Irradiated Human Fibroblasts Research Articles

Anti-inflammatory, cytotoxic and morphological impact of phycocyanin on ultraviolet radiation irradiated human fibroblast cells

Background Recently, the use of natural products as skin photoprotective agents has been in increasing demand. This study investigated the bioactivity of phycocyanin (PC) extracted from Spirulina sp. on human skin fibroblast cell line (CCD-966SK), specifically focusing on apoptosis, necrosis, anti-inflammatory effects, and enzymatic reactions. Methods The first step of this study was cyanobacterial cell culture and the extraction and purification of PC. After that, CCD-966SK cell line was cultivated under normal and UV irradiation. The bioassays included the cytotoxicity measurement, cell viability assay, morphology determination, tumour necrosis factor-α and Interleukin 6 release assays, enzyme activity for superoxide dismutase and glutathione peroxidase as well as malondialdehyde content and the cell-free extract of cyanobacterial stains were assessed. Results The cell viability results showed that as the concentration of the PC increased, the viability of CCD-966SK cell line was reduced, which suggested that the effect of PC on the growth of fibroblast cells was dose dependent. The morphological results indicated that presence of PC in the fibroblast cell culture medium led to a transformation in cell morphology from spindle-shaped to spherical. PC released anti-inflammatory IL-6 and TNF-a cytokines, indicating high inflammation resistance. Furthermore, the findings revealed that PC dramatically reduced the release of superoxide dismutase, glutathione peroxidase, and malondialdehyde from inflammatory cells, with the reduction being more apparent at increasing doses. Conclusions In conclusion, the results indicated that PC inhibit the CCD-966SK cell line by membrane destructor, which led to the increase the leakage of cell constituent and increase enzymes activities.

Human adipose ECM alleviates radiation-induced skin fibrosis via endothelial cell-mediated M2 macrophage polarization

Radiation therapy can lead to late radiation-induced skin fibrosis (RISF), causing movement restriction, pain, and organ dysfunction. This study evaluated adipose-derived extracellular matrix (Ad-ECM) as a mitigator of RISF. Female C57BL/6J mice that were irradiated developed fibrosis, which was mitigated by a single local Ad-ECM injection, improving limb movement and reducing epithelium thickness and collagen deposition. Ad-ECM treatment resulted in decreased expression of pro-inflammatory and fibrotic genes, and upregulation of anti-inflammatory cytokines, promoting M2 macrophage polarization. Co-culture of irradiated human fibroblasts with Ad-ECM down-modulated fibrotic gene expression and enhanced bone marrow cell migration. Ad-ECM treatment also increased interleukin (IL)-4, IL-5, and IL-15 expression in endothelial cells, stimulating M2 macrophage polarization and alleviating RISF. Prophylactic use of Ad-ECM showed effectiveness in mitigation. This study suggests Ad-ECM's potential in treating chronic-stage fibrosis.

Bystander signals from low- and high-dose irradiated human primary fibroblasts and keratinocytes modulate the inflammatory response of peripheral blood mononuclear cells.

Irradiated cells can propagate signals to neighboring cells. Manifestations of these so-called bystander effects (BEs) are thought to be relatively more important after exposure to low- vs high-dose radiation and can be mediated via the release of secreted molecules, including inflammatory cytokines, from irradiated cells. Thus, BEs can potentially modify the inflammatory environment of irradiated cells. To determine whether these modifications could affect the functionality of bystander immune cells and their inflammatory response, we analyzed and compared the in vitro response of primary human fibroblasts and keratinocytes to low and high doses of radiation and assessed their ability to modulate the inflammatory activation of peripheral blood mononuclear cells (PBMCs). Only high-dose exposure resulted in either up- or down-regulation of selected inflammatory genes. In conditioned culture media transfer experiments, radiation-induced bystander signals elicited from irradiated fibroblasts and keratinocytes were found to modulate the transcription of inflammatory mediator genes in resting PBMCs, and after activation of PBMCs stimulated with lipopolysaccharide (LPS), a strong inflammatory agent. Radiation-induced BEs induced from skin cells can therefore act as a modifier of the inflammatory response of bystander immune cells and affect their functionality.

