Transcriptomic Profiling and Pathway Analysis of Mesenchymal Stem Cells Following Low Dose-Rate Radiation Exposure

Low dose-rate radiation exposure can occur in medical imaging, as background from environmental or industrial radiation, and is a hazard of space travel. In contrast with high dose-rate radiation exposure that can induce acute life-threatening syndromes, chronic low-dose radiation is associated with Chronic Radiation Syndrome (CRS), which can alter environmental sensitivity. Secondary effects of chronic low dose-rate radiation exposure include circulatory, digestive, cardiovascular, and neurological diseases, as well as cancer. Here, we investigated 1–2 Gy, 0.66 cGy/h, 60Co radiation effects on primary human mesenchymal stem cells (hMSC). There was no significant induction of apoptosis or DNA damage, and cells continued to proliferate. Gene ontology (GO) analysis of transcriptome changes revealed alterations in pathways related to cellular metabolism (cholesterol, fatty acid, and glucose metabolism), extracellular matrix modification and cell adhesion/migration, and regulation of vasoconstriction and inflammation. Interestingly, there was increased hypoxia signaling and increased activation of pathways regulated by iron deficiency, but Nrf2 and related genes were reduced. The data were validated in hMSC and human lung microvascular endothelial cells using targeted qPCR and Western blotting. Notably absent in the GO analysis were alteration pathways for DNA damage response, cell cycle inhibition, senescence, and pro-inflammatory response that we previously observed for high dose-rate radiation exposure. Our findings suggest that cellular gene transcription response to low dose-rate ionizing radiation is fundamentally different compared to high-dose-rate exposure. We hypothesize that cellular response to hypoxia and iron deficiency are driving processes, upstream of the other pathway regulation.


Introduction
Redox homeostasis has been defined as the balance between cellular generation of nonradical reactive oxygen species (ROS) and cellular antioxidant defenses [1][2][3][4]. Redox homeostasis has been demonstrated to contribute to normal cellular processes, including and differentiate into different phenotypes depending upon their environment [37,38]. MSCs were previously demonstrated to be relatively resistant to radiation-induced damage, requiring 2 Gy to reduce the surviving fraction of cells to 37% [39]. 10 Gy was demonstrated in MSCs to reduce the surviving population to <1% [39]. Because of the importance of MSC in repair following tissue injury, we investigated the effects of chronic low dose-rate (0.66 cGy/h) radiation on MSCs. We confirmed our findings in primary human lung microvascular endothelial cells. Our findings indicate that the pathways regulated by chronic low dose-rate radiation differ greatly compared with those regulated by acute high dose-rate radiation exposure.

Reagents
Chemicals and reagents were purchased from MilliporeSigma (St. Louis, MO, USA) except where indicated.

Cell Culture and Irradiation
Human bone marrow mesenchymal stem cells (MSCs) were purchased from Lonza (Morristown, NJ, USA) and cultured in mesenchymal stem cell growth medium (Lonza). Human lung microvascular endothelial cells (HLMVECs) were purchased from Cell Applications (San Diego, CA, USA) and cultured on plates treated with endothelial cell attachment factor in Microvascular Endothelial Cell Growth Medium (Cell Applications). Cells were grown and irradiated in a humidified environment of 5% CO 2 /95% air at 37 • C, according to the manufacturer's instructions. Cells were used within seven passages for all experiments. For irradiations, cells were plated in 25 ml flasks or in Lab-Tek Flaskette Chamber slides (ThermoFisher Scientific, Waltham, MA, USA), and grown to 60-70% confluence. Cells were irradiated in the Armed Forces Radiobiology Research Institute (AFRRI) Low Level Cobalt Facility at 0.66 cGy/h to reach a total irradiation of 0.3, 0.7, 1, or 2 Gy. Dosimetry for Low Level Cobalt Facility was performed by preliminary mapping of irradiation field inside the cell culture incubator. The irradiation field from the bare source was measured using an A12 ion chamber calibrated at ADCL, University of Wisconsin. The dose rate was measured at the position of each flask (21 points of measurements, 7 flasks on 3 shelves) under the same conditions as actual cell irradiation conditions. To achieve desired dose rate, the 4-times attenuator was mounted and field uniformity was measured at the same positions with ion chamber PTW TN 75 cc. The dose rate on a date of mapping of the field was measured as 0.6646 cGy/h with field uniformity 0.28%. Control dishes were cultured in parallel in a separate incubator without radiation exposure. The 10 Gy irradiation was performed on cells at 70-90% confluence using an RS2000 Biological Irradiator (Rad Source Technologies, Alpharetta, GA, USA) at a dose rate of 1.15 Gy/min (160 kV, 25 mA) for a total dose of 10 Gy, as previously described with previously described dosimetry [40].

