Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Health Phys. 2021 Oct 1;121(4):406–418. doi: 10.1097/HP.0000000000001477

Effect of Radiation on the Essential Nutrient Homeostasis and Signaling of Retinoids in a Non-human Primate Model with Minimal Bone Marrow Sparing

Jianshi Yu 1,†, Weiliang Huang 1,†, Tian Liu 1, Amy E Defnet 1, Stephanie Zalesak-Kravec 1, Ann M Farese 2, Thomas J MacVittie 2, Maureen A Kane 1
PMCID: PMC8549574  NIHMSID: NIHMS1716535  PMID: 34546221

Abstract

High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. Whereas DEARE includes inflammation and fibrosis in multiple tissues, the molecular mechanisms contributing to inflammation and to the development of fibrosis remain incompletely understood. Reports that radiation dysregulates retinoids and proteins within the retinoid pathway indicate that radiation disrupts essential nutrient homeostasis. Active metabolite of vitamin A, retinoic acid (RA), is a master regulator of cell proliferation, differentiation, and apoptosis roles in inflammatory signaling and the development of fibrosis. As facets of inflammation and fibrosis are regulated by RA, we surveyed radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the nonhuman primate after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

Keywords: biological indicators, radiation damage, radiation, ionizing, partial body irradiation

INTRODUCTION

High-dose radiation exposure results in hematopoietic (H) and gastrointestinal (GI) acute radiation syndromes (ARS) followed by delayed effects of acute radiation exposure (DEARE), which include damage to lung, heart, and GI. The H-ARS includes severe myelosuppression where Neulasta® (pegylated granulocyte colony stimulating factor [G-CSF]) or Neupogen® (G-CSF) enhance recovery from radiation-induced myelosuppression (Hankey et al. 2015; Farese et al. 2019). The GI-ARS includes significant loss of crypts, villus flattening, and loss of mucosal integrity (MacVittie et al. 2019b). Chronic GI syndrome includes a persistent impairment of both mucosal barrier function, restitution in the GI tract, and fibrosis (Shea-Donohue et al. 2016; Parker et al. 2019a). Radiation initiates cellular damage that results in chronic oxidative stress, tissue hypoxia, inflammation, and fibro-proliferation in lung leading to the development of fibrosis (Rodemann and Blaese 2007; Ding et al. 2013). DEARE for nonhuman primate (NHP) lung include increased mortality, nonsedated respiratory rate, and incidence of pneumonitis and pleural effusion.(MacVittie et al. 2019a). A hisopathological examination of DEARE in NHP lung has shown evidence of fibrosis, inflammation, and reactive/proliferative changes in pneumocytes.(Parker et al. 2019b). Similar histopathological analysis of NHP heart show DEARE in the heart consisted primarily of myocardial fibrosis (Parker et al. 2019b). The partial body irradiation (PBI) with minimal, 2.5% or 5%, bone marrow (BM) sparing NHP model was developed to mimic intentional or accidental radiation exposures in humans. Such exposures are likely to include bone marrow sparing and to permit the concurrent analysis of coincident short- and long-term damage to organ systems in a time and dose dependent manner (MacVittie et al. 2012).

Vitamin A is essential to numerous physiological processes including cell proliferation, differentiation, immune response, development, reproduction, cellular metabolism, and nervous system function (Berry and Noy 2012; Gudas 2012; Napoli 2017; Wang and Moise 2019; Wang et al. 2019). All-trans retinoic acid (RA) is an active metabolite of vitamin A that regulates gene transcription through the ligand-activated nuclear receptors, retinoic acid receptor (RAR) and retinoid x receptor (RXR), which can also function via non-genomic mechanisms (Germain et al. 2006a; Germain et al. 2006b; Chen and Napoli 2008; Piskunov and Rochette-Egly 2012). RA levels and the individual actions of vitamin A are spatially and temporally regulated by the expression of retinoid-binding proteins, enzymes, and receptors that contribute to RA generation, catabolism, and signaling. Fig. 1 shows the main elements of the retinoid pathway. RA is biosynthesized by a two-step enzymatic conversion of diet-derived vitamin A (retinol) consisting of a rate-limiting oxidation of retinol to retinal by retinol dehydrogenases, which is counterbalanced by retinal reductases which catalyze the conversion of retinal to retinol. Vitamin A can be esterified for storage and then hydrolyzed for mobilization (Napoli 2012; Napoli 2020). RA can also be derived from the cleavage of beta-carotene, which can be cleaved symmetrically into two molecules of retinal by beta-carotene oxygenase 1 (BCO1) (Harrison and Kopec 2020). Asymmetric cleavage of by beta-carotene oxygenase 2 (BCO2) yields apo-carotenals (Raghuvanshi et al. 2015; Harrison and Kopec 2020). RA is catabolized by cytochrome P450 enzymes, including CYP26A1, B1, and C1 (Topletz et al. 2012; Zhong et al. 2018). Glucuronidation by uridine diphosphate glucuronosyltransferases (UGTs) is an additional catabolic pathway for retinoids (Sass et al. 1994; Samokyszyn et al. 2000; Barua and Sidell 2004). Additionally, retinoids are chaperoned by retinoid-binding proteins to protect them from non-specific oxidation and facilitate their transport and metabolism (Napoli 2012).

Figure 1. Retinoid Pathway.

Figure 1.

Open in a new tab

Schematic representation of vitamin A metabolism leading to the formation of bioactive metabolite, all-trans retinoic acid (RA), which is a ligand for nuclear hormone receptors involved in gene transcription. The dietary sources of vitamin A are either preformed retinol and retinyl esters or provitamin A precursors, such as all-trans-β-carotene. Arrows indicate the enzymes involved in retinoid metabolism.

Dysregulation of retinoids and proteins within the retinoid pathway would indicate that radiation disrupts essential nutrient homeostasis (Napoli 2012; Napoli 2020). Previously it was reported that RA is reduced in murine lung within 24h after irradiation (IR) and this deficit persists through 180 days (Jones et al. 2014; Huang et al. 2019a). Similarly, jejunum RA was reduced shortly after IR in both murine and NHP models of the GI-ARS (Huang et al. 2019b; Huang et al. 2020b). Corresponding proteomic analyses of the lung and GI in mouse and primate models indicated a number of retinoid pathway and retinoid-regulated proteins were dysregulated, indicating a potential mechanism of injury involving radiation-induced disruption of essential nutrient signaling (Huang et al. 2019a; Huang et al. 2019b; Huang et al. 2020b). Disruption in RA homeostasis and essential nutrient signaling mediated by vitamin A is significant for a number of reasons. In the small intestine, RA is an important regulator of the immune response, epithelial integrity, and essential for differentiation that maintains the crypt-villus axis (Czarnewski et al. 2017). In the lung and in the heart, RA is important in the development of fibroblasts, their transformation to myofibroblasts, and the development of organ fibrosis (Wang et al. 2020). Here we sought to more thoroughly survey radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the NHP after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5).

MATERIALS AND METHODS

Radiation animal model.