Puerarin Reduces Oxidative Damage and Photoaging Caused by UVA Radiation in Human Fibroblasts by Regulating Nrf2 and MAPK Signaling Pathways.

Fibroblasts account for more than 95% of dermal cells maintaining dermal structure and function. However, UVA penetrates the dermis and causes oxidative stress that damages the dermis and accelerates skin aging. Puerarin, the main active ingredient of Puerariae lobata, has been demonstrated to withstand oxidative stress caused by a variety of factors. However, there are limited findings on whether puerarin protects fibroblasts from UVA-induced oxidative stress damage. The effects of puerarin on human skin fibroblasts (HSF) under UVA-induced oxidative stress were investigated in this study. It is found that puerarin upregulates antioxidant enzymes' mRNA expression level and their content through modulating the KEAP1-Nrf2/ARE signaling pathway, thus improving cell antioxidant capacity and successfully eliminating UVA-induced reactive oxygen species (ROS) and lipid oxidation product malondialdehyde (MDA). Additionally, puerarin blocks the overexpression of human extracellular signal-regulated kinase (ERK), human c-Jun amino-terminal kinase (JNK), and P38, which downregulates matrix metalloproteinase 1 (MMP-1) expression and increases type I collagen (COL-1) expression. Moreover, preliminary research on mouse skin suggests that puerarin can hydrate, moisturize, and increase the antioxidant capacity of skin tissue. These findings suggest that puerarin can protect the skin against photoaging.

Damage prevention effect of milk-derived peptides on UVB irradiated human foreskin fibroblasts and regulation of photoaging related indicators

Long-term exposure to UVB can trigger acute inflammation of the skin and lead to skin photoaging. To scrutinize the anti-photoaging functions of peptides obtained from milk, the physicochemical including molecular weight and amino acid compositions were first analyzed. Totally 267 peptides were screened out and identified by PEAKS X software, and then evaluated through Peptide Ranker and BIOPEP-UMW. Six peptides with the highest antioxidant ability and relative abundance were selected. This study was then conducted in UVB-damaged human foreskin fibroblasts with proadministration of peptides. The results indicated that at concentrations of 0.08–0.10 mg/mL, milk-derived peptides could realize a damage prevention effect through inhibiting the generation of reactive oxygen species (ROS) and lipid peroxidation malondialdehyde (MDA). Also, these peptides were found to promote the photoaging related enzyme activities of superoxide dismutase (SOD) and catalase (CAT), while to block the production of matrix metalloproteinases-1. Through this study, we found that milk-derived peptide mixture is effective in preventing photoaging damage. Milk-derived peptides found in this study could serve as raw materials for future development of antioxidant functional foods.

Insight into Seasonal Change of Phytochemicals, Antioxidant, and Anti-Aging Activities of Root Bark of Paeonia suffruticosa (Cortex Moutan) Combined with Multivariate Statistical Analysis.

Chemical compositions, antioxidants, and anti-aging activities of Cortex Moutan (CM), from different collection periods and different producing areas, were measured and compared in order to obtain excellent CM extracts. The bioactivities of CM extracts were examined by an in vitro antioxidant method and a UVB irradiated human dermal fibroblast (HDF) model. Phytochemical properties were obtained from ultra-fast liquid chromatography quadrupole time-of-flight mass spectrometry (UFLC-Q-TOF-MS) prior to the multivariate statistical analysis. As for the results, the extracts of Heze CM (HZCM) and Luoyang CM (LYCM) collected in June had better in vitro antioxidant activities, significantly increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and reduced the content of malondialdehyde (MDA), compared to other CM extracts. HZCM and LYCM extracts could upregulate the relative expression of SOD and GSH-Px mRNA. The extract of HZCM collected in June could significantly repress the production of matrix metalloproteinase 1 (MMP-1) and improve the production of procollagen type I (PCOL)-I in UVB irradiated HDF. In total, 50 compounds, including 17 monoterpenoids, 19 flavonoids, 13 phenols, and 1 amino acid were identified or tentatively identified in the CM extracts. Gallic acid, p-hydroxybenzoic acid, oxypaeoniflorin, paeoniflorin, 1,2,3,4,6-O-pentagalloyl glucose, and paeonol were predominant compounds in the CM extracts. Taken together, CM collected from April to September had better antioxidant and anti-aging effects for external usage.

