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Investigation of the physiological response of radiation-induced cystitis patients using hyperbaric oxygen

Open AccessPublished:October 28, 2022DOI:https://doi.org/10.1016/j.ctro.2022.10.009

      Highlights

      • We used an acute exposure to hyperbaric oxygen to assess the physiological response of patients with radiation cystitis to a control population.
      • NOS2 and TNFα gene expression differed between the control and cystitis patients.
      • VEGF-A levels were reduced after the dive in plasma from cystitis patients.
      • IL-6 was elevated in the urine of cystitis patients.

      Abstract

      Introduction

      In this pilot study we have taken a novel functional approach to assess whether differences exist in the activity of key genes involved in the response to radiation and oxidative stress between patients with radiation cystitis.

      Materials and methods

      Arm 1 consisted of patients who had previously been treated for prostate cancer and who had received definitive radiation treatment and had subsequently developed cystitis and/or proctitis and were being treated by hyperbaric oxygen (HBO). Arm 2 consisted of patients who had never been treated by radiation but who were scheduled for HBO treatment for another pathology. The genes chosen for the study were HMOX1, NOS2, SOD2, TNFα, IL-6 and TGFβ. Blood and urine was collected pre and post HBO treatment.

      Results

      Gene expression showed a significant difference in NOS2 (p = 0.0178) and TNFα (p = 0.037) between the control and cystitis patients. The plasma levels of VEGF-A were significantly elevated in cystitis patients and there was a strong trend for significant overexpression in urine. Comparing pre and post-dive samples showed little difference in both groups of patients except for VEGF-A which was reduced after the dive in plasma from cystitis patients.

      Conclusions

      This study uncovered some physiological differences in patients with radiation-induced cystitis using HBO treatment as a stimulus to induce mild oxidative stress. Further research is ongoing to assess whether the acute exposure to HBO might be a physiological screening tool to identify patients susceptible to chronic radiation toxicity.