All animal procedures were conducted in accordance with the NIH guidelines for the care and use of laboratory animals and experiments were performed with prior approval from the University of Maryland Institutional Animal Care and Use Committee (IACUC). Tissue was collected from a similar region of the left upper lung, jejunum 2–3 cm, left heart and right heart (ventricle) of male rhesus macaques (Macaca mulatta); EDTA plasma was collected. tissues were snap frozen in liquid nitrogen and stored at −80 °C until assay. Samples were provided by the laboratory of Thomas J. MacVittie, University of Maryland School of Medicine, Department of Radiation Oncology (Baltimore, MD). Description of the animal models, including radiation exposure and dosimetry, medical management (supportive care and health monitoring), as well as collection of tissue have been previously described (MacVittie et al. 2012). NHP were exposed to 12 Gy partial body irradiation with 2.5% bone marrow sparing (PBI/BM2.5) with a peak 6 MV linear accelerator (LINAC)-derived photons with an average energy of 2 MV at 0.80 Gy min-1. Bone marrow sparing was accomplished with tibiae outside the beam field. Tissue was obtained from a natural history study where timed euthanasia was planned at day 4, 8, 15, and 21 after radiation. Additional tissue samples were obtained from animals that were euthanized according to criteria specified in the IACUC protocol. For proteomics, we grouped animals from both planned and for-cause euthanasia at day 4, day 8 and 9, day 11 and 12, day 15, and day 21 and 22 to increase the numbers of n at the timepoints across our study. Two non-irradiated animals were used as baseline controls. Thirty-two samples of each tissue type were analyzed for proteomics in total with the numbers of NHP at each time point (n) as follows: day 0 (baseline), n=2; day 4, n=4; day 8-day9, n=11; day 11-day 12, n=7; day 15, n=5; day 21–22, n=3. For retinoids, we grouped animals from both planned and for-cause euthanasia at day 4–5, day 8–10, day 11–14, day 15–18, and day 21–22 to increase the numbers of n at the timepoints across our study. Two non-irradiated animals were used as naïve group. Forty samples of plasma, thirty-nine samples of jejunum, lung and right heart and thirty-eight samples of left heart were analyzed in total with the numbers of NHP at each time point (n) as follows: day 0 (naïve), n=2; day 4, n=5 for plasma, n=4 for all other tissues; day 8–10, n=12; day 11–14, n=8 for left heart, n=9 for all other tissues; day 15–18, n=9; day 21–22, n=3.

Retinoid quantification.

Tissue samples were stored at −80 °C until processed. Only glass containers, pipettes, and syringes were used to handle retinoids. Extraction of retinoids was performed under yellow lights using a two-step liquid-liquid extraction that has been described in detail previously using 4,4-dimethyl-RA as an internal standard for RA and retinyl acetate as an internal standard for retinol and total retinyl ester (Kane et al. 2005; Kane et al. 2008b; Kane and Napoli 2010; Jones et al. 2015). Briefly, for the extraction of retinoids, 100μL EDTA plasma, 62.12±13.65 mg lung, 104.8±22.7mg jejunum, 91.29±19.21mg left heart and 77.7±16.75mg right heart tissue was homogenized in 2 mL 0.9 % NaCl (normal saline) and two 1000 μL aliquots were extracted as technical duplicates. To each homogenate aliquot, 3 ml of 0.025 M potassium hydroxide (KOH) in ethanol was added to the homogenate followed by addition of 10 ml hexane to the aqueous ethanol phase. The samples were vortexed and centrifuged for 1 to 3 min at 1000 rpm in a Dynac centrifuge (Becton Dickinson) to facilitate phase separation and pellet precipitated protein. The hexane (top) phase containing nonpolar retinoids (retinol and total retinyl esters (RE)) was removed. 4 M hydrochloric acid (HCl) (185 μl) was added to the remaining aqueous ethanol phase, samples were vortexed, and then polar retinoids (RA) were removed by extraction with a second 10 ml aliquot of hexane as described above. Organic hexane phases were evaporated under nitrogen while heating at approximately 30 °C in a water bath (model N-EVAP 112, Organomation Associates, Berlin, MA, USA). All samples were resuspended in 60 μl acetonitrile.

Levels of RA were determined by liquid chromatography-multistage tandem mass spectrometry (LC-MRM3) which is an LC-MS/MS method utilizing two distinct fragmentation events for enhanced selectivity (Jones et al. 2015). RA was measured using a Shimadzu Prominence UFLC XR liquid chromatography system (Shimadzu, Columbia, MD) coupled to an AB Sciex 6500+ QTRAP hybrid triple quadrupole mass spectrometer (AB Sciex, Framingham, MA) using atmospheric pressure chemical ionization (APCI) operated in positive ion mode as previously described (Jones et al. 2015). For the LC separation, the column temperature was controlled at 25 °C, the autosampler was maintained at 10 °C and the injection volume was typically 20 μL. All separations were performed using an Ascentis Express RP-Amide guard cartridge column (Supelco, 50 × 2.1 mm, 2.7 μm) coupled to an Ascentis Express RP-Amide analytical column (Supelco, 100 × 2.1 mm, 2.7 μm). Mobile phase A consisted of 0.1 % formic acid in water, and mobile phase B consisted of 0.1 % formic acid in acetonitrile. Endogenously occurring retinoid isomers including all-trans-RA, 9-cis RA, 13-cis RA, and 9,13-di-cis RA are resolved using a gradient separation at a flow rate of 0.4 mL min−1 with gradient conditions described previously (Jones et al. 2015). The APCI source conditions and MRM3 detection parameters were as previously described where the MRM3 transition for RA was m/z 301.1 → m/z 205.1 → m/z 159.1 and for 4,4-dimethyl RA was m/z 329.2 → m/z 151.2 → m/z 100.0 (Jones et al. 2015).

Levels of retinol and RE were quantified via HPLC-UV according to previously published methodology (Kane et al. 2008a; Kane and Napoli 2010). Retinol and RE were resolved by reverse-phase chromatography (Zorbax SB-C18, 4.6 × 100 mm, 3.5 μm) on a Waters ACQUITY UPLC H-class system and were quantified by UV absorbance at 325 nm. Analytes were separated at 1 ml min−1 with a gradient separation as described previously with a typical injection volume of 30 μl.

The amount of RA, retinol, and RE was normalized per g of tissue or per ml EDTA plasma. Data are expressed as mean ± SEM with n representing retinoid values for individual NHP. Statistical significance was assessed with an ordinary one-way ANOVA, followed by Dunnett’s multiple comparison test compare the mean of each group to naïve group with a single pooled variance by Graphpad Prism software version 7.05. Data were present as mean ± SEM.

Proteomic sample preparation.

For tissue preparation, tissues were homogenized in 50 mM ammonium bicarbonate buffer (pH 7.8) by bead beating using Precellys CK14 lysing kit (Bertin Corp., Rockville, MD) as described previously(Huang 2019). The lysates were further washed, reduced, alkylated and trypsinolyzed in filter as described previously (Huang et al. 2020b). For plasma preparation, two of the most abundant plasma proteins albumin and immunoglobulin G, were depleted by immunoprecipitation using high-capacity anti-albumin and anti-IgG resin cartridges following manufacture’s instruction (R & D Systems). The immunodepleted plasma were washed, reduced, alkylated and trypsinolyzed on filter as described previously (Huang et al. 2020a).

Liquid chromatography-tandem mass spectrometry data acquisition.

Tryptic peptides were separated on a nanoACQUITY UPLC analytical column (BEH130 C18, 1.7 μm, 75 μm x 200 mm, Waters) over a 165-minute linear acetonitrile gradient (3 – 40%) with 0.1 % formic acid on a Waters nano-ACQUITY UPLC system and analyzed on a coupled Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer as previously described (Huang et al. 2019a). Full scans were acquired at a resolution of 240,000, and precursors were selected for fragmentation by collision induced dissociation (normalized collision energy at 35 %) for a maximum 3-second cycle.

Liquid chromatography-tandem mass spectrometry data analysis.

Tandem mass spectra were searched against a UniProt Macaca mulatta reference proteome using Sequest HT algorithm described previously (Eng et al. 2008) and MS Amanda algorithm developed by Dorfer et al. (Dorfer et al. 2014) with a maximum precursor mass error tolerance of 10 ppm. Carbamidomethylation of cysteine and deamidation of asparagine and glutamine were treated as static and dynamic modifications, respectively. Resulting hits were validated at a maximum false discovery rate of 0.01 using a semi-supervised machine learning algorithm Percolator developed by Käll et al. (Kall et al. 2007). Label-free quantifications were performed using Minora, an aligned AMRT (Accurate Mass and Retention Time) cluster quantification algorithm (Thermo Scientific, 2017). Protein abundance ratios between groups were measured by comparing the MS1 peak volumes of peptide ions, whose identities were confirmed by MS2 sequencing as described above.

Bioinformatic and statistical analysis.