Radiation-induced lipoprotein-associated phospholipase A2 increases lysophosphatidylcholine and induces endothelial cell damage

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1β, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.

Effect of different irradiation doses on human and murine fibroblasts used as feeder layer for keratinocytes’ culture

Background & Aim The establishment of human keratinocytes’ culture (KTs) to obtain human Tissue Engineered Skin Substitutes is under research nowadays. Best results have been achieved culturing KTs on a feeder layer of irradiated 3T3 murine fibroblasts (i3T3s). Nevertheless, molecular effects of murine cells on KTs is unknown and for this reason, irradiated human fibroblasts (iHFs) have been studied. The objective of this study is to evaluate the effect of different irradiation doses on cell reducing power and cell proliferation of both fibroblasts. Methods, Results & Conclusion Methods 3T3s were obtained from a commercial vial of 3T3-Swiss Albino. HFs were isolated from human skin biopsies. After culturing and final recovery, both cell types were irradiated to doses of 50 and 100 Gy to constitute the feeder. Non-irradiated cells were used as control. Each condition was cultured 15 days in two 24-well plates (2 cm2/well). In each plate, cell densities cultured in triplicate were: 7,500 cells/cm2 and 10,000 cells/cm2. One plate was used for reducing power analysis; using a dilution of PrestoBlue™ (10%). The other plate was used for cell proliferation analysis; using Trypan Blue solution. N for each plate was 3 and a value of p Results Analysis of each type of feeder (Figs. 1-2) revealed significant differences at Days 10 and 15, between both irradiation doses and non-irradiated cells. In the case of iHFs, irradiation dose had a significant effect on cell reducing power, mainly at Day 15. Analysis of i3T3s revealed that the effect of irradiation on reducing power was extremely significant from Day 3 to 15, but no dose-dependent. A comparative analysis (Fig. 3) indicated that the number of non-irradiated cells after 15 days was higher in the case of 3T3s than HFs (644,620 cells vs 143,740 cells), but cell reducing power was lower in the case of murine fibroblasts (72.33% vs 80.67%). In the case of irradiated cells, iHFs were more resistant to irradiation in both, cell reducing power (27.5% vs 4.92%) and cell proliferation (5,962 cells vs 1,351 cells) at Day 15. Conclusion No differences exist considering initial cell density or irradiation dose in both feeders. Comparative analysis revealed that irradiation process is more effective in 3T3s than in HFs. This study needs to be completed with the incorporation of KTs on the different feeder conditions analyzed. Supported by PI17/02083, SAS PI-0458-2016 and PhD fellowship FI18/00269–Alvaro Sierra-Sanchez).

The Role of Telomerase in Radiation-Induced Genomic Instability.

Findings from previous studies have suggested that the telomerase system is involved in radiation-induced genomic instability. In this study, we investigated the involvement of telomerase in the development and processing of chromosomal damage at different cell cycle stages after irradiation of human fibroblasts. Several response criteria were investigated, including cell survival, chromosomal damage (using the micronucleus assay), G2-induced chromatid aberrations (using the conventional G2 assay as well as a chemically-induced premature chromosome condensation assay) and DNA double-strand breaks (DSBs; using γ-H2AX, 53BP1 and Rad51) in an isogenic pair of cell lines: BJ human foreskin fibroblasts and BJ1-hTERT, a telomerase-immortalized BJ cell line. To distinguish among G1, S and G2 phase, cells were co-immunostained for CENP-F and cyclin A, which are tightly regulated proteins in the cell cycle. After X-ray irradiation at doses in the range of 0.1-6 Gy, the results showed that for cell survival and micronuclei induction, where the overall effect is dominated by the cells in G1 and S phase, no difference was observed between the two cell types; in contrast, when radiation sensitivity at the G2 stage of the cell cycle was analyzed, a significantly higher number of chromatid-type aberrations (breaks and exchanges), and higher levels of γ-H2AX and of Rad51 foci were observed for the BJ cells compared to the BJ1-hTERT cells. Therefore, it can be concluded that telomerase appears to be involved in DNA DSB repair processes, mainly in the G2 phase. These data, taken overall, reinforce the notion that hTERT or other elements of the telomere/telomerase system may defend chromosome integrity in human fibroblasts by promoting repair in G2 phase of the cell cycle.