Transcriptome Profiling by RNA Sequencing
Immediately following irradiation, total RNA was isolated from MSC using the RNeasy Mini Kit with on-column DNase digestion (Qiagen) according to manufacturer's protocol. RNA was quantified spectroscopically (ND-1000 Spectrophotometer, Nano-Drop, Wilmington, DE, USA). The total RNA integrity was assessed using automated capillary electrophoresis with a Fragment Analyzer (Roche, Pleasanton, CA, USA). For all samples with an RNA quality indicator (RQI) > 8.0, a total of >75 ng RNA was used as the input for library preparation using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA). The sequencing libraries were quantified by Real-Time PCR on a Roche LightCycler 480 Instrument II using a KAPA Library Quantification Kit for NGS (Kapa, Wilmington, MA, USA). The size distribution was assessed by automated capillarybased gel electrophoresis with a Fragment Analyzer to confirm the absence of free adapters or adapter dimers. The sequencing libraries were pooled and sequenced on a NovaSeq 6000 Sequencer (Illumina) using a NovaSeq 6000 SP Reagent Kit (300 cycles) within one flowcell lane using an XP workflow with 101 + 8 + 8 + 101 cycle parameters with paired-end reads of 75 bp in length. Raw sequencing reads were demuxed using bcl2fastq2 (v2.20) and aligned to the human reference genome (hg38) with MapSplice (v2.2.2) [43]. Gene-level quantification was performed with HTSeq (v0.9.1) [44] against GENCODE (v28) basic gene annotations. Read alignment statistics and sample quality features were calculated with Samtools and RseQC [45][46][47]. Sequencing quality was verified by manual inspection of sample-wise characteristics: total reads, mapping percentages, pairing percentages, transcript integrity number (TIN), 5 to 3 gene body read coverage slopes, and ribosomal RNA content [48]. The transcript abundance quantitation data were deposited in the NCBI Gene Expression Omnibus (GSE222541). Differential expression analysis was performed with DESeq2 (v1.16.1) [49] on raw gene counts. We defined significant differentially expressed genes (DEGs) between irradiated and control samples as those with a False Discovery Rate (FDR) q-value < 0.05, an absolute fold change > 1.5 (i.e., |log 2 (fold-change)| > 0.585), and mean transcripts per million (TPM) ≥ 1 across samples.

Statistics
Statistical analyses of assays were performed using Graphpad Prism 7 (San Diego, CA, USA) or Excel. For RNA-seq and qPCR analysis, one-way ANOVA with a post-test analysis was used for comparing multiple data sets. For Western blot analysis, two-way ANOVA with either Tukey's or Sidak's post hoc tests for multiple comparisons were used. Our previous studies showed that high-dose/high-dose-rate (10 Gy/0.989-1.15 Gy/min) X-ray irradiation primarily induces accelerated senescence in primary pulmonary artery endothelial cells (PAEC), primary human lung microvascular endothelial cells (HLMVEC), and primary mesenchymal stem cells (MSCs) [22,23,40]. We compared irradiation of MSC at low-and high-dose-rates. The morphology of MSCs following 0.66 Gy/h 60 Co radiation showed that the cells maintained a consistent morphology after 7 days (~1 Gy) and 14 days (~2 Gy) ( Figure 1A). Additionally, cells appeared to increase in density over the time course of the experiment. In contrast, MSCs exposed to 10 Gy X-ray irradiation (1.15 Gy/min) displayed flattened "fried egg" morphology with increased cellular area and reduced cell numbers, consistent with cellular senescence, at 2 weeks post-irradiation ( Figure 1A).
We previously found that low levels of apoptosis were present in 10 Gy (0.989-1.15 Gy/min) X-ray irradiated PAECs, HLMVECs, and MSCs, although in most cases the increase in apoptosis did not reach significance compared with control levels [22,23,40]. We investigated the induction of apoptosis in low-and high-dose-rate irradiation in MSCs using nuclear morphological analysis ( Figure 1B) [40]. Nuclear blebbing is consistent with late apoptotic events. We did not observe any significant increase in nuclear morphological changes in any of the irradiated cells at the time points examined. Effects of high and low dose-rate X-ray irradiation on cell morphology, apoptosis, and γ-H2AX foci in MSCs. MSCs were grown to 60% confluence and exposed to 60Co irradiation at 0.66 cGy/h for 1 Gy (1 week) or 2 Gy (2 weeks). Alternatively, cells were exposed to 10 Gy X-ray irradiation (1.15 Gy/min) and assayed at 3 days and 1 week post-irradiation. Cells were fixed at the indicated times and stained with DAPI, and immunohistochemistry was performed for γ-H2AX. (A). Light microscopy was used to examine cell morphology. Representative images are shown, 20× magnification. (B). DAPI was used to examine the nucluear morphology of the fixed cells at the indicated times. Arrows (10 Gy irradiation) indicate nuclear blebbing, a late apoptotic event. Representative images are shown from each condition, 20× magnification. Nuclei were scored from all cells in random fields to determine percentage of apoptotic nuclei at 3 days (3 d), 1 week (1 w) or 2 weeks (2 w) post-irradiation. Graph shows average of percent apoptosis ± SEM; NS = not significant compared with control (C). (C). γ-H2AX immunohistochemistry was used to detect foci surrounding double-stranded DNA breaks in the fixed cells at the indicated times. Representative images are shown from each condition, 20× magnification. Foci were scored in all cells from random fields to determine numbers of foci per cell. Graph shows average of nuclear foci ± SEM; * indicates p < 0.05 compared with sham-irradiated control cells. Effects of high and low dose-rate X-ray irradiation on cell morphology, apoptosis, and γ-H2AX foci in MSCs. MSCs were grown to 60% confluence and exposed to 60 Co irradiation at 0.66 cGy/h for 1 Gy (1 week) or 2 Gy (2 weeks). Alternatively, cells were exposed to 10 Gy X-ray irradiation (1.15 Gy/min) and assayed at 3 days and 1 week post-irradiation. Cells were fixed at the indicated times and stained with DAPI, and immunohistochemistry was performed for γ-H2AX. (A). Light microscopy was used to examine cell morphology. Representative images are shown, 20× magnification. (B). DAPI was used to examine the nucluear morphology of the fixed cells at the indicated times. Arrows (10 Gy irradiation) indicate nuclear blebbing, a late apoptotic event. Representative images are shown from each condition, 20× magnification. Nuclei were scored from all cells in random fields to determine percentage of apoptotic nuclei at 3 days (3 d), 1 week (1 w) or 2 weeks (2 w) post-irradiation. Graph shows average of percent apoptosis ± SEM; NS = not significant compared with control (C). (C). γ-H2AX immunohistochemistry was used to detect foci surrounding double-stranded DNA breaks in the fixed cells at the indicated times. Representative images are shown from each condition, 20× magnification. Foci were scored in all cells from random fields to determine numbers of foci per cell. Graph shows average of nuclear foci ± SEM; * indicates p < 0.05 compared with sham-irradiated control cells.
We examined DNA damage in the cells after 1 and 2 Gy (0.66 cGy/h, 60 Co) or 10 Gy (1.15 Gy/min, X-ray) exposures. DNA damage initiates signaling pathways that result in the phosphorylation of serine 139 on histone H2AX to form γ-H2AX, which is present in complexes surrounding double-stranded DNA breaks [55]. Immunohistochemistry for γ-H2AX complexes in the nuclei of MSCs following low-and high-dose-rate radiation exposure showed a significant increase in foci at 3 days and 2 weeks following 10 Gy/1.15 Gy/min X-ray irradiation ( Figure 1C). Interestingly, exposure to low dose-rate irradiation did not show a significant increase in γ-H2AX foci compared with basal levels.