      Keywords

      Introduction

      Radiation cystitis (RC) of the bladder is common in prostate cancer patients despite the continuing improvements in pelvic radiation delivery using image-guided treatment, intensity modulated treatment, brachytherapy or protons [
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      Incidence and Morbidity of Radiation-Induced Hemorrhagic Cystitis in Prostate Cancer.
      ]. In a review of the literature, the incidence of radiation cystitis was found to range from 23 to 80% and the incidence of severe hematuria from 5 to 8% [
      • Browne C.
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      A Narrative Review on the Pathophysiology and Management for Radiation Cystitis.
      ]. Late RC is a long-term sequelae of radiation treatment with a mean duration for developing the condition being 31.8 months [
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      A Narrative Review on the Pathophysiology and Management for Radiation Cystitis.
      ] whilst severe hematuria can develop up to fourteen years following radiotherapy treatment [
      • Pavlidakey P.G.
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      Radiation cystitis.
      ,
      • Crew J.P.
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      • Reynard J.M.
      Radiation-induced haemorrhagic cystitis.
      ].
      RC is a term given to a constellation of symptoms which include hematuria, frequency, urgency, and pelvic pain. RC occurs secondary to obliterative endarteritis from hypoxia which causes atrophy and fibrosis of the mucosa. Ulceration of the mucosa leads to the development of fragile telangiectatic blood vessels that easily hemorrhage [
      • Mendenhall W.M.
      • Henderson R.H.
      • Costa J.A.
      • Hoppe B.S.
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      Hemorrhagic radiation cystitis.
      ]. The severity of ranges from grade 1 consisting of minor telangiectasia and microscopic hematuria with minimal increase in frequency, urgency, dysuria or nocturia to grade 3 and 4 where there is severe frequency and dysuria, gross hematuria and severe hemorrhagic cystitis.
      Treatments for RC depend on the severity of hematuria and range from conservative management to transfusion-dependent hemorrhage requiring cystectomy. Algorithms have recently been developed to guide treatment options [
      • Vanneste B.G.L.
      • Van Limbergen E.J.
      • Marcelissen T.A.
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      • et al.
      Development of a Management Algorithm for Acute and Chronic Radiation Urethritis and Cystitis.
      ]. One approach for patients with severe hematuria refractory to conventional management has been to use hyperbaric oxygen (HBO) [
      • Degener S.
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      • Zumbé J.
      • Roth S.
      • et al.
      Long-term experience of hyperbaric oxygen therapy for refractory radio- or chemotherapy-induced haemorrhagic cystitis.
      ,
      • Hughes A.J.
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      Hyperbaric oxygen in the treatment of refractory haemorrhagic cystitis.
      ,
      • Ribeiro de Oliveira T.M.
      • Carmelo Romão A.J.
      • Gamito Guerreiro F.M.
      • Matos Lopes T.M.
      Hyperbaric oxygen therapy for refractory radiation-induced hemorrhagic cystitis.
      ]. A recent randomized phase 2/3 trial of radiation-induced cystitis treated with hyperbaric oxygen therapy (RICH-ART) concluded that HBO treatment relieves the symptoms of late radiation cystitis and is safe and well tolerated [
      • Oscarsson N.
      • Müller B.
      • Rosén A.
      • Lodding P.
      • Mölne J.
      • Giglio D.
      • et al.
      Radiation-induced cystitis treated with hyperbaric oxygen therapy (RICH-ART): a randomised, controlled, phase 2–3 trial.
      ]. In HBO treatment, 100% O2 is administered in a pressurized chamber (usually ∼2 atmospheres) for 5–7 days/week with a daily duration of 60–90 min and in 30–45 sessions. The aim of HBO treatment is to reverse the process of radiation‐induced cellular hypoxia in the bladder tissue through better diffusion of oxygen within the tissues and intracellular generation of reactive species of oxygen and nitrogen [
      • Thom S.R.
      Hyperbaric oxygen: its mechanisms and efficacy.
      ]. This results in activation of intracellular signaling pathways leading to the induction of neovascularization through VEGF expression [
      • Lee C.-C.
      • Chen S.-C.
      • Tsai S.-C.
      • Wang B.-W.
      • Liu Y.-C.
      • Lee H.-M.
      • et al.
      Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells.
      ], and therefore angiogenesis and granulation tissue formation, fibroblast proliferation, and optimization of the cellular immune functions. The overall and complete response to HBO treatment have varied from 64.8% to 100% and 20% to 100%, respectively [
      • Villeirs L.
      • Tailly T.
      • Ost P.
      • Waterloos M.
      • Decaestecker K.
      • Fonteyne V.
      • et al.
      Hyperbaric oxygen therapy for radiation cystitis after pelvic radiotherapy: Systematic review of the recent literature.
      ].
      Accumulated damage from RC may be irreversible and hence early diagnosis or, better still, predicting patients who are susceptible to develop RC is essential to improve the quality of life of cancer survivors. However, the evaluation and prediction of normal tissue radiosensitivity is an ongoing field of study without definitive conclusions. There is no doubt that individual radiation sensitivity is partly determined genetically which has led to studies of whether it can be inferred from the reaction of cells exposed ex vivo to ionizing radiation or from analyses of the genotype [
      • Rajaraman P.
      • Hauptmann M.
      • Bouffler S.
      • Wojcik A.
      Human individual radiation sensitivity and prospects for prediction.
      ]. Ex vivo assays have centered on measuring a variety of DNA damage assays (chromosome aberrations, micronuclei, γH2AX foci, apoptosis) in isolated peripheral blood lymphocytes or fibroblasts [
      • Rajaraman P.
      • Hauptmann M.
      • Bouffler S.
      • Wojcik A.
      Human individual radiation sensitivity and prospects for prediction.
      ]. Association of genotype to adverse effects of radiotherapy has been studied in blood and saliva using both a targeted and genome-wide approach. Meta-analyses of these studies identified SNPs in ATM and TGFβ1 associated with late radiation toxicity [
      • Andreassen C.N.
      • Rosenstein B.S.
      • Kerns S.L.
      • Ostrer H.
      • De Ruysscher D.
      • Cesaretti J.A.
      • et al.
      Individual patient data meta-analysis shows a significant association between the ATM rs1801516 SNP and toxicity after radiotherapy in 5456 breast and prostate cancer patients.
      ,
      • Barnett G.C.
      • Elliott R.M.
      • Alsner J.
      • Andreassen C.N.
      • Abdelhay O.
      • Burnet N.G.
      • et al.
      Individual patient data meta-analysis shows no association between the SNP rs1800469 in TGFB and late radiotherapy toxicity.
      ]; although these results have not been consistently validated [
      • Barnett G.C.
      • Thompson D.
      • Fachal L.
      • Kerns S.
      • Talbot C.
      • Elliott R.M.
      • et al.
      A genome wide association study (GWAS) providing evidence of an association between common genetic variants and late radiotherapy toxicity.
      ,
      • Fachal L.
      • Gómez-Caamaño A.
      • Barnett G.C.
      • Peleteiro P.
      • Carballo A.M.
      • Calvo-Crespo P.
      • et al.
      A three-stage genome-wide association study identifies a susceptibility locus for late radiotherapy toxicity at 2q24.1.
      ,
      • Kerns S.L.
      • Dorling L.
      • Fachal L.
      • Bentzen S.
      • Pharoah P.D.P.
      • Barnes D.R.
      • et al.
      Meta-analysis of Genome Wide Association Studies Identifies Genetic Markers of Late Toxicity Following Radiotherapy for Prostate Cancer.
      ,
      • Kerns S.L.
      • Fachal L.
      • Dorling L.
      • et al.
      Radiogenomics Consortium Genome-Wide Association Study Meta-Analysis of Late Toxicity After Prostate Cancer Radiotherapy.
      ,
      • Dorling L.
      • Barnett G.C.
      • Michailidou K.
      • et al.
      Patients with a High Polygenic Risk of Breast Cancer do not have An Increased Risk of Radiotherapy Toxicity.
      ]. Recently, a deep learning approach was used to validate genetic risk factors for various late toxicities after prostate cancer radiation treatment [
      • Massi M.C.
      • Gasperoni F.
      • Ieva F.
      • Paganoni A.M.
      • Zunino P.
      • Manzoni A.
      • et al.
      A Deep Learning Approach Validates Genetic Risk Factors for Late Toxicity After Prostate Cancer Radiotherapy in a REQUITE Multi-National Cohort.
      ]. This promising approach did identify 9 significant SNPs associated with urinary toxicities.
      In this pilot study we have taken a novel functional approach to assess whether differences exist in the activity of key genes involved in the response to radiation and oxidative stress between patients with radiation cystitis compared to those without bladder pathologies. HBO was used as a tool to invoke an oxidative stress response in patients undergoing their first “dive” and downstream genes involved in increased growth factor response and diminished inflammatory response were assessed before and after the “dive” in plasma and urine.