Pathway, upstream regulator, and statistical analysis were performed as described previously (Huang et al. 2019b). Proteins showing at least a 2-fold change (FC) with a false discovery rate (FDR) adjusted ANOVA p-value < 0.05 were considered significantly changed and used for further analysis. Ingenuity Pathway Analysis (IPA) was used to predict canonical pathways and upstream regulators according to the proteins that were significantly different using a Benjamini-Hochberg corrected Fisher’s exact test p-value < 0.05, and z-score was used to infer the activity change direction.

RESULTS

Retinoic acid is reduced after radiation in multiple tissues

Retinoids were quantified in plasma, lung, heart (right and left ventricle), and jejunum. RA is the active metabolite of vitamin A (retinol (ROL)), retinol is the dietary-derived vitamin, and RE is the storage form of vitamin A. We had previously observed reductions in RA after radiation (Jones et al. 2014; Huang et al. 2019a; Huang et al. 2019b) and, thus, the objective of this study was to systematically measure retinoids in a natural history study in NHP after 12 Gy PBI/BM2.5. Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. Fig. 2 shows data for plasma, lung, heart, and jejunum RA. Jejunum and lung had the highest levels of RA and radiation caused 40–50% decreases in the amount of active metabolite RA.

Figure 2. RA is reduced after radiation in plasma, jejunum, lung, left ventricle, and right ventricle.

Figure 2.

Open in a new tab

RA quantification in NHP plasma and tissues after 12 Gy PBI/BM2.5. Days after radiation dose are noted. Data are mean ± SEM. For some points, the error bars are contained within the data symbol. Statistical analysis was done by using one-way ANOVA followed by Dunnett’s multiple comparisons test between groups as compared to Day 0 (naïve). ns not significant, *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. p value notation was also color coded: black for jejunum, brown for lung, green for right heart, blue for left heart and red for plasma. RA is expressed as picomol/mL for plasma and picomol/gram for solid tissues.

Vitamin A (retinol) and retinyl ester are largely unchanged after radiation in multiple tissues

Retinol is the substrate for RA biosynthesis and an essential diet-derived nutrient. Vitamin A (retinol) is esterified for storage, where the majority of retinyl esters are retinyl palmitate (90+%) with minor contributions from stearate, oleate. The RE quantified here is expressed as total RE, which is a sum of these esterified forms. Fig. 3 shows retinol and Fig. 4 shows RE after radiation where there was no significant difference in either retinol or RE when compared to naïve group in all tissue type. For any of the retinoid species, if animals were delineated into those meeting euthanasia criteria (cause) and those scheduled a priori for euthanasia on a given day (scheduled), we saw no significant differences between the retinoid levels in the scheduled and for cause groups after IR; both groups showed similar decreases in RA after IR and similar (not changed) values for ROL and RE.

Figure 3. Retinol is not significantly altered after radiation in plasma, jejunum, lung, left ventricle, and right ventricle.

Figure 3.

Open in a new tab

All-trans retinol (ROL) quantification in NHP plasma and tissues after 12 Gy PBI/BM2.5. Days after radiation dose are noted. Data are mean ± SEM. For some points, the error bars are contained within the data symbol. Statistical analysis was done by using one-way ANOVA followed by Dunnett’s multiple comparisons test between groups as compared to Day 0 (naïve). Adjusted p value of each comparison is greater than 0.05. Retinol is expressed as nanomol/mL for plasma and nanomol/gram for solid tissues.

Figure 4. Retinyl esters (RE) are not significantly altered after radiation in plasma, jejunum, lung, left ventricle, and right ventricle.

Figure 4.

Open in a new tab

RE in NHP plasma and tissues after 12 Gy PBI/BM2.5. Days after radiation dose are noted. Data are mean ± SEM. For some points, the error bars are contained within the data symbol. Statistical analysis was done by using one-way ANOVA followed by Dunnett’s multiple comparisons test between groups as compared to Day 0 (naïve). Adjusted p value of each comparison is greater than 0.05. RE is expressed as nanomol/mL for plasma and nanomol/gram for solid tissues.

Expression of retinoid pathway proteins is altered by radiation across multiple tissues

To understand the mechanism of this global reduction in RA across all the tissues that were interrogated, we conducted quantitative proteomic profiling. We used a liquid chromatography tandem-mass spectrometry-based quantitative proteomic workflow to characterize the proteome of each tissue and plasma (Huang et al. 2020a; Huang et al. 2020b). It should be noted that proteomics is a direct sampling technique with no amplification. As such, peptides in a complex mixture, like a tissue digest, compete for ionization and lower abundant proteins can sometimes fail to be detected. Not detected should not be interpreted as not expressed here, but more appropriately as below the limit of quantification. Although this proteomic approach has limitations for detection, it was still fruitful for informing on retinoid pathway protein expression in jejunum (Table 1) and heart (Table 2) after 12 Gy PBI/BM2.5 radiation. Table 1 and Table 2 shows retinoid pathway proteins that were detected in jejunum and heart, respectively, with arrows to indicate the direction of change alongside the numerical fold change. Some proteins are not detected in baseline, and are increased by radiation to detectable levels, for example, retinol-binding protein, type 4 (RBP4) and BCO2. Some proteins in the retinoid pathway were detected, but unaffected by radiation and notated as not significantly changed (NS) (Table 1, Table 2).

Table 1.

Retinoid pathway proteins changed in NHP jejunum after 12 Gy PBI/BM2.5
RA biosynthesis pathway Gene day 4 day 8–9 day 11–12 day 15 day 21–22
Chaperone RBP4 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
TTR ns-NS ↑2.09-NS ns-NS ns-NS ns-NS
RBP1 ↑2.22-* ↑2.52-NS ↑2.49-NS ↑2.12-NS ↑2.36-NS
ROL→RAL DHRS4 ns-NS ↓2.53-NS ↓4.55-NS ↓4.08-* ns-NS
ROL→ dihydroretinol RETSAT ns-NS ↓3.51-NS ↓3.98-NS ↓2.44-NS ns-NS
RAL→RA ALDH1A1 ns-NS ↓2.79-NS ↓2.94-NS ↓2.92-NS ↓2.38-NS
ALDH1A2 ns-NS ↑2.92-NS ↑2.80-NS ↑2.05-NS ns-NS
ALDH1A3 ns-NS ↓29.41-* ↓12.20-* ↓2.69-NS ↓2.26-NS
RA glucuronidation UGT1A1 ns-NS ↓5.05-NS ↓10.10-* ↓3.88-* ↓2.31-NS
UGT2B15 ns-NS ↓11.49-* ↓16.67-* ↓8.47-* ↓2.83-*
β-Carotene→apo-carotenals BCO2 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
ROL→RE, retinoid storage DGAT1 ns-NS ns-NS ↓3.76-NS ns-NS ns-NS
RE→ROL CES2 ns-NS ↓8.70-* ↓4.81-* ↓2.98-NS ↓2.38-NS
CES1 ns-NS ↓2.91-NS ↓2.93-NS ↓2.71-NS ns-NS
AADAC ns-NS ↓2.68-NS ↓8.70-* ↓2.97-NS ↓3.19-*
LIPE ns-NS ↑3.93-* ns-NS ns-NS ns-NS
RAL→ROL reductase AKR1C1 ns-NS ↓4.03-NS ↓5.05-NS ↓4.63-* ↓3.53-*
RDH13 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
 
Open in a new tab

Notation has two parts: fold change – adjusted p value significance

Fold change notations

↑

up reguated more than 2-fold, followed by the numerical fold change

↓

down regulate more than 2-fold, followed by the numerical fold change

NDb: not detected in baseline. Consider as up regulated significantly.

ns: fold change is less than 2-fold. Consider as not significant.

Adjusted p value notations

*

p value of the fold change is less than 0.05, consider as statistically significant.

NS p value of the fold change is greater than 0.05, consider as statistically not significant.

Table 2.