Genome-Wide Transcriptional Responses to Low Dose-Rate Radiation
To expand the understanding of overall gene expression changes in primary MSCs in response to low dose-rate radiation, we used comprehensive transcriptome profiling by RNA-seq. Gene expression profiles from sham-irradiated (control) MSCs were compared with MSCs irradiated at 0.66 cGy/h for 1 Gy total (~1 week) and 2 Gy total (~2 weeks). Comparative differential expression analysis identified 862 genes differentially expressed between 1 Gy irradiation samples and matched controls (q-value < 0.05, absolute fold change > 1.5) (Table S1). For 2 Gy irradiation, comparative differential expression analysis identified 725 differential genes (q-value < 0.05, absolute fold change > 1.5) (Table S2). A heatmap of the differentially expressed genes (DEGs) of all samples is shown in Figure 2A. At 1 Gy, 472 genes were downregulated compared with the matched control, and 390 genes were upregulated. At 2 Gy, 427 genes were downregulated compared with the matched control, and 298 genes were upregulated. A comparison of the gene sets from 1-and 2-Gy-regulated genes showed an overlap of~39% of genes that were regulated at both doses of radiation ( Figure 2B).
GO analyses were focused on terms relevant to cellular biological processes (BPs) and not disease states. The BP graphs show clusters of pathways with enrichment scores ≥ 1.6 ( Figure 3, Supplemental Tables S3-S6). DAVID analysis showed that following 1 Gy low dose-rate irradiation (1 week, 0.66 cGy/h), the largest changes (both up-and downregulation) in BP terms were vasoconstriction and blood pressure, metabolic processes (including glucose, cholesterol, fatty acid/lipid metabolism, and cellular response to starvation), proliferation, cellular response to hypoxia/reactive oxygen species and iron ion responses, apoptosis, and adhesion, and extracellular matrix modification ( Figure 3A,B). In contrast, following 2 Gy low dose-rate irradiation (2 weeks, 0.66 cGy/h), DAVID analysis showed positive regulation of proliferation, continued metabolic processes (cholesterol and fatty acid biosynthesis/metabolism, response to starvation), continued cellular response to hypoxia, extracellular matrix modification (especially collagen), and apoptotic signaling ( Figure 3C,D). Metascape (https://metascape.org accessed on 10 August 2022) [54] was used to create an image of clustered GO terms present in 1300 genes with the lowest qvalue (up-and down-regulated, from both 1 Gy and 2 Gy) ( Figure 3E). Relationships were identified between the pathway functions, notably the signaling genes for MAPK, protein phosphorylation and enzyme-linked receptors with genes that regulate cellular adhesion and locomotion, vascular development, tissue morphogenesis, and skeletal system development. Additional links were identified between regulation of genes for extracellular matrix organization, supramolecular fiber organization, and overall changes in genes encoding the core proteins making up the extracellular matrix (ECM; NABA core matrisome) and the ECM-associated proteins (NABA-matrisome-associated). GOrilla analysis of the ranked DEGs with q value ≤ 10 −5 also identified enrichment in cellular processes, including cholesterol, lipid, sterol, and alcohol metabolism, protein catabolism, polysaccharide metabolism, responses to hormones and oxidative stress, extracellular matrix reorganization and cell motility, and developmental processes, including cell differentiation, and regulation of tissue remodeling, including vascular smooth muscle and bone remodeling (data not shown). GOrilla analysis also showed major changes in pathways regulating proliferation, inflammation, and programmed cell death (data not shown).
olism, polysaccharide metabolism, responses to hormones and oxidative stress, extracellular matrix reorganization and cell motility, and developmental processes, including cell differentiation, and regulation of tissue remodeling, including vascular smooth muscle and bone remodeling (data not shown). GOrilla analysis also showed major changes in pathways regulating proliferation, inflammation, and programmed cell death (data not shown).   . GO term cluster, KEGG pathway enrichment, and Metascape analyses of differentially expressed genes in MSCs following chronic low dose-rate irradiation. MSCs were grown to 60% confluence and exposed to 60 Co irradiation at 0.66 cGy/h for 1 Gy (1 week) or 2 Gy (2 weeks). Control cells were cultured under identical conditions for 1 or 2 weeks. Irradiated and control cells were lysed at the same time, and RNA was prepared for RNAseq. Pathway regulation was compared for all conditions. A,B. 1 Gy irradiation, upregulated pathways (A) and downregulated pathways (B). C,D. 2 Gy irradiation, upregulated pathways (C) and downregulated pathways (D). (E). Clustered GO terms using Metascape using genes with q < 0.05 to visualize pathway relationships. . GO term cluster, KEGG pathway enrichment, and Metascape analyses of differentially expressed genes in MSCs following chronic low dose-rate irradiation. MSCs were grown to 60% confluence and exposed to 60 Co irradiation at 0.66 cGy/h for 1 Gy (1 week) or 2 Gy (2 weeks). Control cells were cultured under identical conditions for 1 or 2 weeks. Irradiated and control cells were lysed at the same time, and RNA was prepared for RNAseq. Pathway regulation was compared for all conditions. A,B. 1 Gy irradiation, upregulated pathways (A) and downregulated pathways (B). C,D.

