western blot staining of HDFs treated with HF-LED-RL demonstrated no significant decrease in procol1A1 levels immediately following irradiation. A representative western blot is shown. After normalizing to GAPDH, we found 320 J/cm2 LED-RL resulted in 91% procol1A1 and 640 J/cm2 LED-RL resulted in 97% procol1A1, relative to matched controls, respectively. (d) Western blot staining of HDFs treated with HF-LED-RL demonstrated a decrease in procol1A1 levels at 2 hours following irradiation. A representative western blot is shown. After normalizing to GAPDH, we found 320 J/cm2 LED-RL resulted in 55% procol1A1 and 640 J/cm2 LED-RL resulted in 51% procol1A1, relative to matched controls, respectively. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 3

High Fluence LED-RL inhibits human dermal fibroblast collagen production.HDFs were irradiated, fixed, and stained with picrosirius red (a collagen-specific dye). Picrosirius red was then eluted and measured using spectrophotometry. HF-LED-RL irradiated groups demonstrated a dose-dependent decrease in collagen levels with 320 J/cm2 = 83.2 ± 2.0% of matched control (p=0.002), 640 J/cm2 = 54.1 ± 6.3% of matched control (p < 0.001). (b) HDFs treated with HF-LED-RL and stained with picrosirius red demonstrated a decrease in staining intensity compared with matched controls when viewed with light microscopy at 40x magnification. (A, top left panel) Control for 320 J/cm2 LED-RL. (B, top right panel) Treatment group irradiated with 320 J/cm2 LED-RL. (C, bottom left panel) Control for 640 J/cm2 LED-RL. (D, bottom right panel) Treatment group irradiated with 640 J/cm2 LED-RL. (c) Western blot staining of HDFs treated with HF-LED-RL demonstrated no significant decrease in procol1A1 levels immediately following irradiation. A representative western blot is shown. After normalizing to GAPDH, we found 320 J/cm2 LED-RL resulted in 91% procol1A1 and 640 J/cm2 LED-RL resulted in 97% procol1A1, relative to matched controls, respectively. (d) Western blot staining of HDFs treated with HF-LED-RL demonstrated a decrease in procol1A1 levels at 2 hours following irradiation. A representative western blot is shown. After normalizing to GAPDH, we found 320 J/cm2 LED-RL resulted in 55% procol1A1 and 640 J/cm2 LED-RL resulted in 51% procol1A1, relative to matched controls, respectively. Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05

Figure 4

HF-LED-RL inhibits human dermal fibroblast migration speed through modulation of the PI3K/AKT pathway. (a) HDF migration speed following HF-LED-RL irradiation was assessed used time-lapse video microscopy. HF-LED-RL fluences of 320 and 640 J/cm2 decreased HDF migration speed to 86.6% (p < 0.001) and 74.5% (p<0.001) relative to matched controls, respectively. (b) High Fluence LED-RL significantly decreases the migrations speed of HDFs for up to 12 hours. 320 and 640 J/cm2 of LED-RL resulted in significantly decreased migration speed relative to matched control for 12-hours after irradiation when measured at 4 hour intervals. Pictured above, relative cellular migration speed for HDFs treated with 320 J/cm2 at 0-4 hours was 81.4 ± 4.8% (p<0.05) and at 12-16 hours was 99.2 ± 4.8%. HDFs treated with 640 J/cm2 at 0-4 hours was 72.8 ± 7.1% (p<0.05) and at 12-16 hours was 98.3 ± 6.5%. (c) HDFs treated with 320 and 640 J/cm2 LED-RL demonstrated increased AKT phosphorylation (S473) compared to matched controls at 4 hours post-irradiation. A representative western blot is shown. (d) AKT phosphorylation was no longer increased at 12 hours post-irradiation. A representative western blot is shown. (e) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) did not significantly alter HDF migration speed when compared with control. Pretreating HDFs with 30 μM LY294002 for 30 minutes resulted in a relative speed of 95.2 ± 4.7% (p = 0.48) compared to untreated control. (f) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) prevented the inhibitory effects of 640 J/cm2 LED-RL on HDF migration speed. Primary HDFs with or without Ly294002 were treated with HF-LED-RL and their migration speed was assessed used time-lapse video microscopy. 640 J/cm2 LED-RL significantly decreased migration speed (73.3 ± 4.9%, p < 0.0001) versus matched control. Pretreating HDFs with 30 μM LY294002 for 30 minutes blocked the inhibitory effects of 640 J/cm2 LED-RL on migration resulting in a relative speed of 94.4 ± 4.8% (p = 0.62) relative to matched control pretreated with 30 μM LY294002.Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05.

