Laser Irradiation Alters the Expression Profile of Genes Involved in the Extracellular Matrix In Vitro

The extracellular matrix (ECM) forms the basis of every phase in wound healing. Healing may be impaired if some of these components are destroyed. Photobiostimulation has demonstrated a stimulatory response in biological processes.This study aimed to evaluate various genes involved in the ECM, in response to laser irradiation. Isolated human skin fibroblasts were used in three different cell models, namely, normal, normal wounded, and diabetic wounded. Cells were irradiated with 5 J/cm using a continuous wave diode laser emitting at a wavelength of 660 nm and incubated for 48 h. Nonirradiated (0 J/cm) normal and diabetic wounded cells served as the control. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR) was used to determine the expression of 84 genes in a PCR array.There was a significant upregulation of 29 genes in the normal cells, 32 genes in the normal wounded cells, and 18 genes in the diabetic wounded cells as well as a downregulation of 19 genes (normal), 6 genes (normal wounded), and 31 genes (diabetic wounded). Low intensity laser irradiation (LILI) stimulates gene expression in various cell adhesion molecules (CAMs) and extracellular proteins at 660 nm in wounded fibroblasts in vitro.


Introduction
Components of the extracellular matrix (ECM) have been shown to be useful in wound healing [1,2].They form the core of every wound healing phase, and healing may be impaired if any of these components is destroyed [3].In addition, they play a role in angiogenesis, tissue remodelling, and rapid scaffold breakdown [4].The interaction between the ECM and various cells is very important for proper functioning of the cell [5][6][7][8].This interaction could be direct or indirect.Directly, cellular receptors stimulate the ECM or indirectly, through the structural components of the ECM produced by glycoproteins.Cellular activities directed by these interactions are required for wound healing.
For proper cell survival and gene expression in normal wound healing, the environment needs to be at equilibrium with the activity of growth factors, fibroblast interaction, and mechanical forces to ensure normal tissue remodelling [9].Fibroblasts produce most of the molecules in the ECM including proteases, integrins, cytokines, and growth factors during tissue repair which are responsible for late phase tissue remodelling and eventually scarring [10,11].However, the situation is different in chronic wounds due to the disruption of the regular healing process because of tissue damage, biochemical and cellular imbalances, or an underlying pathological state such as diabetes and venous insufficiency.Venous leg ulcers (VLUs) are amongst the major problems in public health and have become an economic burden in most health care services.It is commonly associated with pain, reduces the quality of life, and is even associated with death.It may also cause tiredness and depression [12][13][14].The prevalence of diabetic foot ulcers is approximately 1-2% worldwide [15]; it occurs at any age [16] with an incidence of 3-5% over 65 years [17].
Photobiostimulation, or photobiomodulation, is a noninvasive type of treatment that modulates the treatment of wounds through various cellular or biological processes.It is effective in the visible and near infrared (NIR) spectral range.It functions at wavelengths of 500-1 100 nm and a power output of 10-200 mW [18].The use of photobiomodulation in wound healing has greatly ameliorated various cellular processes affecting different phases of wound healing.Studies have demonstrated the stimulatory effects of photobiomodulation in wounded cell models [19] at 660 nm [20].Studies have also shown that it enhances diabetic wound healing in both rats and mice [21][22][23].Photobiostimulation in the visible and NIR spectral range has been demonstrated to regulate gene expression in human and animal cell cultures, even though its effect was not consistent in all irradiated cells [24].Studies from different areas showed variations in the gene expression profile of 50 cultures of fibroblasts [25].Few studies have exploited the relationship of laser irradiation and gene expression of the ECM in fibroblasts in vitro.Due to previous studies which showed an increase in collagen type I (Col-I) in response to laser irradiation at 660 nm [20], this study aimed to determine the effect of laser irradiation at 660 nm on the gene expression profile of the ECM and its cell adhesion molecules.