Focused Heatmap Analysis of Chronic Low-Dose Radiation Gene Regulation
According to the GO analysis by DAVID and GOrilla, we evaluated gene regulation in the pathways and processes found to be most affected by chronic low-dose radiation. The focused analyses included genes selected by the DAVID and GOrilla, and included additional genes that we curated through literature searches for genes involved in each process that were also regulated in our study. qPCR and/or Western blotting was used to validate the pathways identified by RNAseq.

Alteration of Cellular Metabolism
Chronic low-dose radiation affected a number of metabolic pathways in the MSCs, including downregulation of cholesterol synthesis, upregulation of glycolysis over oxidative phosphorylation, and a reduction in fatty acid biosynthesis and modification ( Figure 4A). qPCR was performed to validate at least one gene in each pathway ( Figure 4B). Both 1 Gy and 2 Gy low dose-rate radiation showed downregulation of almost all genes encoding cholesterol synthesis enzymes, including for the synthesis of squalene from acetyl-CoA and for the synthesis of cholesterol from squalene ( Figure 4A, left panel). These genes include the enzyme for the initiation of cholesterol synthesis (acetyl-CoA acetyltransferase 2, ACAT2), the rate-limiting enzyme (3-hydroxy-3-methylglutaryl-CoA synthase 1, HMGCS1), through to the final enzyme in the pathway (24-dehydrocholesterol reductase, DHCR24). In addition to the downregulation of cholesterol synthesis enzymes, we also observed the suppression of two major regulators of cholesterol biosynthesis: insulin-induced gene (INSIG1) and sterol regulatory element binding transcription factor-1 and -2 (SREBF1 and SREBF2). Finally, we observed downregulation of Niemann-Pick Type C disease 1 (NPC1), which regulates intracellular cholesterol transport and esterification.
Low-dose chronic radiation also resulted in the regulation of metabolic pathways that favored glucose metabolism over mitochondrial oxidative phosphorylation ( Figure 4A, middle panel). Genes associated with increased glycolysis were upregulated: 3-phosphate dehydrogenase (GAPDH), pyruvate dehydrogenase kinases (PDK1, 3, and 4), aldo-keto reductase family 1 member C3 (AKR1C3), leptin (LEP), phosphofructo-2-kinase/fructose-2,6bisphosphatase 3 (PFKFB3), proprotein convertase subtilisin (PCSK9), FOXO1, and NUAK2. PCSK9, which can negatively regulate glucose metabolism, was decreased five-fold. PDK1, -3, and -4, were each upregulated~two-fold, inhibiting pyruvate dehydrogenase, and reducing the production of acetyl-coenzyme A from pyruvate. The upregulation of the PKDs can result in decreased activity of the tricarboxylic acid (TCA) cycle, decreased oxidative phosphorylation, and increasing the production of lactate as a final downstream function of glycolysis. We also observed an increase in lactate dehydrogenase, which catalyzes the conversion of pyruvate to lactate, again suggesting that pyruvate is being diverted away from the TCA cycle. FOXO1, a transcription factor responsible for increased gluconeogenesis, was reduced. Nu [novel] AMPK-related protein kinase-2 (NUAK2), which is responsive to increased AMP/decreased ATP, low glucose, and oxidative or endoplasmic reticulum stress, signals to suppress cell death by glucose starvation, was increased.
There was a general decrease in genes encoding enzymes for fatty acid (FA) metabolism and processing. Low-density lipoprotein receptor (LDLR), which can take up lipids from the environment, was decreased. There were decreases in folliculin (FLCN) and folliculininteracting proteins (FNIP1 and FNIP2) regulators of AMP-dependent protein kinase (AMPK), a master regulator of FA metabolism, antioxidant responses, and mitochondrial and lysosome biogenesis. Decreases were observed in a number of types of FA acid synthesis enzymes: fatty acid synthase (FASN), a central regulator of lipid metabolism; acetyl-CoA carboxylase, which catalyzes the rate-limiting step in long-chain FA biosynthesis; acyl-CoA synthetase family member 2 (ACSF2), which enables medium-chain FA ligase activity; desaturase enzymes (FADS1 and 2), which produce highly unsaturated FA (HUFA); and stearoyl-coenzyme A desaturase (SCD), which synthesizes monounsaturated