Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells

Abstract

Objectives: This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density.

Materials and methods: Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays.

Result: Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible.

Conclusions: These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ.

Keywords: Akt 1; OPG; RANKL; TGF-beta; infrared; low-level laser treatment; mesenchymal stem cell; microarray; protein array.

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Figures

Figure 1

Custom LED array for even illumination of cell cultures A) Photograph of array and culture plates. B) Schematic illustration. Culture plates(6 well, Life Technologies, Grand Island, NY) were removed from culture incubators and exposed to light in a sterile hood at fixed distances. Energy density varied according to time of light exposure based on calculations by P Matthews. Power density was confirmed with laser meter(Ophir, Jerusalem, Isreal).

Figure 2

Test of light effects on cell proliferation. BrdU staining of (A) illuminated(visible red 0.5 joules/cm2) vs. (B) unlit control hBMSC cell cultures. Human bone marrow primary cell culture were seeded at the same initial cell density, then manually counted at 12 hour time points for 60 hours when the cultures were assayed for BrdU labeling.

Figure 3

(A) Venn Diagram comparing two wavelengths of light: IR- infrared (830 nm), and VR- visible red (633 nm) at 4 energy densities ranging from 0.5 to 2.0 J/cm2 (B) Three-Dimensional Venn Diagram showing overlap among the four energy densities (0.5, 1.0, 1.5, 2.0; J/cm2) for 830 nm(IR-infrared) and 633 nm(VR-visible red) wavelengths of light.

Figure 4

Microarray analysis of candidate genes: A) RANKL, B) OPG, C) MMP10 and D) TIMP

Figure 4

Microarray analysis of candidate genes: A) RANKL, B) OPG, C) MMP10 and D) TIMP

Figure 5

Pathway analysis illustrating (A) convergence of TGF beta 1 in VR and (B)emergence of Akt 1 pathways in IR networks. The 126 IR genes, 2,320 VR genes were uploaded into Ingenuity System’s Pathway Analysis 9.0. Red=upregulation; Green=downregulation

Figure 5

Pathway analysis illustrating (A) convergence of TGF beta 1 in VR and (B)emergence of Akt 1 pathways in IR networks. The 126 IR genes, 2,320 VR genes were uploaded into Ingenuity System’s Pathway Analysis 9.0. Red=upregulation; Green=downregulation

Figure 6

Protein array data illustrating components of the TGF beta and Akt 1 pathways. A) TGF beta and related proteins. B). The Y axis is the signal log2 ratio that compares illuminated cultures with control cultures. No change from control cultures=0. A two-fold increase corresponds to a Signal Log Ratio of 1.

Figure 6

Protein array data illustrating components of the TGF beta and Akt 1 pathways. A) TGF beta and related proteins. B). The Y axis is the signal log2 ratio that compares illuminated cultures with control cultures. No change from control cultures=0. A two-fold increase corresponds to a Signal Log Ratio of 1.

Figure 7

Principle component analysis of epithelial(NHEK-Neo) vs. mesenchymal cell (hBMSC) cultures stimulated by LLLT under serum and serum-free conditions. Each axis measures variance between data points of gene sets identified by the Affymetrix HuEx 1.0 ST microarrays for the three different variables: mesenchymal vs. epithelial cell type, treated(IR 2.0 J/cm2) vs. control and serum vs. serum-free media. Large distances between data points suggest dissimilarity(Partek Inc, St. Louis, MO).