Retinoid pathway proteins changed in NHP heart after 12 Gy PBI/BM2.5
RA biosynthesis pathway Gene left heart
 right heart
day 4 day 8–9 day 11–12 day 15 day 21–22 day 4 day 8–9 day 11–12 day 15 day 21–22
Chaperone TTR ns-NS ns-NS ↓2.25-NS ↓2.04-NS ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS
RBP4 ↓ ND-* ns-NS ↓ ND-* ↓ ND-* ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS
RBP1 ↓3.45-* ns-NS ↓3.53-* ↓2.82-NS ns-NS ↑3.18-* ns-NS ns-NS ns-NS ns-NS
APOA1 ↓2.09-NS ↓2.05-NS ↓2.94-NS ↓2.25-NS ↓2.08-NS ns-NS ↓2.01-NS ns-NS ns-NS ↓2.04-NS
ROL→ dihydroretinol RETSAT ns-NS ↓2.75-* ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS ↓2.75-*
ROL→RAL RDH14 ns-NS ns-NS ns-NS ↓2.91-NS ↓2.75-NS ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
RAL→RA ALDH1A1 ↓3.76-* ns-NS ↓3.83-* ns-NS ↓3.37-NS ns-NS ns-NS ns-NS ns-NS ns-NS
ALDH1A2 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
β-Carotene→apo-carotenals BCO2 ↓ ND-* ↓2.06-NS ns-NS nv-NV ns-NS ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* nv-NV
RE→ROL CES1 ↓ ND-* ↓2.87-* ↓ ND-* ↓ ND-* ↓ ND-* ↑3.62-* ns-NS ns-NS ↑2.99-* ↑2.45-*
LIPE ↓14.93-* ↓3.15-* ↓4.07-* ↓3.11-* ↓ ND-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
Open in a new tab

Notation has two parts: fold change – adjusted p value significance

Fold change notations

↑

up reguated more than 2-fold, followed by the numerical fold change

↓

down regulate more than 2-fold, followed by the numerical fold change

NDb: not detected in baseline. Consider as up regulated significantly.

ND: not detected in this time after radiation. Consider as down regulated significantly.

nv: no value in fold change.

ns: fold change is less than 2-fold. Consider as not significant.

Adjusted p value notations

*

p value of the fold change is less than 0.05, consider as statistically significant.

NS p value of the fold change is greater than 0.05, consider as statistically not significant.

NV: no p value associate with this comparison.

Expression of fibrosis related proteins were altered by radiation across multiple tissues.

Because we observed a reduction in all tissue and plasma RA and because dysregulation of RA signaling has been widely reported to be involved in the development and progression of fibrosis (Wang et al. 2019), we interrogated the proteomic profiles for proteins that were related to fibrosis. Table 3 – Table 6 show the proteomic results for plasma (Table 3), jejunum (Table 4), lung (Table 5), and heart (Table 6). Upregulation of collagens and fibrinogens are seen throughout all of the tissues. Some proteins in the left and right ventricle of heart had opposite trends (Table 6). Also of note, inflammatory protein serum amyloid A1 (SAA1) was increased in expression across all the tissues and plasma.

Table 3.

Fibrosis related proteins changed in NHP plasma after 12 Gy PBI/BM2.5
Gene day 4 day 8–9 day 11–12 day 15 day 21–22
ACTA2 ↓2.00-NS ↓10.42-* ↓7.30-* ↓6.80-* ↓3.21-NS
COL1A1 ↑2.14-NS ns-NS ns-NS ns-NS ↑3.35-NS
COL3A1 ↑6.11-NS ↑8.53-NV ↑20.96-NV ↑4.39-NV ↓ND-NV
FGA ↑2.07-NS ↑2.14-NS ↑2.34-* ↑3.41-* ↑2.70-*
FGB ns-NS ns-NS ns-NS ↑2.17-* ns-NS
FGG ↑2.06-NS ns-NS ↑2.02-* ↑2.58-* ↑2.33-NS
KRT1 ns-NS ns-NS ns-NS ↑2.98-* ↑4.40-*
KRT5 ↓2.30-NS ns-NS ns-NS ns-NS ns-NS
KRT10 ns-NS ↑6.09-* ↑4.59-* ↑5.44-* ↑24.51-*
SAA1 ↑7.71-NS ↑52.79-* ↑61.86-* ↑44.30-* ↑11.60-NS
Open in a new tab

Notation has two parts: fold change – adjusted p value significance

Fold change notations

↑

up reguated more than 2-fold, followed by the numerical fold change

↓

down regulate more than 2-fold, followed by the numerical fold change

NDb: not detected in baseline. Consider as up regulated significantly.

ND: not detected in this time after radiation. Consider as down regulated significantly.

nv: no value in fold change.

ns: fold change is less than 2-fold. Consider as not significant.

Adjusted p value notations

*

p value of the fold change is less than 0.05, consider as statistically significant.

NS p value of the fold change is greater than 0.05, consider as statistically not significant.

NV: no p value associate with this comparison.

Table 6.

Fibrosis related proteins changed in NHP heart after 12 Gy PBI/BM2.5
Gene name left heart-day 4 left heart-day 8–9 left heart-day 11–12 left heart-day 15 left heart-day 21–22 right heart-day 4 right heart-day 8–9 right heart-day 11–12 right heart-day 15 right heart-day 21–22
COL1A1 ↓2.51-NS ↓2.10-NS ↓2.21-NS ↓2.62-NS ↓4.46-* ↑3.72-* ↑2.63-* ↑2.05-NS ↑2.54-NS ns-NS
COL3A1 ↓12.82-* ↓3.38-* ↓3.52-* ↓4.78-* ↓4.41-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
COL4A1 ns-NS ns-NS ns-NS ns-NS ns-NS ↑5.84-* ↑4.39-* ↑3.71-* ↑3.08-* ↑2.97-*
FGA ns-NS ↑2.75-* ↑2.00-NS ↑2.70-* ns-NS ↑3.84-* ↑4.02-* ↑5.53-* ↑5.56-* ↑2.94-*
FGB ns-NS ns-NS ↑2.10-NS ns-NS ns-NS ↓3.01-* ↓3.10-* ↓2.82-* ↓2.11-NS ↓4.44-*
FGG ns-NS ↑2.18-* ↑2.46-NS ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS
KRT1 ns-NS ↓2.18-NS ↓2.12-NS ns-NS ↓2.67-NS ns-NS ns-NS ns-NS ns-NS ns-NS
KRT5 nv-NV nv-NV nv-NV nv-NV nv-NV ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
KRT8 ns-NS ns-NS ↑3.14-* ns-NS ↓3.05-NS ↓2.91-* ns-NS ↓2.33-NS ns-NS ↓2.75-*
LAMA2 ns-NS ns-NS ns-NS ns-NS ns-NS ns-NS ↑2.10-* ↑2.09-* ns-NS ns-NS
MFAP2 nv-NV nv-NV nv-NV nv-NV nv-NV ↑5.09-* ↑2.72-* ↑4.22-* ↑3.55-* ↑3.60-*
MFAP4 ↓2.06-NS ns-NS ns-NS ns-NS ns-NS ↑6.72-* ↑2.76-* ↑3.42-* ↑3.74-* ↑3.91-*
SAA1 ↑10.81-* ↑30.10-* ↑30.50-* ↑34.87-* ↑9.05-* ↑28.52-* ↑82.42-* ↑94.89-* ↑99.23-* ↑25.12-*
Open in a new tab

Notation has two parts: fold change – adjusted p value significance

Fold change notations

↑

up reguated more than 2-fold, followed by the numerical fold change

↓

down regulate more than 2-fold, followed by the numerical fold change

NDb: not detected in baseline. Consider as up regulated significantly.

ND: not detected in this time after radiation. Consider as down regulated significantly.

nv: no value in fold change.

ns: fold change is less than 2-fold. Consider as not significant.

Adjusted p value notations

*

p value of the fold change is less than 0.05, consider as statistically significant.

NS p value of the fold change is greater than 0.05, consider as statistically not significant.

NV: no p value associate with this comparison.

Table 4.