FA. Enzymes for the processing of FA were decreased, including for FA desaturation (FA desaturase-1 and 2, FADS1 and 2) and transport (FA binding protein 3, FABP3). Additionally, enzymes for FA degradation were also reduced: HADH, ASAH1, PLA1A, and GBA. (HUFA); and stearoyl-coenzyme A desaturase (SCD), which synthesizes monounsaturated FA. Enzymes for the processing of FA were decreased, including for FA desaturation (FA desaturase-1 and 2, FADS1 and 2) and transport (FA binding protein 3, FABP3). Additionally, enzymes for FA degradation were also reduced: HADH, ASAH1, PLA1A, and GBA. RNAseq was used to identify gene expression changes in primary human MSCs following 1 or 2 Gy (0.66 cGy/h) 60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM N = 3 biological replicates with two technical repeats; * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001, respectively, compared with sham irradiated control cells.  60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM N = 3 biological replicates with two technical repeats; * indicates p < 0.05, *** indicates p < 0.001, **** indicates p < 0.0001, respectively, compared with sham irradiated control cells.

Regulation of Proliferation and Cell Division
We observed mixed regulation of genes related to cell proliferation. Focused heatmaps of proliferation and cell cycle genes are shown in Figure 5A; three of the regulated genes were validated by qPCR ( Figure 5B). However, the cell numbers and morphology determined by light microscopy suggested that the MSCs continued to proliferate over the course of the 1 and 2 Gy exposures, without significant apoptosis or accelerated senescence (see Figure 1). We observed upregulation of proliferation-inducing genes. Upregulated growth factors included fibroblast growth factor 1 (FGF1), transform in growth factor A (TGFA), and vascular endothelial growth factor A (VEGFA). Upregulated transcription factors included Odd-skipped related transcription factor (OSR1) and c-Jun (JUN). We also observed downregulation of other growth factors, such as colony stimulating factor (CSF1) and pleiotrophin (PTN), as well as some transcription factors, such as transcription factor AP4 (TFAP4). There was also mixed regulation of factors that regulate apoptosis, including the upregulation of Baculovirus inhibitor of apoptosis repeat containing 5 (BIRC5), which suppresses apoptosis and promotes proliferation, but also the downregulated proteins that inhibit apoptosis, such as SFRP4 and IFIT3.

Regulation of Apoptosis, Cell Death, and Autophagy
A variety of forms of cell death and autophagy are induced in cells in response to high-dose acute ionizing radiation, and we previously observed the upregulation of proapoptotic and pro-senescence pathways [5,40]. In contrast, we did not observe significant levels of apoptosis at any time points following chronic low-dose radiation (see Figure 1), and GO analysis showed apoptotic pathway regulation predominantly favoring the inhibition of apoptosis by a number of mechanisms ( Figure 6). The regulation of this group of genes was confirmed by qPCR of EGR1 and DUSP1 and Western blotting of Egr1 and DUSP1 ( Figure 6B,C).  60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM N = 3 biological replicates with two technical repeats; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, respectively, compared with sham-irradiated control cells. (C). Western blot data showing regulation of AKT and MAPK (phosphorylated and total). Western blots were performed on N = 3 biological repeats. Bar graphs show average band densities normalized to β-actin. Graphs show means ± SEM; * indicates p < 0.05 and ** indicates p < 0.01, respectively, compared with sham-irradiated control cells.
Interestingly, the downregulation of these proteins occurred without the induction of apoptosis or accelerated senescence (See Figure 1). The phosphorylation of Akt and p42/p44 MAPK are associated with proliferation and cell survival, and we observed significant increases in both phosphorylated Akt and p42/p44 MAPK in the 1 and 2 Gy exposures ( Figure 5C). This suggests that the mixed regulation of proliferation and cell cycle genes favors cell survival and proliferation, or potentially, cell cycle arrest without senescence.

