Gene expression of fibroblasts following light irradiation
Authors: Zhang Y. , Fong C. C. , Tsang C. H. , Yang M. 
Published: September 2010

Introduction

Low‐intensity red laser light therapy has been used to treat various medical problems including wound repair processes and various dermatological diseases. [1–3]. Fibroblasts are important cellular component of wound healing, and it has been reported that red light irradiation stimulated fibroblasts proliferation and promoted procollagen synthesis and release of basic fibroblast growth factor (bFGF) [1–5]. While the evidence of the beneficial effects of low‐intensity red laser light irradiation is mounting, the molecular mechanisms of biomodulation of low‐intensity laser remain poorly understood. In this study, the effects of red light irradiation on fibroblast cell proliferation were studied and the gene expression profiles of human fibroblasts upon irradiation were examined by using cDNA microarrays. Functional analysis of the differentially expressed genes was carried out in order to understand how low‐intensity red light irradiation modulates the behavior of fibroblasts at the molecular level.

Methods and materials

Red light source

An instrument equipped with red light‐emitting (628 nm) diodes (Healthtech Ltd Co., Shanghai, China) was used. The irradiation distance is 0.75 cm.

Cell culture and proliferation assay

Normal human fibroblasts of Hs27 cell line (ATCC, Manassas, VA, USA) were cultured at 37oC in a humidified atmosphere containing 5% CO2, with Dubach's minimal essential medium supplemented with 5% fetal bovine serum, 100 units penicillin, 100 μg streptomycin, and 2 mM glutamine (Gibco BRL, CA, USA). The cells were irradiated with different doses (0, 0.44, 0.88, 2, 4.4, and 8.68 J cm–²) for 3 days and the cell numbers were counted at day 3.

RNA isolation

Fibroblast cells were cultured in 14 cm tissue culture plates and irradiated three times a day with each irradiation at an energy dose of 0.88 J cm–². The cells were allowed to culture for 8 h after the last irradiation. The cells were collected and total RNA was isolated with TRIZOL reagent (Gibco BRL). The total RNA from untreated cells was used as control.

cDNA microarray hybridization

A home‐made cDNA microarray containing 10 k human genes and EST (Human clone set Unigen v.2.0; Incyte Genomics, CA, USA) was used for the study. Total RNA from control or irradiated samples were reverse transcripted to cDNA in the presence of Cy3‐dUTP and Cy5‐dUTP (Amersham Pharmacia Biotech, NJ, USA). Hybridization was carried out at 65oC overnight. Following washing and drying, the slide was scanned with a microarray scanner (ScanArray 4000; GSI Lumonics, MA, USA). Fluorescence ratios (Cy5 over Cy3) were used to determine the relative level of gene expression. Genes showed a greater than 2‐fold induction or repression (Cy5/Cy3 ratios above 2 or below 0.5) were selected for further cluster analysis.

Results and discussion

Effect of red light on human fibroblast proliferation

Cell proliferation assay was first carried out to study the response of HS 27 fibroblasts to different doses of low‐intensity red light irradiation. The cells were irradiated daily with different energy doses of red light‐emitting diodes at 628 nm wavelength for 3 days and the changes in cell number were recorded. The response of the HS 27 fibroblasts to the irradiation showed a curve effect and the cells grew faster than the control at energy doses of 0.44, 0.88, and 2 J cm–², with an optimal proliferation effect at 0.88 J cm–² (Fig. 1). When the energy dose was increased to over 4 J cm–², the cells grew at equal or slightly slower rates than control cells. The growth rate was increased to 25–35% when the cells were cultured in DMEM with 5% FCS.

Expresion profile of Hs17 newborn foreskin fibroblast irradiated by red light using a 10 K human cDNA microarray.

image

Effects of red light on gene expression profile in human fibroblasts

The gene expression profile of human fibroblasts irradiated with red light was obtained by hybridizing the fluorescently‐labeled cDNA from human fibroblasts with and without red light irradiation with a cDNA microarray containing 9982 human genes and EST. A typical microarray image was shown in Fig. 1. Among them, 68 genes were up regulated and 43 genes were down regulated. These genes were classified into 10 clusters according to their functions.

Functional clustering of the genes affected by red light

Cluster 1: Genes related to cell proliferation

The first cluster consists of 9 genes related to cell proliferation and most of them were up‐regulated. Eight of the genes play stimulatory roles in cell proliferation. The down regulation of cullin 1, an inhibitory regulator of the cell cycle, may also enhance cell growth.

Cluster 2: Antioxidant‐related genes

The second cluster includes two genes and both of them were up‐regulated.

Cluster 3: Genes related to apoptosis or stress response

The third cluster consists of five apoptosis or stress related genes. Most of them were down‐regulated following red light irradiation. The up‐regulation of JAK binding protein may also increase the survival ability of the cells.