Fibrosis related proteins changed in NHP jejunum after 12 Gy PBI/BM2.5
Gene name day 4 day 8–9 day 11–12 day 15 day 21–22
COL1A1 ↑3.08-* ↑7.67-* ↑3.28-NS ns-NS ns-NS
COL3A1 ↑15.93-* ↑88.30-* ↑34.97-* ↑15.43-* ↑19.24-*
COL4A1 ↑2.66-* ↑7.96-* ↑6.42-* ↑3.90-* ns-NS
COL4A2 ns-NS ↑4.07-* ↑3.03-NS ns-NS ns-NS
COL6A3 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
FGA ↑3.93-* ↑10.00-* ↑8.10-* ↑6.29-* ↑3.95-*
FGB ↑2.89-* ↑6.99-* ↑6.52-* ↑3.99-* ↑2.66-NS
FGG ↑3.46-* ↑8.18-* ↑6.88-* ↑4.36-* ↑3.06-NS
MFAP4 ns-NS ↑4.21-* ↑3.37-NS ↑2.42-NS ↑3.16-*
MMP2 nv-NV ↑ NDb-* ↑ NDb-* nv-NV nv-NV
TIMP1 ns-NS ↑2.72-NS ↑2.79-NS ns-NS ↑2.08-NS
MSLN ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* nv-NV
SAA1 ↑9.64-* ↑40.97-* ↑41.47-* ↑23.71-* ↑12.36-*
YAP1 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
Open in a new tab

Notation has two parts: fold change – adjusted p value significance

Fold change notations

↑

up reguated more than 2-fold, followed by the numerical fold change

↓

down regulate more than 2-fold, followed by the numerical fold change

NDb: not detected in baseline. Consider as up regulated significantly.

ND: not detected in this time after radiation. Consider as down regulated significantly.

nv: no value in fold change.

ns: fold change is less than 2-fold. Consider as not significant.

Adjusted p value notations

*

p value of the fold change is less than 0.05, consider as statistically significant.

NS p value of the fold change is greater than 0.05, consider as statistically not significant.

NV: no p value associate with this comparison.

Table 5.

Fibrosis related proteins changed in NHP lung after 12 Gy PBI/BM2.5
Gene name lung-day 4 lung-day 8–9 lung-day 11–12 lung-day 15 lung-day 21–22
COL1A1 ns-NS ns-NS ns-NS ns-NS ↑2.09-*
COL4A1 ns-NS ns-NS ns-NS ns-NS ↑2.09-*
TIMP1 ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-* ↑ NDb-*
TIMP3 ↓ ND-* ns-NS ns-NS ns-NS ns-NS
FGA ns-* ↑3.41-* ↑3.92-* ↑3.67-* ns-NS
FGB ns-NS ↑2.60-* ↑2.70-* ↑2.20-* ns-NS
FGG ns-NS ↑2.51-* ↑2.85-* ↑2.46-* ns-NS
KRT5 ns-NS ns-NS ns-NS ↑2.17-* ↑3.69-*
SAA1 ↑28.86-* ↑138.17-* ↑199.32-* ↑132.29-* ↑33.97-*
YAP1 ↑2.05-NS ↑2.45-* ↑2.08-* ns-NS ns-NS
Open in a new tab

Notation has two parts: fold change – adjusted p value significance

Fold change notations

↑

up reguated more than 2-fold, followed by the numerical fold change

↓

down regulate more than 2-fold, followed by the numerical fold change

NDb: not detected in baseline. Consider as up regulated significantly.

ND: not detected in this time after radiation. Consider as down regulated significantly.

nv: no value in fold change.

ns: fold change is less than 2-fold. Consider as not significant.

Adjusted p value notations

*

p value of the fold change is less than 0.05, consider as statistically significant.

NS p value of the fold change is greater than 0.05, consider as statistically not significant.

NV: no p value associate with this comparison.

DISCUSSION

RA is a master regulator of cell proliferation, differentiation, and apoptosis and an essential diet-derived nutrient (Napoli 2020). It has roles in inflammatory signaling and the development of fibrosis (Taylor et al. 2015; Wang et al. 2019). As facets of inflammation and fibrosis are regulated by RA, we postulated that the radiation-induced reduction in RA could be associated with changes in protein expression regulated by or associated with RA that were relevant to the development of fibrosis and/or inflammation.

In regards to retinol (vitamin A) (Fig. 2) and RE (storage) (Fig. 3), we did not expect to see a dramatic change in ROL or RE after radiation over the timescale of this study. The animals are nutritionally replete before IR, so they would have adequate storage of vitamin A and are provided a vitamin A containing diet. Vitamin A deficiency usually takes a longer period to manifest, as it takes time to deplete stored reserves of vitamin A before achieving deficiency. Accordingly, the data shows no significant difference for ROL and RE when compared to the naïve group in all tissue types measured. This finding is significant because it indicates the decrease in RA is not due to a lack of substrate to biosynthesize RA. There are sufficient levels of ROL and RE, but depleted levels of RA that indicate that there is either a defect in the biosynthesis of RA from ROL or an increased degradation of RA after IR.

Jejunum and lung had the highest levels of RA and experienced significant decreases in the abundance of RA after radiation that progressively declined over time. The reduction in RA in jejunum after IR observed here is consistent with a decrease in jejunum RA after radiation as well as in other models of GI damage where the epithelium of the gut is damaged (Byrareddy et al. 2016; Huang et al. 2019b). As jejunum had the highest levels of RA, it seems to be a major contributor to the plasma pool of circulating RA consistent with previously observed correlation between the levels of jejunum RA and the levels of plasma RA(Huang et al. 2020a). The level of reduction in jejunum RA would likely effect epithelial barrier integrity as well as the differentiation and identity of immune-relevant cell populations within the small intestine (Czarnewski et al. 2017). In jejunum, decreased dehydrogenase/reductase 4 (DHRS4) (retinol dehydrogenase), increased retinol dehydrogenase 13 (RDH13) (retinal reductase) and reduced aldehyde dehydrogenase (ALDH), ALDH1A1 and ALDH1A3, likely contribute to the reduction in RA. ALDH1A2 is enriched in immune cells and the upregulation of ALDH1A2 is a sign of inflammatory response after radiation in jejunum (Cassani et al. 2012; Czarnewski et al. 2017). The reduction in RA in lung after IR agrees with our previous observations that lung RA in mouse. In those studies, mouse lung RA is decreased shortly after IR (24h) and this reduction persists through 180 days post-IR, never recovering back to baseline levels days (Jones et al. 2014; Huang et al. 2019a). This persistent reduction in RA represents a major molecular disruption in cellular signaling that could be an initiating precursor to fibrosis as well as barrier to full repair by therapeutic agents. RA has been shown to be a significant factor in the development of various types of fibrosis (Wang et al. 2019).

Radiation damage occurred in the left heart first and was more severe than the right heart. The left ventricle is the thickest of the heart’s chambers and is responsible for pumping oxygenated blood to tissues. During cardiomyopathy and hypertrophy, left ventricle function is greatly affected. Over time, the rest of the chambers can become affected. RA is important for heart development and heart failure (Wang et al. 2018a; Wang et al. 2018b; Yang et al. 2021). In the left heart, reduced RA may be due to reduced retinol-binding protein, type 1 (RBP1) (chaperone) and RDH14 (retinol dehydrogenase). BCO2 was upregulated in right heart and also in jejunum; asymmetric cleavage of beta-carotene yields apo-carotenals that have been reported to be upregulated in various conditions of cellular stress and able to mediate signaling and biological activity (Harrison and Quadro 2018; Harrison and Kopec 2020).