      Materials and methods

      Patient population and HBO treatment

      Approval for the study was obtained from the William Beaumont Hospital Institutional Review Board (IRB# 2014-312). The study was approved to collect 12 patients in each arm. Arm 1 consisted of patients who had previously been treated for prostate cancer with definitive radiation treatment either by external beam or a combination of external beam and high-dose brachytherapy (Table 1) who had subsequently developed cystitis and/or proctitis and were being treated by HBO. All patients were diagnosed by cystoscopies and all patients had hematuria. In addition to hematuria some had dysuria (2), frequency (1), nocturia (1) and urethral strictures (2). Arm 2 was originally designed to be prostate cancer patients who had never experienced cystitis after radiation treatment and who were willing to undergo a single “dive” in the hyperbaric oxygen chamber. However, this was not feasible and it was decided to recruit patients who had never been treated by radiation but who were scheduled for HBO treatment for another pathology (Table 1). None of the patients in the control group had urinary symptoms. All patients were studied at the time of their first dive. Blood and urine were collected immediately prior to the dive. Subjects underwent a standard hyperbaric oxygen treatment; the total dive time was 90 min. Vital signs (temperature, blood pressure and pulse) were monitored during and after the HBOT to ensure subject safety. Following the treatment, additional blood and urine samples were collected. Table 1 shows the demographics of the patients who had successful data analysis. There were 10 patients in the radiation cystitis group and 11 in the non-cystitis group. Other patients who were consented were omitted due to issues with blood or urine draws and ineligibility.
      Table 1Clinicopathological features of the study patients. EBRT is external beam radiation treatment, HDR is high dose rate brachytherapy.
      Non-Radiation Subjects
      ID#AgeSexDiagnosisFirst RxDiabetic
      162mChronic osteomyelitis10/9/2017yes
      248fFailed skin graft11/1/2017no
      383mDiabetic ulcer lower extremity11/30/2017yes
      448mCrush injury12/18/2017no
      568mFailed skin graft2/19/2018no
      674mDiabetic ulcer lower extremity5/9/2018yes
      790fChronic osteomyelitis5/30/2018no
      865fFailed skin graft6/4/2018no
      965mDiabetic ulcer lower extremity6/25/2018yes
      1056mDiabetic ulcer lower extremity7/11/2018yes
      1150mFailing skin flap8/22/2018yes
      Radiation-Treated Subjects
      ID#AgeSexDiagnosisRTCancerFirst Rx
      167mRadiation cystitis51 Gy/10FProstate1/25/2016no
      283mRadiation cystitis/proctitisEBRT unknownProstate