Methodology
2.1.Cell Culture.This study was performed on human skin fibroblasts isolated from a consenting adult undergoing abdominoplasty (Linksfield, Sandringham, Johannesburg) (University of Johannesburg Academic Ethics Committee Clearance Reference number 01/06).Cells were seeded into 3.4 cm diameter tissue culture flasks at a density of 6 × 10 5 and routinely cultured according to standard techniques [26].Different cell models, namely, normal (N), normal wounded (NW), and diabetic wounded (DW), were used.To establish an in vitro diabetic model, 17 mM/L D-glucose was added to the media with a base concentration of 5.6 mM/L Dglucose.Thirty minutes prior to irradiation, a sterile 1 mL disposable pipette was used to scratch the monolayer of cells in a streaking motion (i.e., creating a central scratch (CS)).This creates a cell-free zone on either side of the central scratch [27,28].

Laser Irradiation.
Cells were irradiated with 5 J/cm 2 using a continuous wave diode laser emitting at a wavelength Table 3: Gene expression profile in normal (N) cells irradiated at 660 nm.Total RNA from nonirradiated N cells and irradiated N cells were characterised in triplicate.A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation.Fold differences are indicated as upregulation (↑) or downregulation (↓).X denotes the nonsignificant genes in that particular cell model.

Gene symbol
Gene  (for irradiated normal and normal wounded) and diabetic wounded cells (for irradiated diabetic wounded cells) served as the control groups.Cells were irradiated from above, with the culture dish lid off, in 1 mL culture media and in the dark to omit nuisance variables suggestive of polychromatic light that would interfere with the laser effect.The power output was measured using a power meter (FieldMate, 0398D05) at bench level prior to each irradiation, and the readings were used to determine the irradiation time.The temperature of the culture media during irradiation was measured every 2 min and remained less than 32 ∘ C. Cells were incubated for 48 h, and the profile of genes involved in the ECM and cell adhesion molecules were assessed using a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) array.

RNA Isolation and Purity.
Isolation of total RNA from the cells was performed on the Qiagen QIAcube (Whitehead Scientific, Cape Town, South Africa) using the RNeasy Mini Kit (Whitehead Scientific, Cape Town, South Africa, Qiagen, 74104) including QIAshredder homogenizers (Whitehead Scientific, Cape Town, South Africa, Qiagen, 79654).After incubation, cell cultures were detached with TrypLE Express (1 mL/25 cm 2 ) (Life Technologies, Gibco, Invitrogen, 12605-021) and washed with phosphate buffered saline (PBS) to eliminate traces of culture media and then resuspended in 600 L of a guanidine-thiocyanate-containing buffer (RLT buffer) to disrupt the cells, inactivate RNases, and release cellular contents.Within 30 min, 30 L of total RNA was eluted and quantified.The concentration of RNA was established using the Quant-iT RNA Assay Kits (Life Technologies, Johannesburg, South Africa, Invitrogen, Q32852) with the Invitrogen Qubit 2.0 fluorometer (Life Technologies, Johannesburg, South Africa).The ratio between absorbance 260 and 280 nm (A 260 nm/A 280 nm) was used to estimate the sample purity using a UV/Vis spectrophotometer (Separation Scientific, Johannesburg, South Africa, PerkinElmer, Victor 3 ).

cDNA Synthesis.
According to the protocol, a twostep procedure was used to synthesise cDNA using the QuantiTect Reverse Transcription Kit (Whitehead Scientific, Cape Town, South Africa, Qiagen, 205311).Traces of possible contaminating genomic DNA (gDNA) was eliminated from 1 g purified RNA sample using the gDNA Wipeout Buffer for 2 min at 42 ∘ C. RNA was then reverse-transcribed using a reverse transcription (RT) master mix.Six microliters of RT master mix was added to the reaction mixture to give a final volume of 20 L.The mixture was then incubated for 30 min at 42 ∘ C and thereafter 3 min at 95 ∘ C to terminate the reaction.One microliter of sample was used to estimate the purity as stated earlier.Samples were stored on ice to proceed directly with real-time qPCR or stored at −20 ∘ C.