A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Collagens were significant upregulated after radiation in several tissue. Collagen deposition (especially collagen I, collagen III) adds stiffness to tissues and contributes to the development of pulmonary fibrosis. Mechanical forces have been shown to be important to myofibroblast activation and matrix deposition in lung (Liu et al. 2010; Balestrini et al. 2012; Huang et al. 2012; Wells 2013). Increased matrix stiffness from the deposition of fibrillary collagens increases alpha-smooth muscle actin (α-SMA) as well as transforming growth factor-beta (TGF-β). TGF-β is regulated by RA and is an important and potent pro-fibrotic factor and inducer of epithelial-to-mesenchymal transition (EMT) that undergoes activation due to mechanical tension (Glick et al. 1991; Wells 2013). Fibrinogen, microfibril-associated glycoprotein 4 (MFAP4), several keratin, and SAA1 were upregulated. Elevated levels of SAA1 have been observed after radiation exposure and SAA1 has been reported to have potential biodosimetry and biomarker utility for radiation exposure in multiple species (Ossetrova and Blakely 2009; Bazan et al. 2014; Nylund et al. 2014; Blakely et al. 2018; Ossetrova et al. 2018; Sproull et al. 2019). Keratin-5 (Krt5) is expressed in human airway basal cells (Rock et al. 2010). Fibrinogen is encoded by three genes: FGA, FGB, FGG where each gene is transcribed and translated separately to produce proteins containing different numbers of amino acids. Fibrinogen is increased during injury, infection, and inflammation (Thompson et al. 1989). Interleukin-6 (IL6) stimulates fibrinogen expression both in vivo and in vitro, including in the lung epithelium (Rokita et al. 1993; Duan and Simpson-Haidaris 2006). FGA, FGB, and FGG are also regulated by Forkhead box protein O1 (FOXO1), a significant transcription factor in the epithelial-to-mesenchymal transition(Wang et al. 2013). Matrix metalloproteinase-2 (MMP2), tissue inhibitor of metalloproteinases 1 (TIMP1), mesothelin (MSLN) and yes-associated protein 1 (YAP) were turned on after radiation. Increased YAP in jejunum and lung is of interest because YAP1 is a downstream effector of the Hippo pathway that regulates target gene expression. Hippo signaling is crucial to tissue homeostasis and regeneration of intestinal epithelial cells (Chang et al. 2019). Constitutively active YAP1 leads to contact inhibition in epithelial cells as well as mislocalized Paneth cells and reduced intestinal stem cell numbers resulting in loss of cellular proliferation in crypts (Zhao et al. 2010; Barry et al. 2013). Dysregulation of YAP/ Tafazzin (TAZ) signaling causes pulmonary fibrosis (Zhu et al. 2020).

In animal models, treatment with RA reversed pulmonary fibrosis in a model of radiation-induced pulmonary fibrosis by inhibiting proliferation and reducing collagen synthesis. Tabata et al. reported RA reduced radiation-induced increases in TGF-β1 production through the p38 mitogen-activated protein kinase (p38MAPK)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Based on in vitro studies, Tabata et al. also reported that RA reduced radiation-induced production of IL-6, TGF-β1, α-SMA, collagen 1A1, and activated NF-κB p65 in lung fibroblasts (Tabata et al. 2006a; Tabata et al. 2006b). RA administration has also been shown to be protective towards cell injury and extracellular matrix (ECM) accumulation by reducing collagen I mRNA, and inhibiting TGF-β and connective tissue growth factor (CTGF) expression (Davis et al. 1990; Ye and Dan 2010). Administration of RA to vitamin A-deficient (VAD) animals restored the concentration of parenchymal elastic fibers and lung mechanical properties (McGowan et al. 2005; McGowan and Holmes 2007). Additionally, RA treatment of VAD animals reduced the amount of collagen IV and downregulated inflammatory cytokine expression, including IL-1α, IL-1β, and TNFα (Esteban-Pretel et al. 2010).

CONCLUSIONS

Here we sought to more thoroughly survey radiation-induced changes in retinoids as well as proteins related to and targets of the retinoid pathway in the NHP after high dose radiation with minimal bone marrow sparing (12 Gy PBI/BM2.5). Retinoic acid was decreased in plasma as well as in lung, heart, and jejunum over time, indicating a global disruption of RA homeostasis after IR. A number of proteins associated with fibrosis and with RA were significantly altered after radiation. Together these data indicate that a local deficiency of endogenous RA presents a permissive environment for fibrotic transformation.

Footnotes

The authors declare no conflicts of interest.