Cluster 4: Metabolism‐related genes

Twenty genes that mediate metabolism are grouped in the fourth cluster and can be further categorized into 4 sub‐clusters, including genes related to protein and amino acid metabolism, sugar metabolism, lipid metabolism, and respiratory chain and energy metabolism. In the sugar metabolism sub‐cluster, all of the genes were down regulated. In the lipid metabolism subcluster, both apolipoprotein C‐III, and lysophospholipase II were up regulated. All three genes in the sub‐cluster related to energy metabolism and respiratory chain were up‐regulated.

Cluster 5: Genes related to ion channels, transport proteins and membrane potential

There are four up‐regulated genes in this cluster. Up‐regulation of the ATP‐binding cassette (ABCA1) transporter gene may prevent the cells from over‐accumulation of cholesterol and phospholipids. The change in the expression of the other 3 ion channel related genes may be required for cell proliferation.

Cluster 6: Genes related to cytoskeleton, cell motility, extracellular matrix, migration, aggregation, and adhesion

There are 17 genes in this cluster which can be further divided into three sub‐clusters. Most genes in the cluster are functional and/or structural constituents of cell proliferation upon mitogenic stimulation.

Cluster 7: Genes related to DNA synthesis and repair

All 3 genes in this cluster are up‐regulated.

Cluster 8– 10: Genes related to other functions

These clusters include genes related to transcriptional factors (cluster 8), immune/inflammation and cytokine (cluster 9), and genes with known and unknown functions (cluster 10). The roles in which these genes play in fibroblasts irradiated by low‐intensity red light are not clear.

Molecular mechanisms of the effects of red light irradiation on fibroblasts

Among the 10 gene clusters, seven of them were directly or indirectly involved in cell proliferation. Irradiation of low‐intensity red light stimulates cell growth directly through regulation of the expression of cell proliferation related genes and indirectly through regulation of the expression of cell migration and remodeling genes, DNA synthesis and repair genes, ion channel and membrane potential genes, and cell metabolism genes. The irradiation of red light also enhances cell proliferation by suppression of cell apoptosis.

Cell proliferation is a very important factor of wound healing. Irradiation of low‐intensity laser or red light accelerated the healing of skin wound in animal models and in human by increasing the proliferation of cellular components for wound healing, promoting the synthesis of collagen content and improving microcirculation of wounded areas [1, 6]. Fibroblast is one of the important cellular components of wound healing. Several genes related to cell proliferation played major roles in fibroblast proliferation stimulated by red light irradiation. Many of these genes participate in either or both of the p38 MAPK signaling pathway and the PDGF signaling pathway. Because PDGF can increase the transcription and secretion of VEGF by beta‐PDGFR‐expressing endothelial cells [7], the up‐regulation of PDGF not only plays a role in the proliferation of fibroblasts but also takes part in the stimulation of the growth of endothelial cells. The proliferation of endothelial cells can be regarded as a basis for the improvement of microcirculation by red light irradiation. Figure 2 shows the participation of various differentially expressed genes in the signaling pathways that accelerate the proliferation of fibroblast and endothelial cells, eventually leading to wound healing.

The participation of various differentially expressed genes in the signaling pathways that accelerate the proliferation of fibroblast and endothelial cells, eventually leading to wound healing. MAPK11: Mitogen‐activated protein kinase 11, SRF: Serum response factor, PD6F: Platelet derived growth factor, BCR: Breakpoint cluster region, VEGF: Vascular endothelial growth factor.

image

References

1. Schindl, A., Schindl, M., Pernerstorfer‐Schon, H. and Schindl L. Red light laser therapy: a review. J. Investig. Med. 48, 312–326 (2000).

2. Mester, E., Mester, A.F. and Mester A. The biomedical effects of laser application. Lasers Surg. Med. 5, 31–39 (1985).

3. Conlan, M.J., Rapley, J.W. and Cobb, C.M. Biostimulation of wound healing by low‐energy laser irradiation. A review. J. Clin. Periodontol. 23, 492–426 (1996).

4. Lubart, R., Friendmann, H., Peled, I. and Grossmann, N. Light effect on fibroblast proliferation. Laser Ther. 5, 55–57 (1993).

5. Yu, W., Naim, J.O. and Lanzafame, R.J. The effect of laser irradiation on the release of bFGF from 3T3 fibroblasts. Photochem. Photobiol. 59, 167–70 (1994).

6. Reddy, G.K., Stehno‐Bittel, L. and Enwemeka, C.S. Laser photostimulation of collagen production in healing rabbit Achilles tendons. Lasers Surg. Med. 22, 281–7 (1998).

7. Wang, D., Huang, H.J., Kazlauskas, A. and Cavenee, W.K. Induction of vascular endothelial growth factor expression in endothelial cells by platelet‐derived growth factor through the activation of phosphatidylinositol 3‐kinase. Cancer Res. 59, 1464–72 (1999).