Gene Expression Profiling.
Real-time qPCR was performed using the SABiosciences RT 2 profiler PCR array (Whitehead Scientific, Cape Town, South Africa, PAHS-01321Z) which profiled 84 genes (

Results
Irradiation of N cells with 660 nm resulted in the significant upregulation of 29 genes and downregulation of 19 genes (Table 3).Irradiation of NW cells with 660 nm resulted in the significant upregulation of 32 genes and downregulation of 6 genes (Table 4).Irradiation of DW cells with 660 nm resulted in the upregulation of 18 genes and downregulation of 31 genes (Table 5).A summary of the results is presented in Table 6.

Discussion and Conclusion
ECM components are very useful in different aspects of wound healing.The ECM interacts with various cells and growth factors in cell proliferation, influencing migration, cell differentiation, and regulating several biological responses [1,5,6,29,30].The effects shown by different ECM components depend on the stage of the wound and are determined by the interactions between the cells and growth factors [29].There is great need for gene expression profiling in the ECM following laser irradiation to be exploited.In this study, 84 genes related to the ECM were studied in various models.Photoirradiation was shown to stimulate gene expression 48 h after incubation in irradiated N, NW, and DW cells as compared to their respective controls.The genes, either up-or downregulated, are functionally grouped depending on their pathways in the ECM.
In the present study, four main CAM families were mediated following irradiation at 660 nm.They include cadherins, integrins, selectins, and immunoglobulin CAM (Ig-CAM).The cadherin family are mainly calcium-dependent glycoproteins containing an extracellular domain, a transmembrane domain, and an intracellular domain [31].Cadherins and integrins form the main cell-surface transmembrane receptors and are involved in modulating cell-cell and cell-matrix adhesion.They function in various cellular events, namely, cell migration, proliferation, survival, differentiation, and modulation of gene expression profiling [32,33].In irradiated N cells, CTNND2 was upregulated and CDH1, CTNNA1, and CTNND1 were downregulated at 660 nm; irradiated NW cells showed downregulation of CTNND2; and DW cells showed an upregulation of CDH1 and CTNND2, while CTNND1 was downregulated.
Selectins consist of an extracellular domain with a calcium-dependent lectin domain, an epidermal growth factor domain, and a hydrophobic transmembrane domain [35,36].Selectins expressed in response to laser irradiation in N cells included CLEC3B, while NW cells showed an upregulation in SELE, SELL, and SELP and in DW cells there was an upregulation in SELL.
Ig-CAM contains an extracellular domain with FN repeats, a transmembrane domain, and an intracellular domain [31,35].These domains bind with proteins of the ECM, namely, collagen, LMN, and FN, as well as certain integral cell-surface proteins [31].Members of the Ig-CAM family expressed in response to LILI were CD44, FN1, NCAM1, PECAM1, SGCE, THBS1, THBS2, SPP1, VTN, VCAM1, and CNTN1.In irradiated N cells, CD44, FN1, NCAM1, THBS1, and THBS2 were upregulated, while SGCE, VCAM1, and SPP1 were downregulated following irradiation at 660 nm as compared to nonirradiated N cells.In irradiated NW cells, Table 5: Gene expression profile in diabetic wounded (DW) cells irradiated at 660 nm.Total RNA from nonirradiated DW cells and irradiated DW cells were characterised in triplicate.A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation.Fold differences are indicated as upregulation (↑) or downregulation (↓).X denotes the nonsignificant genes in that particular cell model.