REFERENCES

  1. Balestrini JL, Chaudhry S, Sarrazy V, Koehler A, Hinz B. The mechanical memory of lung myofibroblasts. Integr Biol (Camb) 4: 410–21; 2012. [DOI] [PubMed] [Google Scholar]
  2. Barry ER, Morikawa T, Butler BL, Shrestha K, de la Rosa R, Yan KS, Fuchs CS, Magness ST, Smits R, Ogino S, Kuo CJ, Camargo FD. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493: 106–10; 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barua AB, Sidell N. Retinoyl beta-glucuronide: a biologically active interesting retinoid. J Nutr 134: 286S–289S; 2004. [DOI] [PubMed] [Google Scholar]
  4. Bazan JG, Chang P, Balog R, D’Andrea A, Shaler T, Lin H, Lee S, Harrison T, Shura L, Schoen L, Knox SJ, Cooper DE. Novel human radiation exposure biomarker panel applicable for population triage. Int J Radiat Oncol Biol Phys 90: 612–9; 2014. [DOI] [PubMed] [Google Scholar]
  5. Berry DC, Noy N. Signaling by vitamin A and retinol-binding protein in regulation of insulin responses and lipid homeostasis. Biochim Biophys Acta 1821: 168–76; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blakely WF, Bolduc DL, Debad J, Sigal G, Port M, Abend M, Valente M, Drouet M, Herodin F. Use of Proteomic and Hematology Biomarkers for Prediction of Hematopoietic Acute Radiation Syndrome Severity in Baboon Radiation Models. Health Phys 115: 29–36; 2018. [DOI] [PubMed] [Google Scholar]
  7. Byrareddy SN, Arthos J, Cicala C, Villinger F, Ortiz KT, Little D, Sidell N, Kane MA, Yu J, Jones JW, Santangelo PJ, Zurla C, McKinnon LR, Arnold KB, Woody CE, Walter L, Roos C, Noll A, Van Ryk D, Jelicic K, Cimbro R, Gumber S, Reid MD, Adsay V, Amancha PK, Mayne AE, Parslow TG, Fauci AS, Ansari AA. Sustained virologic control in SIV+ macaques after antiretroviral and alpha4beta7 antibody therapy. Science 354: 197–202; 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cassani B, Villablanca EJ, De Calisto J, Wang S, Mora JR. Vitamin A and immune regulation: role of retinoic acid in gut-associated dendritic cell education, immune protection and tolerance. Mol Aspects Med 33: 63–76; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang YC, Wu JW, Wang CW, Jang AC. Hippo signaling-mediated mechanotransduction in cell movement and cancer metastasis. Front Mol Biosci 6: 157; 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen N, Napoli JL. All-trans-retinoic acid stimulates translation and induces spine formation in hippocampal neurons through a membrane-associated RARalpha. FASEB J 22: 236–45; 2008. [DOI] [PubMed] [Google Scholar]
  11. Czarnewski P, Das S, Parigi SM, Villablanca EJ. Retinoic acid and Its role in modulating intestinal innate immunity. Nutrients 9; 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davis BH, Kramer RT, Davidson NO. Retinoic acid modulates rat Ito cell proliferation, collagen, and transforming growth factor beta production. J Clin Invest 86: 2062–70; 1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ding NH, Li JJ, Sun LQ. Molecular mechanisms and treatment of radiation-induced lung fibrosis. Curr Drug Targets 14: 1347–56; 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dorfer V, Pichler P, Stranzl T, Stadlmann J, Taus T, Winkler S, Mechtler K. MS Amanda, a universal identification algorithm optimized for high accuracy tandem mass spectra. J Proteome Res 13: 3679–84; 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Duan HO, Simpson-Haidaris PJ. Cell type-specific differential induction of the human gamma-fibrinogen promoter by interleukin-6. J Biol Chem 281: 12451–7; 2006. [DOI] [PubMed] [Google Scholar]
  16. Eng JK, Fischer B, Grossmann J, Maccoss MJ. A fast SEQUEST cross correlation algorithm. J Proteome Res 7: 4598–602; 2008. [DOI] [PubMed] [Google Scholar]
  17. Esteban-Pretel G, Marin MP, Renau-Piqueras J, Barber T, Timoneda J. Vitamin A deficiency alters rat lung alveolar basement membrane: reversibility by retinoic acid. J Nutr Biochem 21: 227–36; 2010. [DOI] [PubMed] [Google Scholar]
  18. Farese AM, Bennett AW, Gibbs AM, Hankey KG, Prado K, Jackson W 3rd, MacVittie TJ. Efficacy of neulasta or neupogen on H-ARS and GI-ARS mortality and hematopoietic recovery in nonhuman primates after 10-Gy irradiation with 2.5% bone marrow sparing. Health Phys 116: 339–353; 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Germain P, Chambon P, Eichele G, Evans RM, Lazar MA, Leid M, De Lera AR, Lotan R, Mangelsdorf DJ, Gronemeyer H. International Union of Pharmacology. LX. Retinoic acid receptors. Pharmacol Rev 58: 712–25; 2006a. [DOI] [PubMed] [Google Scholar]
  20. Germain P, Chambon P, Eichele G, Evans RM, Lazar MA, Leid M, De Lera AR, Lotan R, Mangelsdorf DJ, Gronemeyer H. International Union of Pharmacology. LXIII. Retinoid X receptors. Pharmacol Rev 58: 760–72; 2006b. [DOI] [PubMed] [Google Scholar]
  21. Glick AB, McCune BK, Abdulkarem N, Flanders KC, Lumadue JA, Smith JM, Sporn MB. Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat. Development 111: 1081–6; 1991. [DOI] [PubMed] [Google Scholar]
  22. Gudas LJ. Emerging roles for retinoids in regeneration and differentiation in normal and disease states. Biochim Biophys Acta 1821: 213–21; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hankey KG, Farese AM, Blaauw EC, Gibbs AM, Smith CP, Katz BP, Tong Y, Prado KL, MacVittie TJ. Pegfilgrastim Improves Survival of Lethally Irradiated Nonhuman Primates. Radiat Res 183: 643–55; 2015. [DOI] [PubMed] [Google Scholar]
  24. Harrison EH, Kopec RE. Enzymology of vertebrate carotenoid oxygenases. Biochim Biophys Acta Mol Cell Biol Lipids: 158653; 2020. [DOI] [PMC free article] [PubMed]
  25. Harrison EH, Quadro L. Apocarotenoids: emerging roles in mammals. Annu Rev Nutr 38: 153–172; 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Huang W, Yu J, Jones JW, Carter CL, Jackson IL, Vujaskovic Z, MacVittie TJ, Kane MA. Acute Proteomic Changes in the Lung After WTLI in a Mouse Model: Identification of Potential Initiating Events for Delayed Effects of Acute Radiation Exposure. Health Phys 116: 503–515; 2019a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Huang W, Yu J, Jones JW, Carter CL, Pierzchalski K, Tudor G, Booth C, MacVittie TJ, Kane MA. Proteomic evaluation of the acute radiation syndrome of the gastrointestinal tract in a murine total-body irradiation model. Health Phys 116: 516–528; 2019b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Huang W, Yu J, Liu T, Defnet AE, Zalesak S, Farese AM, MacVittie TJ, Kane MA. Proteomics of Non-human Primate Plasma after Partial-body Radiation with Minimal Bone Marrow Sparing. Health Phys 119: 621–632; 2020a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Huang W, Yu J, Liu T, Tudor G, Defnet AE, Zalesak S, Kumar P, Booth C, Farese AM, MacVittie TJ, Kane MA. Proteomic Evaluation of the Natural History of the Acute Radiation Syndrome of the Gastrointestinal Tract in a Non-human Primate Model of Partial-body Irradiation with Minimal Bone Marrow Sparing Includes Dysregulation of the Retinoid Pathway. Health Phys 119: 604–620; 2020b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Huang WY J; Jones JW; Carter CL; Jackson IL; Vujaskovic Z; MacVittie TJ; Kane MA Acute proteomic changes in lung after whole thorax lung irradiation in a mouse model: identification of potential initiating events for delayed effects of acute radiation exposure. Health Phys 116: 503–515; 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Huang X, Yang N, Fiore VF, Barker TH, Sun Y, Morris SW, Ding Q, Thannickal VJ, Zhou Y. Matrix stiffness-induced myofibroblast differentiation is mediated by intrinsic mechanotransduction. Am J Respir Cell Mol Biol 47: 340–8; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Jones JW, Pierzchalski K, Yu J, Kane MA. Use of fast HPLC multiple reaction monitoring cubed for endogenous retinoic acid quantification in complex matrices. Anal Chem 87: 3222–30; 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jones JW, Scott AJ, Tudor G, Xu PT, Jackson IL, Vujaskovic Z, Booth C, MacVittie TJ, Ernst RK, Kane MA. Identification and quantitation of biomarkers for radiation-induced injury via mass spectrometry. Health Phys 106: 106–19; 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4: 923–5; 2007. [DOI] [PubMed] [Google Scholar]
  35. Kane MA, Chen N, Sparks S, Napoli JL. Quantification of endogenous retinoic acid in limited biological samples by LC/MS/MS. Biochem J 388: 363–9; 2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kane MA, Folias AE, Napoli JL. HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues. Anal Biochem 378: 71–9; 2008a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kane MA, Folias AE, Wang C, Napoli JL. Quantitative profiling of endogenous retinoic acid in vivo and in vitro by tandem mass spectrometry. Anal Chem 80: 1702–8; 2008b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kane MA, Napoli JL. Quantification of endogenous retinoids. Methods Mol Biol 652: 1–54; 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Liu F, Mih JD, Shea BS, Kho AT, Sharif AS, Tager AM, Tschumperlin DJ. Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression. J Cell Biol 190: 693–706; 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. MacVittie TJ, Bennett A, Booth C, Garofalo M, Tudor G, Ward A, Shea-Donohue T, Gelfond D, McFarland E, Jackson W 3rd, Lu W, Farese AM. The prolonged gastrointestinal syndrome in rhesus macaques: the relationship between gastrointestinal, hematopoietic, and delayed multi-organ sequelae following acute, potentially lethal, partial-body irradiation. Health Phys 103: 427–53; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. MacVittie TJ, Farese AM, Parker GA, Jackson W 3rd. The time course of radiation-induced lung injury in a nonhuman primate model of partial-body irradiation with minimal bone marrow sparing: clinical and radiographic evidence and the effect of neupogen administration. Health Phys 116: 366–382; 2019a. [DOI] [PubMed] [Google Scholar]