Regulation of Apoptosis, Cell Death, and Autophagy
A variety of forms of cell death and autophagy are induced in cells in response to high-dose acute ionizing radiation, and we previously observed the upregulation of proapoptotic and pro-senescence pathways [5,40]. In contrast, we did not observe significant levels of apoptosis at any time points following chronic low-dose radiation (see Figure 1), and GO analysis showed apoptotic pathway regulation predominantly favoring the inhibition of apoptosis by a number of mechanisms ( Figure 6). The regulation of this group of genes was confirmed by qPCR of EGR1 and DUSP1 and Western blotting of Egr1 and DUSP1 ( Figure 6B,C).  Gene regulation was observed in the pathways for intrinsic and extrinsic apoptosis, p53-pathway-regulated apoptosis, and Wnt signaling-induced apoptosis. Two pro-apoptotic master regulators were downregulated: forkhead box protein O1 (FOXO1) and nephroblastoma-overexpressed protein (NOV). Three anti-apoptotic regulators were upregulated: early growth response 1 (EGR1), leptin (LEP), and eukaryotic elongation factor  60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM from N = 3 biological replicates with two technical repeats. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, respectively, compared with sham-irradiated control cells. (C). Western blot data showing regulation of Egr1 and DUSP1. Western blots were performed on N = 3 biological repeats. Bar graphs show average band densities normalized to β-actin.

Regulation of Pathways for Extracellular Matrix, and Cell Attachment and Migration
GO analysis of the gene changes showed significant radiation-induced alterations in pathways related to synthesis, breakdown, and organization of the extracellular matrix (ECM), as well as for cell attachment to the extracellular matrix ( Figure 7A). Two genes from these pathways, keratin 34 (KRT34) and neuregulin 1 (NRG1), were validated by qPCR ( Figure 7B 8, 14, 16, 18, 34, 80, and 81, which also can increase cellular stiffness and promote motility. We did not observe upregulation of any proteins associated with inhibition of motility, but we did observe the downregulation of several proteins that would increase cellular adhesion: secreted phosphoprotein 1 (SPP1), desmoplakin (DSP), and metastasis suppressor protein 1 (MTSS1).   60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM from N = 3 biological replicates with two technical repeats. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, respectively, compared with sham-irradiated control cells.

Regulation of Pathways for Vascular Constriction and Inflammation
GO analysis of the transcriptome revealed alterations in pathways for vascular constriction and inflammation ( Figure 8A). Three of these genes were validated using qPCR: OXTR, HMOX1, and NPR3, which showed similar alterations as the transcriptomic data ( Figure 8B). The regulation of vascular constriction pathways showed an increase in the number of genes associated with increased vasoconstriction: 5-hydroxytrypamine receptor 2A (HTR2A), adrenoreceptor alpha 1B and 1D (ADRA1B and 1D), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2, also called cyclooxygenase-2), gap junction alpha 5 (GJA5), leptin (LEP), apelin (APLN), and endothelin 1 (EDN1), which has a specific role in pulmonary hypertension. At the same time, there was a decrease in expression of several genes associated with vasodilation: superoxide dismutase 2 (SOD2) and heme oxygenase 1 (HMOX1).  60 Co irradiation. Control cell were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance i applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supportin heatmaps of pathways. Data show averages ± SEM from N = 3 biological replicates with two tech nical repeats. * indicates p < 0.05 compared with sham-irradiated control cells.

Regulation of PATHWAYs for Cell Response to Hypoxia and Iron Homeostasis
GO analysis revealed the regulation of pathways related to cellular response to hy poxia as well as to iron homeostasis and iron-binding proteins ( Figure 9A). Four of th genes in these groups were validated by qPCR ( Figure 9B). Interestingly, the regulation o genes to hypoxia included a number of genes that are also regulated by redox stress [56 and a number of genes related to iron homeostasis and iron-binding can also be regulated  60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM from N = 3 biological replicates with two technical repeats. * indicates p < 0.05 compared with sham-irradiated control cells.
3.3.6. Regulation of PATHWAYs for Cell Response to Hypoxia and Iron Homeostasis GO analysis revealed the regulation of pathways related to cellular response to hypoxia as well as to iron homeostasis and iron-binding proteins ( Figure 9A). Four of the genes in these groups were validated by qPCR ( Figure 9B). Interestingly, the regulation of genes to hypoxia included a number of genes that are also regulated by redox stress [56], and a number of genes related to iron homeostasis and iron-binding can also be regulated by redox stress [57].
Surprisingly, we observed downregulation of a number of enzymes that would mitigate redox stress, including superoxide dismutase 1 and 3 (SOD1, 3) and glutathione peroxidase 1 and 4 (GPX1, 4). We also observed the downregulation of the oxidative stressactivated transcription factor nuclear factor erythoid-derived 2-like 1 and 2 (NFE2L1 and 2), as well as its regulator, kelch-like ECH-associate protein 1 (KEAP1). In contrast, we Figure 9. Heatmaps of gene expression changes for pathways involved in cellular response to hypoxia or redox stress and iron homeostasis and iron-binding proteins. (A). RNAseq was used to identify gene expression changes in primary human MSCs following 1 or 2 Gy (0.66 cGy/h) 60 Co irradiation. Control cells were cultured under identical conditions for 1 or 2 weeks. GO analysis was performed using DAVID and heatmaps were generated using genes with q < 0.05. Rows are centered and unit variance is applied to rows. (B). qPCR gene regulation using log base 2 scale fold change of genes supporting heatmaps of pathways. Data show averages ± SEM from N = 3 biological replicates with two technical repeats. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, respectively, compared with sham-irradiated control cells.