Gene symbol Gene description
Gene  FN1, PECAM1, THBS1, and THBS2 were upregulated, while SGCE and VTN were downregulated at 660 nm as compared to nonirradiated N cells.In irradiated DW cells, CNTN1 and VCAM1 were upregulated, while CD44, FN1, THBS1, and SPP1 were downregulated at 660 nm as compared to nonirradiated DW cells.Other adhesion molecules expressed in response to LILI were HAS1, VCAN, TNC, KAL1, and CTGF.In this study, KAL1 was upregulated while TNC, VCAN, and CTGF were downregulated in irradiated N cells.
In irradiated NW cells, HAS1 and KAL1 were upregulated and TNC was downregulated.In irradiated DW cells, KAL1 was upregulated at 660 nm as compared to nonirradiated DW cells.ECM proteins including collagen, LMN, EI, proteoglycans, and FN have both adhesive and structural functions.The ECM maintains skin integrity and homeostasis and interacts with several structural and extracellular proteins.Collagen is encoded for by more than 42 genes [29,37].Some of the collagen molecules are formed through the interaction between FN and integrins [38,39].Collagens are extracellular proteins produced mainly by fibroblasts, divided into two main classes, namely, the nonfibril forming (collagens types IV, VI, VII, and XI) and the fibril forming collagens distinguished by their triple helix (collagens types I, II, III, V, and XI).Their main function is to maintain the structural integrity of various tissues and to strengthen and reorganise the ECM [40].In this study, COL1A1, COL4A2, COL5A1, COL6A1, COL6A2, COL8A1, and COL12A1 were upregulated in irradiated N cells.In irradiated NW cells, COL1A1, COL5A1, COL7A1, COL8A1, and COL12A1 were upregulated, while in DW cells COL11A1 and COL14A1 were upregulated and COL5A1, COL6A1, COL6A2, COL7A1, COL12A1, and COL16A1 were downregulated.
LMNs are basement membrane proteins made up of three nonidentical chains.They are associated with cell adhesion, differentiation, migration, matrix organisation, and signal transduction.LAMA1 was upregulated in irradiated N cells, while LAMA2 and LAMA3 were downregulated.In irradiated DW cells LAMA3 was upregulated and LAMA1, LAMB3, and LAMC1 were downregulated.Other matrix associated proteins, such as secreted protein, acidic, and cysteine-rich (encoded for by SPARC), spastic paraplegin 7 (encoded for by SPG7), and extracellular matrix protein 1, were also evaluated.SPARC is associated with cell structure organisation, cell migration, and ECM synthesis [41,42].SPG7 is involved in the breakdown of incorrectly folded proteins intracellular motility, membrane trafficking, and organelle biogenesis [43].
ECM1 is part of a cluster of genes involved in epidermal differentiation.Irradiation of N and NW cells to 660 nm resulted in an upregulation of SPARC and SPG7 and a downregulation in ECM1.Analysis of the gene profile of irradiated DW cells revealed a significant downregulation of SPARC and SPG7.Significantly increased gene expression of the constituents of the basement membrane was observed at 660 nm in N and NW cells, while DW cells showed a decrease in gene regulation, with most of the genes downregulated.
These results demonstrated changes in gene expression within the different irradiated N, NW, and DW cell models.The genetic profile seen in the N cell model is a normal response of fibroblast cells to laser irradiation at 660 nm with 5 J/cm 2 .On the other hand, cells in the NW and DW models have been stressed and compromised in some way, and the genetic profile seen in these cells is a response of wounded/stressed fibroblast cells to laser irradiation at 660 nm.Mechanical modulation of these cells would increase upregulation of ECM components, ECM-specific receptors, and enhanced expression of several cytokines and growth factors in a time-dependent manner [49][50][51].The present study showed that DW cells had a significantly downregulated gene expression profile as compared to N and NW cells when irradiated at 660 nm.The downregulation of most of the genes in DW cells is probably due to the dysfunctioning of the ECM exhibited in chronic wounds as a result of Table 6: Summary of the various genes significantly expressed and their pathways in normal (N), normal wounded (NW), and diabetic wounded (DW) cells.Fold differences are indicated as upregulation (↑) or downregulation (↓).X denotes the nonsignificant genes in that particular cell model.
Gene symbol hyperglycaemia.Also in chronic wounds, the inflammatory phase is normally delayed, which promotes increased levels of proteases such as MMPs, causing destruction of the ECM, and damages growth factors as well as receptors essential in the healing process.This also results in a lack of integrins which bind to FN to enhance migration, and hence the decrease in migration [52,53].Furthermore MMP3, MMP7, MMP9, MMP11, and MMP13 were upregulated in DW cells irradiated at 660 nm.This is possibly due to the fact that degraded collagen molecules do not interact properly enabling a disorganised and weak ECM, increasing the levels of some MMPs in chronic wounds [54].This corresponds with the decrease in Col-I seen in these cells [21].However, there was a significant upregulation of some of the collagens and other essential ECM proteins, which is in line with the increase in collagen seen in irradiated DW cells [20].
In conclusion, photobiomodulation at a wavelength of 660 nm enhances gene expression of proteins involved in the ECM.The profile is dependent on the culture conditions and stressors placed on the cells.Increased glucose concentration in the culture media was associated with impaired gene regulation, which could be accountable for the poor response of these cells seen in wound healing.Previous studies have not exploited the role of LILI in gene expression of proteins in the ECM using fibroblast cells in vitro.This study was able to show the gene profile in normal and diabetic wound healing in vitro.The results also confirm the very important role exhibited by cell adhesion molecules (CAMs), integrins, ECM proteins, proteases, and inhibitors in wound healing.Therefore LILI mediated gene expression in wounded fibroblasts through paracrine and autocrine interactions to enhance wound healing.Further work on the molecular advances of gene modulation and their receptors will elucidate the therapeutic importance of LILI.