  42. MacVittie TJ, Farese AM, Parker GA, Jackson W 3rd, Booth C, Tudor GL, Hankey KG, Potten. The Gastrointestinal subsyndrome of the acute radiation syndrome in rhesus macaques: a systematic review of the lethal dose-response relationship with and without medical management. Health Phys 116: 305–338; 2019b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. McGowan SE, Holmes AJ. Vitamin A deficiency alters pulmonary parenchymal collagen and tissue mechanics. Respir Physiol Neurobiol 156: 312–9; 2007. [DOI] [PubMed] [Google Scholar]
  44. McGowan SE, Takle EJ, Holmes AJ. Vitamin A deficiency alters the pulmonary parenchymal elastic modulus and elastic fiber concentration in rats. Respir Res 6: 77; 2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Napoli JL. Physiological insights into all-trans-retinoic acid biosynthesis. Biochim Biophys Acta 1821: 152–67; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Napoli JL. Cellular retinoid binding-proteins, CRBP, CRABP, FABP5: Effects on retinoid metabolism, function and related diseases. Pharmacol Ther 173: 19–33; 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Napoli JL. Post-natal all-trans-retinoic acid biosynthesis. Methods Enzymol 637: 27–54; 2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Nylund R, Lemola E, Hartwig S, Lehr S, Acheva A, Jahns J, Hildebrandt G, Lindholm C. Profiling of low molecular weight proteins in plasma from locally irradiated individuals. J Radiat Res 55: 674–82; 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ossetrova NI, Blakely WF. Multiple blood-proteins approach for early-response exposure assessment using an in vivo murine radiation model. Int J Radiat Biol 85: 837–50; 2009. [PubMed] [Google Scholar]
  50. Ossetrova NI, Stanton P, Krasnopolsky K, Ismail M, Doreswamy A, Hieber KP. Biomarkers for radiation biodosimetry and injury assessment after mixed-field (neutron and gamma) radiation in the mouse total-body irradiation model. Health Phys; 2018. [DOI] [PubMed]
  51. Parker GA, Li N, Takayama K, Booth C, Tudor GL, Farese AM, MacVittie TJ. Histopathological features of the development of intestine and mesenteric lymph node injury in a nonhuman primate model of partial-body irradiation with minimal bone marrow sparing. Health Phys 116: 426–446; 2019a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Parker GA, Li N, Takayama K, Farese AM, MacVittie TJ. Lung and heart injury in a nonhuman primate model of partial-body irradiation with minimal bone marrow sparing: histopathological evidence of lung and heart injury. Health Phys 116: 383–400; 2019b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Piskunov A, Rochette-Egly C. A retinoic acid receptor RARalpha pool present in membrane lipid rafts forms complexes with G protein alphaQ to activate p38MAPK. Oncogene 31: 3333–45; 2012. [DOI] [PubMed] [Google Scholar]
  54. Raghuvanshi S, Reed V, Blaner WS, Harrison EH. Cellular localization of beta-carotene 15,15’ oxygenase-1 (BCO1) and beta-carotene 9’,10’ oxygenase-2 (BCO2) in rat liver and intestine. Arch Biochem Biophys 572: 19–27; 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rock JR, Randell SH, Hogan BL. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling. Dis Model Mech 3: 545–56; 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Rodemann HP, Blaese MA. Responses of normal cells to ionizing radiation. Semin Radiat Oncol 17: 81–8; 2007. [DOI] [PubMed] [Google Scholar]
  57. Rokita H, Neta R, Sipe JD. Increased fibrinogen synthesis in mice during the acute phase response: co-operative interaction of interleukin 1, interleukin 6, and interleukin 1 receptor antagonist. Cytokine 5: 454–8; 1993. [DOI] [PubMed] [Google Scholar]
  58. Samokyszyn VM, Gall WE, Zawada G, Freyaldenhoven MA, Chen G, Mackenzie PI, Tephly TR, Radominska-Pandya A. 4-hydroxyretinoic acid, a novel substrate for human liver microsomal UDP-glucuronosyltransferase(s) and recombinant UGT2B7. J Biol Chem 275: 6908–14; 2000. [DOI] [PubMed] [Google Scholar]
  59. Sass JO, Forster A, Bock KW, Nau H. Glucuronidation and isomerization of all-trans- and 13-cis-retinoic acid by liver microsomes of phenobarbital- or 3-methylcholanthrene-treated rats. Biochem Pharmacol 47: 485–92; 1994. [DOI] [PubMed] [Google Scholar]
  60. Shea-Donohue T, Fasano A, Zhao A, Notari L, Yan S, Sun R, Bohl JA, Desai N, Tudor G, Morimoto M, Booth C, Bennett A, Farese AM, MacVittie TJ. Mechanisms involved in the development of the chronic gastrointestinal syndrome in nonhuman primates after total-body irradiation with bone marrow shielding. Radiat Res 185: 591–603; 2016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Sproull M, Shankavaram U, Camphausen K. Comparison of Proteomic Biodosimetry Biomarkers Across Five Different Murine Strains. Radiat Res 192: 640–648; 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tabata C, Kadokawa Y, Tabata R, Takahashi M, Okoshi K, Sakai Y, Mishima M, Kubo H. All-trans-retinoic acid prevents radiation- or bleomycin-induced pulmonary fibrosis. Am J Respir Crit Care Med 174: 1352–60; 2006a. [DOI] [PubMed] [Google Scholar]
  63. Tabata C, Kubo H, Tabata R, Wada M, Sakuma K, Ichikawa M, Fujita S, Mio T, Mishima M. All-trans retinoic acid modulates radiation-induced proliferation of lung fibroblasts via IL-6/IL-6R system. Am J Physiol Lung Cell Mol Physiol 290: L597–606; 2006b. [DOI] [PubMed] [Google Scholar]
  64. Taylor RN, Kane MA, Sidell N. Pathogenesis of Endometriosis: Roles of Retinoids and Inflammatory Pathways. Semin Reprod Med 33: 246–56; 2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Thompson C, Blumenstock FA, Saba TM, Feustel PJ, Kaplan JE, Fortune JB, Hough L, Gray V. Plasma fibronectin synthesis in normal and injured humans as determined by stable isotope incorporation. J Clin Invest 84: 1226–35; 1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Topletz AR, Thatcher JE, Zelter A, Lutz JD, Tay S, Nelson WL, Isoherranen N. Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases. Biochem Pharmacol 83: 149–63; 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Wang H, Meyer CA, Fei T, Wang G, Zhang F, Liu XS. A systematic approach identifies FOXA1 as a key factor in the loss of epithelial traits during the epithelial-to-mesenchymal transition in lung cancer. BMC Genomics 14: 680; 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Wang S, Huang W, Castillo HA, Kane MA, Xavier-Neto J, Trainor PA, Moise AR. Alterations in retinoic acid signaling affect the development of the mouse coronary vasculature. Dev Dyn 247: 976–991; 2018a. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Wang S, Moise AR. Recent insights on the role and regulation of retinoic acid signaling during epicardial development. Genesis 57: e23303; 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wang S, Yu J, Jones JW, Pierzchalski K, Kane MA, Trainor PA, Xavier-Neto J, Moise AR. Retinoic acid signaling promotes the cytoskeletal rearrangement of embryonic epicardial cells. FASEB J: fj201701038R; 2018b. [DOI] [PMC free article] [PubMed]
  71. Wang S, Yu J, Kane MA, Moise AR. Modulation of retinoid signaling: therapeutic opportunities in organ fibrosis and repair. Pharmacology & Therapeutics: In revision; 2019. [DOI] [PMC free article] [PubMed]
  72. Wang S, Yu J, Kane MA, Moise AR. Modulation of retinoid signaling: therapeutic opportunities in organ fibrosis and repair. Pharmacol Ther 205: 107415; 2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Wells RG. Tissue mechanics and fibrosis. Biochim Biophys Acta 1832: 884–90; 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Yang N, Parker L, Yu J, Jones JW, Liu T, Papanicolaou KN, Talbot CC Jr., Margulies KB, O’Rourke B, Kane M, Foster DB. Cardiac retinoic acid levels decline in heart failure. JCI Insight; 2021. [DOI] [PMC free article] [PubMed]
  75. Ye Y, Dan Z. All-trans retinoic acid diminishes collagen production in a hepatic stellate cell line via suppression of active protein-1 and c-Jun N-terminal kinase signal. J Huazhong Univ Sci Technolog Med Sci 30: 726–33; 2010. [DOI] [PubMed] [Google Scholar]
  76. Zhao B, Li L, Tumaneng K, Wang CY, Guan KL. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP). Genes Dev 24: 72–85; 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Zhong G, Ortiz D, Zelter A, Nath A, Isoherranen N. CYP26C1 Is a Hydroxylase of Multiple Active Retinoids and Interacts with Cellular Retinoic Acid Binding Proteins. Molecular pharmacology 93: 489–503; 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Zhu T, Ma Z, Wang H, Jia X, Wu Y, Fu L, Li Z, Zhang C, Yu G. YAP/TAZ affects the development of pulmonary fibrosis by regulating multiple signaling pathways. Mol Cell Biochem 475: 137–149; 2020. [DOI] [PubMed] [Google Scholar]

RESOURCES