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI.

Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.

Low-level PDT treatment modulated photoaging mediated by UVA irradiation through regulating Bach2.

To investigate low-level ALA-PDT (Aminolevulinic acid photodynamic therapy) effects on photorejuvenation in vitro and in vivo, exploring the basic mechanism of Bach2 involved in PDT treatment in photoaging. Photoaging model was established by UVA chronic irradiation in human fibroblasts and mice skins. Cell viability was determined by MTS assay and cell senescence was detected by SA-β-gal activity. PDT treatment and Bach2 knockdown with adenovirus in fibroblasts were confirmed by Western blot. UVA chronic irradiation induced photoaging in vitro and in vivo. Treatment of low-level PDT reduced photoaging by decreasing SA-β-gal activity and cell senescence-related proteins levels of p16 and p21 in fibroblasts. Moreover, low-level PDT treatment accompany with Bach2 accumulation increased in fibroblasts and in mice skin tissues. Bach2 knockdown with adenovirus induced cell senescence and Bach2 depletion with PDT treatment some extent decreased SA-β-gal activity, but was with no significant change of Bach2 itself and p16 protein levels in fibroblasts. Low-level PDT treatment decreased skin photoaging which might be through up-regulating Bach2.

Adipocyte Stem Cells Ameliorate Total Body Irradiation-Induced Hematopoietic Syndrome and Late Radiation Fibrosis

Radiation therapy to the head and neck, chest wall or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. In vitro Transwell co-cultures contained a bottom layer of: 1) irradiated human foreskin fibroblasts (HFFs), 2) mouse cell lines derived from fibrosis biopsies; the upper layer contained freshly prepared mouse or human ASCs. We quantitated fibrosis-related gene transcripts in lower layer cells and regulatory cytokines in upper layer cells by quantitative real time (qRT) PCR. Female C57BL/6J mice received 9.25 Gy by TBI, treatment group (n=12) received ASC while control (n=12) received I.P saline injection at 24 h. Other female C57BL/6J mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electrons. Irradiated and contralateral unirradiated flank tissue was tested for fibrosis related gene transients at day 14 post irradiation (PI). Subgroups had irradiated and control sites injected with ASCs from gender mismatched luciferase+ GFP+ mice, and were imaged in real time. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. Transwell coculture revealed significant down regulation of profibrotic genes (TGF β, and Col 1-4) in irradiated cells. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was prominent. Addition of human recombinant HGF to irradiated HFFs significantly down regulated pro-fibrotic gene transcripts. Irradiated HFF revealed high expression of HGF receptor c-Met. Intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (p = 0.047). RF was detected in vivo at day 14 and increased by day 28, and confirmed by histological staining for collagen by Masson’s Trichrome. At day 14, there was upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), Collagen 1 (400 fold), Collagen3 (500 fold) and collagen4 (500 fold) in irradiated compared to non-irradiated tissue. At day 28, irradiation-induced a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral non-irradiated limb. A single 1 x 106 ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). ASCs ameliorate TBI acute lethality. HGF secreted by ASCs reduces RF in vitro and in vivo. HGF and other secreted and cell contact regulators in adipose tissue based cell therapy of both the hematopoietic syndrome and radiation fibrosis are being elucidated.