Table 2
). Ninety-two microliters of PCR water (Diethyl Pyrocarbonate, DEPC free) was added to thawed cDNA (19 L) giving a final volume of 111 L.One hundred and two microliters of diluted cDNA was added to the ready-to-use 2x SABiosciences RT 2 qPCR master mix (330521), and then 1 248 L of PCR water was added to give a total volume of 2 700 L.Components were mixed and 25 L of the experimental cocktail was dispensed into each well of the 96-well plate.The sealed PCR plates were centrifuged at 1000 g (Separation Scientific, Johannesburg, South Africa, Thermo Scientific, Heraeus Labofuge 400) for 1 min to remove any bubbles and run in the preset real-time thermocycler (Anatech, Randburg, South Africa, Stratagene

Table 4 :
Gene expression profile in normal wounded (NW) cells irradiated at 660 nm.Total RNA from nonirradiated N cells and irradiated NW cells were characterised in triplicate.A fold difference >1 is considered as gene upregulation, while a fold difference <1 is considered as gene downregulation.Fold differences are indicated as upregulation (↑) or downregulation (↓).X denotes the nonsignificant genes in that particular cell model.

Table 4 :
Continued.Mx3000p).The thermocycler profile setting was 10 min at 95 ∘ C for 1 cycle and 15 s at 95 ∘ C and 1 min at 60 ∘ C for 40 cycles.The software was also programmed to do a melt or dissociation curve at the end of the run to ensure the amplification of a single product for each gene.The threshold cycle (  ) values were imported into an Excel spreadsheet (Available from the SABiosciences website: http://www.sabiosciences.com/)which normalised the results against the 5 housekeeping genes (ACTB, B2M, GAPDH, HPRTI, and RPLPO).In addition, the relative gene expression (ΔΔ  ) and fold change (2 −ΔΔ  ) were also calculated.Prior to data analysis, all the   values of the controls were examined to ensure proper functioning of the PCR array and preceding steps (positive PCR control,   value of 20 ± 2; genomic DNA control,   value of >35).A fold change of >1 was reported as fold upregulation and a fold change <1 was reported as fold downregulation.
2.6.Statistical Analysis.Experiments were repeated three times ( = 3).Student's -test was analysed based on the replicate fold change for each gene in both the test and the control groups by the SABiosciences Excel-based data analysis template and reported as significant if  < 0.05.Results are represented in Tables3 to 6.