Effect of Chronic Low Dose-Rate Irradiation on Primary Human Lung Microvascular Endothelial Cells
The vascular endothelium is hypothesized to be a primary mediator of radiation injuries [58]. We therefore investigated the effect of low dose-rate radiation on primary human lung microvascular endothelial cells in culture using the same targets identified in MSCs (Figure 10). We found similar regulation for genes in cholesterol and fatty acid biosynthesis (HMGCS1 and MSMO1), for cell survival and suppression of accelerated senescence (EGR1 and CDKN1A and MAPK and Akt activation), for reduced antioxidant signaling (SOD2), and we observed a trend for extracellular matrix modification (COL1A2).
Antioxidants 2023, 12, x FOR PEER REVIEW 24 of 30 Figure 10. Response of human lung microvascular endothelial cells (HLMVECs) to chronic low dose-rate irradiation. HLMVECs were grown to 50% confluence and exposed to 60 Co irradiation at 0.64 cGy/h for 1 Gy (1 week) or 2 Gy (2 weeks). Control cells were cultured under identical conditions. Irradiated and control cells were lysed at the same time, and either frozen for protein analysis or placed in RNAlater. (A). qPCR gene regulations are represented on a log base 2 fold change scale, using N = 3 biological replicates with two technical repeats. Graphs show means ± SEM; * indicates p < 0.05 and ** indicates p < 0.01, respectively, compared with sham-irradiated control cells. (B). Western blots of phosphorylated and total MAPK and Akt, and Egr1. Western blots were performed on N = 3 biological repeats. Bar graphs show average band densities normalized to β-actin.