Grafting of an autologous tissue-engineered human corneal epithelium to a patient with limbal stem cell deficiency (LSCD)

PurposeIn this study, we evaluated the feasibility of recovering the corneal surface integrity in a patient suffering from unilateral LSCD through the transplantation of cultured autologous corneal epithelial cells. MethodsHuman corneal epithelial cells (HCECs) were isolated from a limbal biopsy of the contralateral eye of a patient with unilateral LSCD and cultured in monolayer in the presence of an irradiated human fibroblasts feeder layer (iHFL). To produce a cultured autologous corneal epithelium (CACE), HCECs were seeded on a fibrin substrate and maintained in culture until confluence. The in vitro obtained CACE was then used to treat the affected eye of the patient. Two years later, a successful penetrating keratoplasty was performed. ResultsEfficient restoration of the corneal epithelium was achieved following transplantation of CACE indicating probable re-colonization of the cornea by stem cells. Corneal transparency was restored after removing the scarred stroma by performing a penetrating keratoplasty. ConclusionCACE produced in vitro was shown to restore a normal corneal surface capable of sustaining a viable and clear penetrating keratoplasty and reestablished a near normal vision in a unilateral LSCD patient.

Adipose-Derived Stem Cell Therapy Ameliorates Ionizing Irradiation Fibrosis via Hepatocyte Growth Factor-Mediated Transforming Growth Factor-β Downregulation and Recruitment of Bone Marrow Cells.

Radiation therapy to anatomic regions, including the head and neck, chest wall, and extremities, can produce radiation-induced fibrosis (RIF). To elucidate the cellular and molecular mechanism(s) involved in RIF, female C57BL/6J mice were irradiated to the right flank to 35 Gy in single fraction using 6 Mv electrons. Radiation fibrosis was detected by day 14, was increased by day 28, and confirmed by Masson's trichrome histological staining for collagen. Biopsied tissue at day 14 showed an increase in expression of fibrosis-related genes including transforming growth factor-β (TGF-β) and collagens 1-6. A single adipose-derived stem cell (ASC) injection on day 28 at the irradiated site decreased by day 40: epithelial thickness, collagen deposition, and significantly improved limb excursion compared with irradiated controls. Noncontact transwell coculture of ASCs above a monolayer of irradiated human foreskin fibroblasts downregulated fibrosis-related genes TGF-β, connective tissue growth factor, interleukin-1, NF-kB, tumor necrosis factor, and collagens 1-6. Hepatocyte growth factor (HGF) secreted by ASCs was identified as a novel mechanism by which ASCs exert antifibrotic effects by downregulating fibrotic gene expression in irradiated cells and recruiting bone marrow cells to the irradiated site. In conclusion, these data indicate a mechanistic role of HGF secreted by ASCs in reducing RIF. Stem Cells 2019;37:791-802.

The Effects of Ultraviolet Light-Emitting Diodes with Different Wavelengths on Periodontopathic Bacteria In Vitro.

Objective: The aim of this study was to examine effects of recently developed ultraviolet light-emitting diodes (UV LEDs) wavelengths on in vitro growth and gene expression of cultural periodontopathic bacteria, and on viability of experimental gingival fibroblasts. Materials and methods: Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans, and Streptococcus oralis were irradiated by UV LEDs (265, 285, 310, 365, and 448 nm) at 600 mJ/cm2 and grown anaerobically in vitro. The colony forming units were counted after 1 week. Cell morphology was observed using a scanning electron microscope (SEM). Quantitative real-time polymerase chain reaction was performed to investigate gene expression changes by 310 nm irradiation. Viability of the irradiated human gingival fibroblasts was evaluated using WST-8 assay. Results: Both 265 and 285 nm resulted in the complete death of bacteria and fibroblasts, whereas 310 nm caused partial killing and suppression of bacterial growth and much less damage to the fibroblasts in vitro. Both 365 and 448 nm resulted in no significant change. SEM showed that P. gingivalis cells gradually degraded from day 2 or 3 and were severely destructed on day 5 for 265, 285, and 310 nm. The 310 nm irradiation transiently suppressed the transcripts of SOS response- and cell division-relative genes. Conclusions: Both 265 and 285 nm may induce powerful bactericidal effects and severe fibroblast phototoxicity, and 310 nm may induce partial killing or growth suppression of bacterial cells with much less fibroblast phototoxicity. UV lights may have potential for bacterial suppression, with situations dependent on wavelength, in periodontal and peri-implant therapy.