Figure 4

HF-LED-RL inhibits human dermal fibroblast migration speed through modulation of the PI3K/AKT pathway. (a) HDF migration speed following HF-LED-RL irradiation was assessed used time-lapse video microscopy. HF-LED-RL fluences of 320 and 640 J/cm2 decreased HDF migration speed to 86.6% (p < 0.001) and 74.5% (p<0.001) relative to matched controls, respectively. (b) High Fluence LED-RL significantly decreases the migrations speed of HDFs for up to 12 hours. 320 and 640 J/cm2 of LED-RL resulted in significantly decreased migration speed relative to matched control for 12-hours after irradiation when measured at 4 hour intervals. Pictured above, relative cellular migration speed for HDFs treated with 320 J/cm2 at 0-4 hours was 81.4 ± 4.8% (p<0.05) and at 12-16 hours was 99.2 ± 4.8%. HDFs treated with 640 J/cm2 at 0-4 hours was 72.8 ± 7.1% (p<0.05) and at 12-16 hours was 98.3 ± 6.5%. (c) HDFs treated with 320 and 640 J/cm2 LED-RL demonstrated increased AKT phosphorylation (S473) compared to matched controls at 4 hours post-irradiation. A representative western blot is shown. (d) AKT phosphorylation was no longer increased at 12 hours post-irradiation. A representative western blot is shown. (e) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) did not significantly alter HDF migration speed when compared with control. Pretreating HDFs with 30 μM LY294002 for 30 minutes resulted in a relative speed of 95.2 ± 4.7% (p = 0.48) compared to untreated control. (f) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) prevented the inhibitory effects of 640 J/cm2 LED-RL on HDF migration speed. Primary HDFs with or without Ly294002 were treated with HF-LED-RL and their migration speed was assessed used time-lapse video microscopy. 640 J/cm2 LED-RL significantly decreased migration speed (73.3 ± 4.9%, p < 0.0001) versus matched control. Pretreating HDFs with 30 μM LY294002 for 30 minutes blocked the inhibitory effects of 640 J/cm2 LED-RL on migration resulting in a relative speed of 94.4 ± 4.8% (p = 0.62) relative to matched control pretreated with 30 μM LY294002.Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05.

Figure 4

HF-LED-RL inhibits human dermal fibroblast migration speed through modulation of the PI3K/AKT pathway. (a) HDF migration speed following HF-LED-RL irradiation was assessed used time-lapse video microscopy. HF-LED-RL fluences of 320 and 640 J/cm2 decreased HDF migration speed to 86.6% (p < 0.001) and 74.5% (p<0.001) relative to matched controls, respectively. (b) High Fluence LED-RL significantly decreases the migrations speed of HDFs for up to 12 hours. 320 and 640 J/cm2 of LED-RL resulted in significantly decreased migration speed relative to matched control for 12-hours after irradiation when measured at 4 hour intervals. Pictured above, relative cellular migration speed for HDFs treated with 320 J/cm2 at 0-4 hours was 81.4 ± 4.8% (p<0.05) and at 12-16 hours was 99.2 ± 4.8%. HDFs treated with 640 J/cm2 at 0-4 hours was 72.8 ± 7.1% (p<0.05) and at 12-16 hours was 98.3 ± 6.5%. (c) HDFs treated with 320 and 640 J/cm2 LED-RL demonstrated increased AKT phosphorylation (S473) compared to matched controls at 4 hours post-irradiation. A representative western blot is shown. (d) AKT phosphorylation was no longer increased at 12 hours post-irradiation. A representative western blot is shown. (e) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) did not significantly alter HDF migration speed when compared with control. Pretreating HDFs with 30 μM LY294002 for 30 minutes resulted in a relative speed of 95.2 ± 4.7% (p = 0.48) compared to untreated control. (f) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) prevented the inhibitory effects of 640 J/cm2 LED-RL on HDF migration speed. Primary HDFs with or without Ly294002 were treated with HF-LED-RL and their migration speed was assessed used time-lapse video microscopy. 640 J/cm2 LED-RL significantly decreased migration speed (73.3 ± 4.9%, p < 0.0001) versus matched control. Pretreating HDFs with 30 μM LY294002 for 30 minutes blocked the inhibitory effects of 640 J/cm2 LED-RL on migration resulting in a relative speed of 94.4 ± 4.8% (p = 0.62) relative to matched control pretreated with 30 μM LY294002.Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05.

Figure 4

HF-LED-RL inhibits human dermal fibroblast migration speed through modulation of the PI3K/AKT pathway. (a) HDF migration speed following HF-LED-RL irradiation was assessed used time-lapse video microscopy. HF-LED-RL fluences of 320 and 640 J/cm2 decreased HDF migration speed to 86.6% (p < 0.001) and 74.5% (p<0.001) relative to matched controls, respectively. (b) High Fluence LED-RL significantly decreases the migrations speed of HDFs for up to 12 hours. 320 and 640 J/cm2 of LED-RL resulted in significantly decreased migration speed relative to matched control for 12-hours after irradiation when measured at 4 hour intervals. Pictured above, relative cellular migration speed for HDFs treated with 320 J/cm2 at 0-4 hours was 81.4 ± 4.8% (p<0.05) and at 12-16 hours was 99.2 ± 4.8%. HDFs treated with 640 J/cm2 at 0-4 hours was 72.8 ± 7.1% (p<0.05) and at 12-16 hours was 98.3 ± 6.5%. (c) HDFs treated with 320 and 640 J/cm2 LED-RL demonstrated increased AKT phosphorylation (S473) compared to matched controls at 4 hours post-irradiation. A representative western blot is shown. (d) AKT phosphorylation was no longer increased at 12 hours post-irradiation. A representative western blot is shown. (e) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) did not significantly alter HDF migration speed when compared with control. Pretreating HDFs with 30 μM LY294002 for 30 minutes resulted in a relative speed of 95.2 ± 4.7% (p = 0.48) compared to untreated control. (f) Pretreatment with 30 μM LY294002 (antagonist of AKT phosphorylation) prevented the inhibitory effects of 640 J/cm2 LED-RL on HDF migration speed. Primary HDFs with or without Ly294002 were treated with HF-LED-RL and their migration speed was assessed used time-lapse video microscopy. 640 J/cm2 LED-RL significantly decreased migration speed (73.3 ± 4.9%, p < 0.0001) versus matched control. Pretreating HDFs with 30 μM LY294002 for 30 minutes blocked the inhibitory effects of 640 J/cm2 LED-RL on migration resulting in a relative speed of 94.4 ± 4.8% (p = 0.62) relative to matched control pretreated with 30 μM LY294002.Data are representative of three repeat experiments in two different HDF strains. Similar temporal results were obtained in the two strains examined. Error bars represent mean ± SEM; *P<0.05.