Discussion
The potential for accidental radiation exposure has increased with the increasing use of medical and industrial radiation, and with the potential increase in nuclear energy generation and the potential for military use of radioactive weapons. Current proposals for space travel will also result in chronic radiation exposure. Therefore, an increased understanding of the cellular effects of chronic low dose-rate radiation is needed. Here, we demonstrate in primary human MSCs that exposure to 0.3-2 Gy gamma radiation (0.66 cGy/h) did not result in growth arrest, the induction of significant apoptosis, or accelerated senescence. GO analysis of the transcriptomic changes showed that low dose-rate radiation exposure resulted in changes in gene expression related to cellular metabolic activity, pro-survival and proliferation signaling, alterations in the extracellular matrix and cellular motility, increased expression of factors related to vascular constriction, and increased signaling related to hypoxia and iron deficiency. We confirmed our findings using targeted qPCR and Western blotting in primary HLMVEC. Noticeably absent from the identified DAVID and Metascape pathway analyses were the upregulation of DNA damage responses, strong pro-apoptotic and senescence pathway activation, oxidative stress-related signaling, and marked pro-inflammatory activation pathways, that our laboratory previously identified as major pathway responses to acute exposure to 10 Gy irradiation (1.15 Gy/min) [40]. Together, these data suggest that acute high-dose/high-dose-rate radiation exposure and chronic low dose-rate radiation exposure activate distinct signaling events, resulting in fundamentally different cellular outcomes.
Several previous studies investigated the sensitivity and responses of MSCs to radiation. One study utilized 40-2000 mGy (dose-rate not disclosed), and showed increased apoptosis, senescence, and autophagy, with evidence of DNA damage response at 1-48 h post-irradiation [7]. A second study exposed MSCs to 1-9 Gy radiation at 300 cGy/min, and examined the cells from 12 h to several weeks after irradiation [39]. This study also showed DNA damage response and loss of viability. A third study performed comprehensive analysis of transcriptome changes in MSCs exposed to 0.01 to 1 Gy (0.79 Gy/min), using microarrays to identify pathway regulation over a short time course from 1-48 h post-irradiation [59]. In contrast with our study, which exposed cells to 0.66 cGy/h, the irradiation of the MSCs in all of these studies was performed as acute exposures. Our study did not identify significant DNA damage response, apoptosis or senescence, although it is possible that these processes occurred at a low level earlier in the time course of the exposure, and that a percentage of the remaining cells recovered and proliferated over the time course. Our analysis at the 1-and 2-week time points suggests that the cells continued to proliferate (due to cellular density) and maintained normal (non-senescent) morphology. A previous study using very-low-dose X-ray irradiation of fibroblasts also identified an initial cell cycle pause, followed by resumed proliferative response [60]. The authors concluded that the early pause in cell cycle could have been associated with DNA repair [60]. Future work to investigate very early responses of MSCs to chronic low-dose radiation is needed to determine whether an early response may include a transient pause in the cell cycle.
In our experiments, MSCs responded to chronic low dose-rate radiation by downregulating cholesterol and lipid biosynthesis, as well as downregulating some enzymes in the pathway for oxidative phosphorylation while upregulating enzymes for glycolysis. A shift to aerobic glycolytic metabolism was previously demonstrated in vivo following fractionated high-dose radiation (5 Gy/day × 3 days, 702 cGy/min) [61]. Whether the changes observed in the chronic low-dose exposure is also aerobic or anaerobic glycolysis requires further investigation. With regard to changes in lipid and cholesterol metabolism, a number of reports showed increased cholesterol biosynthesis following acute, high-dose radiation exposure, which has been hypothesized to be a potential mechanism for radiation-induced cardiovascular disease and carcinogenesis [62]. In contrast, relatively low-dose radiation (25-50 mGy, 1.0 mGy/min) was shown to reduce atherosclerosis lesions in a murine model predisposed to atherosclerosis, although the mechanism of this is unknown [63].
At both 1 and 2 Gy exposures, we observed changes in the expression of ECM proteins, as well as proteins involved in cellular attachment and motility. The specific alterations in the expression of the ECM proteins and cellular intermediate filaments could have the combined effect of increasing cellular stiffness and increasing motility [64]. In some cases, the changes in proteins that increase cell motility, including some keratins, have also been linked to increased cellular proliferation [65]. Further studies of MSCs following chronic radiation are required to determine whether cellular motility is altered and also to measure specific changes in ECM stiffness.
Although GO analysis of altered gene expression identified a number of altered pathways, analysis of potential pathway hierarchies led us to hypothesize that hypoxia and iron deficiency pathways lie upstream of the other pathways that are regulated. Hypoxia can be associated with increased cellular proliferation and inhibition of apoptosis, in some cases through AQP1 regulation [66]. Additionally, cellular responses to hypoxia, and activation of hypoxia-inducible factors (HIFs), can lead to downstream regulation of cholesterol and lipid metabolism, changes in ECM protein synthesis and cell migration, increased cell survival and proliferation gene expression, and changes in glycolysis [61,67]. In vivo, pulmonary hypoxia leads to decreased cholesterol synthesis, and favors certain types of pulmonary vascular constriction [68]. In agreement with the potent regulation of hypoxia (directly or indirectly), GO analysis identified at least 15 genes known to be activated by hypoxia-inducible factors. Interestingly, we also found downregulation of genes associated with oxidative protective mechanisms, including SOD and Nrf2, suggesting that redox stress is not strongly regulated by chronic low dose-rate radiation at the time points that were studied.
Radiation-induced oxygen depletion in aqueous environments was identified over 40 years ago [69,70]. High-dose radiation (15 Gy, 67 cGy/min) induces hypoxia-related gene expression at 24 h post-irradiation [71], and recent advances in FLASH radiotherapy have rekindled interest in this effect [72]. The biological effects of radiation that would lead to cellular iron deficiency or iron-deficiency-like signaling are not known. Although our laboratory has described the effects of radiation on iron in vivo, these effects are initiated by red blood cell and reticulocyte hemolysis [73,74].
We also observed an increase in pathway regulation related to iron deficiency. High levels of iron can inhibit activation of HIFs, whereas low iron can increase HIF activation [75]. Iron deficiency can independently modulate glycolysis, regulate genes involved in cholesterol and lipid metabolism, and increase Egr-1 signaling [76,77]. Studies have shown that hypoxia can affect iron homeostasis and absorption under some conditions [78]. The link between radiation and cellular iron is not known. In cultured breast cancer cells, radiation-induced autophagic cell death was associated with iron accumulation, increased levels of transferrin receptor, and increased ferritin following acute exposure to 1-8 Gy X-ray irradiation (1.0 Gy/min) [79]. One possibility is that autophagy-induced release of cellular iron occurs in chronic low-dose radiation at an early time point, perhaps during an adaptation phase. Such events could result in iron deficiency, but future studies with early time points are needed to determine whether some cellular adaptation occurred early during the irradiation.
Studies of the biological response to low levels of radiation have revealed that there is a large uncertainty in determining actual health risks [80,81]. Several studies have suggested that there may be a non-linear biological response to radiation at low doses, with some data showing complex cellular responses that are not always detrimental [80]. According to the hormesis hypothesis, the dose-response relationship can be non-linear, in which low-dose stress can result in an optimal outcome [82]. Some animal model data from low-dose radiation studies do support a non-linear response [80]. However, the factors that determine in vivo responses to radiation, in animal models or in humans, are not sufficiently understood to predict individual outcomes of low level radiation exposure [35]. Further understanding of pathway regulation by low-dose radiation may allow the identification of markers to predict biological outcomes and also to identify countermeasures for adverse effects of low-dose radiation.   Data Availability Statement: All transcriptomics data will be made available upon request.