Adipocyte Stem Cells Ameliorate Total Body Irradiation Induced Hematopoietic Syndrome and Late Radiation Fibrosis

Radiation therapy to the head and neck, chest wall or extremities can result in late radiation fibrosis (RF). Several case reports suggest that injection of autologous adipose tissue stem cells can ameliorate RF. We sought to elucidate the cellular and molecular mechanism(s) involved. In vitro Transwell co-cultures contained a bottom layer of: 1) irradiated human foreskin fibroblasts (HFFs), 2) mouse or 3) human cell lines derived from fibrosis biopsies; the upper layer contained freshly prepared mouse or human ASCs. We quantitated fibrosis-related gene transcripts in lower layer cells and regulatory cytokines in upper layer cells by quantitative real time (qRT) PCR. Female C57BL/6J mice (n=12) received 9.25 Gy by TBI, treatment group (n=12) received ASC while control (n=12) received I.P. saline injection at 24 h. Other female C57BL/6J mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electrons. Irradiated and contralateral unirradiated flank tissue was tested for fibrosis related gene transients at days 1, 14, and 21 post-irradiation (PI). Subgroups had irradiated and control sites injected with ASCs from gender-mismatched luciferase+ GFP+ mice, and were imaged in real time. Fibrosis was quantitated by histologic staining for collagen and range of limb motion measurements. Transwell ASCs demonstrated significant down regulation in lower level cells of pro-fibrotic genes (including collagen 1-4, and TGF β) in unirradiated acutely irradiated and irradiation- fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was prominent. Addition of human recombinant HGF to irradiated HFFs significantly down regulated pro-fibrotic gene transcripts. Intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (p = 0.047). RF was detected in vivo at day 14 and increased by day 28, and confirmed by histological staining for collagen by Masson’s Trichrome. At day 14, there was upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), Collagen 1 (400 fold), Collagen3 (500 fold) and collagen4 (500 fold) in irradiated compared to non-irradiated tissue. At day 28, irradiation induced a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p < 0.0001) degrees in the contralateral non-irradiated limb. Single 1 x 106 ASCs injection day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). ASCs ameliorate TBI acute lethality. HGF secreted by ASCs reduces RF in vitro and in vivo. HGF and other secreted and cell contact regulators in adipose tissue based cell therapy of both the hematopoietic syndrome and radiation fibrosis are being elucidated.

Abstract 980: Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF)

Abstract Radiation therapy doses above 50Gy to head and neck, breast cancer and soft tissue sarcomas can result in the rare late complication of RIF, which can significantly impair function. Several published case studies suggest injection of fibrotic sites with autologous adipose tissue stem cells can ameliorate RIF. In this study, we elucidated the molecular mechanism in an in vitro co-cultivation system and in vivo using a mouse model of hind limb fibrosis. In vitro Transwell co-cultures contained a bottom layer of irradiated human foreskin fibroblasts (HFFs), mouse or human cell lines derived from clinical fibrosis biopsies, and an upper layer containing freshly prepared ASCs. We quantitated levels of fibrosis -related genes in the lower layer cells and regulatory cytokines in the upper layer cells using quantitative real-time (qRT) PCR. In vivo studies: Female C57BL/6 mice were irradiated to the right flank to 35Gy in single fraction with 6Mv electron beam. The irradiated and contralateral unirradiated flank tissue was monitored for expression of fibrosis related genes at days 1 and 14 post irradiation (PI). Fibrosis was confirmed by histologic staining for collagen and range of limb motion measurements. Irradiated fibrotic sites were injected at day 28 with ASCs from luciferase+ GFP+ mice. RIF was uniformly detected at day 14 and amplified by day 28, and confirmed by histologic staining for collagen by Masson's Trichrome. At day 14 PI there was clear upregulation in biopsied tissue of fibrosis -related genes: TGF β (500 fold), CTGF (60 fold), collagen 1 (400 fold), collagen3 (500 fold) and collagen4 (500 fold) compared to nonirradiated contralateral hind limb tissue. Irradiation induced by day 28 a reduction in limb excursion with a range of limb extension of 11.4 ± 2.7 degrees compared to 57.0 ± 2.5 (p &amp;lt; 0.0001) degrees in the contralateral nonirradiated limb. A single ASCs injection at day 28 significantly restored the limb excursion to 42.5 ± 2.5 degrees (p = 0.0013). To measure possible systemic radiation mitigatory effects of ASCs PI, a single intraperitoneal injection of 1 million ASCs at 24 hrs after 9.25 Gy total body irradiation significantly increased mouse survival at 30 days (P = 0.047). In transwell co-cultures ASCs demonstrated significant downregulation of profibrotic genes (including collagen 1-4, and TGF β) in both acutely irradiated and irradiation-fibrosis tissue derived cell lines. Among the genes expressed in upper layer ASCs, hepatocyte growth factor (HGF) was a key secreted mediator in co-culture models. Addition of human recombinant HGF to irradiated HFFs significantly downregulated profibrotic gene transcripts. Thus, HGF secreted by ASCs is involved in the reduction of RIF in vivo and with in vitro models of radiation fibrosis. Further studies should elucidate the mechanism of HGF and other secreted and cell contact regulators of adipose tissue-based cell therapy of radiation fibrosis. Citation Format: Asim Ejaz, Micheal Epperly, Renne Fisher, Xichen Zhang, Moriah Johngrass, Asher Schusterman, Lauren Kokai, Joel Greenberger, Peter Rubin. Molecular basis of adipose-derived stem cell (ASCs) therapy for management of radiation-induced fibrosis (RIF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 980.

Genetic and epigenetic changes in clonal descendants of irradiated human fibroblasts

To study delayed genetic and epigenetic radiation effects, which may trigger radiation-induced carcinogenesis, we have established single-cell clones from irradiated and non-irradiated primary human fibroblasts. Stable clones were endowed with the same karyotype in all analyzed metaphases after 20 population doublings (PDs), whereas unstable clones displayed mosaics of normal and abnormal karyotypes. To account for variation in radiation sensitivity, all experiments were performed with two different fibroblast strains. After a single X-ray dose of 2 Gy more than half of the irradiated clones exhibited radiation-induced genome instability (RIGI). Irradiated clones displayed an increased rate of loss of chromosome Y (LOY) and copy number variations (CNVs), compared to controls. CNV breakpoints clustered in specific chromosome regions, in particular 3p14.2 and 7q11.21, coinciding with common fragile sites. CNVs affecting the FHIT gene in FRA3B were observed in independent unstable clones and may drive RIGI. Bisulfite pyrosequencing of control clones and the respective primary culture revealed global hypomethylation of ALU, LINE-1, and alpha-satellite repeats as well as rDNA hypermethylation during in vitro ageing. Irradiated clones showed further reduced ALU and alpha-satellite methylation and increased rDNA methylation, compared to controls. Methylation arrays identified several hundred differentially methylated genes and several enriched pathways associated with in vitro ageing. Methylation changes in 259 genes and the MAP kinase signaling pathway were associated with delayed radiation effects (after 20 PDs). Collectively, our results suggest that both genetic (LOY and CNVs) and epigenetic changes occur in the progeny of exposed cells that were not damaged directly by irradiation, likely contributing to radiation-induced carcinogenesis. We did not observe epigenetic differences between stable and unstable irradiated clones. The fact that the DNA methylation (DNAm) age of clones derived from the same primary culture varied greatly suggests that DNAm age of a single cell (represented by a clone) can be quite different from the DNAm age of a tissue. We propose that DNAm age reflects the emergent property of a large number of individual cells whose respective DNAm ages can be highly variable.

Low-dose irradiation of human fibroblasts leads to delayed acceleration of proliferation of their progeny

We have demonstrated for the first time that a single exposure to γ-radiation at a dose of 3 cGy on HELF-104 human embryonic lung fibroblasts in early passages leads to the delayed stimulation of proliferation of the progeny of irradiated cells by 30–37 days. Moreover, the general changes in dynamics of proliferation after irradiation with low doses (3 and 5 cGy) are more pronounced than after high-dose irradiation (2 Gy). We suggest that this effect may play an important role in the formation of the specific effects of low doses of ionizing radiation, as detected by integral endpoints at higher levels of organization of living matter.

Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station

Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37°C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37°C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.