      Rectal
      10/18/2016

      10/19/2016
      no
      385mRadiation cystitis79.2 Gy/44FProstate11/2/2016no
      474mRadiation cystitis79.2 Gy/44FProstate1/3/2017no
      580mRadiation cystitis/proctitis79.2 Gy/44FProstate4/26/2017no
      682mRadiation cystitisEBRT unknownProstate recurrence5/9/2017no
      771mRadiation cystitisEBRT and HDR unknownProstate5/30/2017no
      865mRadiation cystitis/proctitisEBRT unknownProstate3/13/2018no
      980mRadiation cystitisEBRT unknownProstate7/17/2017no
      1074mRadiation cystitis76 Gy/38FProstate4/23/2018yes

      Strategy to select genes of interest

      Selection of genes to be studied was based on Pathway Studio (Elsevier) using an input of cystitis, radiation and oxygen (Fig. 1). The genes chosen for the study were HMOX1, NOS2, SOD2 and TNFα based on pathway analysis and IL-6 and TGFβ based on their known association with radiation-induced fibrosis.
      Figure thumbnail gr1
      Fig. 1Pathway analysis to select genes of interest highlighted by the black arrows.

      Gene expression

      2.5 ml of blood was collected in a PAXgene blood RNA tube (Qiagen, Valencia, CA) before and after the dive and RNA extracted using the manufacturer’s protocol. RNA was quantified using a Nanodrop 2000 spectrophotometer (ThermoFisher, Waltham, MA) and RNA integrity was assessed in a Bioanalyzer 2100 (Agilent, Santa Clara, CA). Complementary DNA (cDNA) was then synthesized by reverse transcription from 40 to 100 ng RNA using the Superscript™ IV VILO™ Master Mix (Invitrogen, Carlsbad, CA) using the manufacturer’s protocol. The expression levels of all studied genes were detected and identified by real-time PCR using a TaqMan Universal Master Mix (ThermoFisher)with the following genes; VEGF (Hs00900055_m1), SOD2 (Hs00167309_m1), HMOX1 (Hs01110250_m1), NOS2 (Hs01075529_m1), TNFα (Hs00174128_m1), IL-6 (Hs00174131_m1) and TGFβ (Hs00248373_m1). GAPDH control reagents (ThermoFisher) was used as the reference gene. Real-time PCR runs were performed using a Viia7 Real-Time PCR Detection System (ThermoFisher). The conditions of PCR included 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles of 95 °C 15 s and 1 min at 60 °C. The experiments were performed in duplicate for each sample. Expression levels were calculated using QuantStudio software which sets the best threshold and the cycle at which each amplification curve crossed that threshold was then assigned as the CT for that sample. The ΔCT was calculated by subtracting the CT from the gene from the CT of the housekeeping gene (GAPDH), The data were presented as a ratio of the post-dive ΔCT compared to the pre-dive ΔCT.

      Protein expression

      For protein expression in plasma and urine, 3 ml of blood was collected in BD Vacutainer tubes and urine collected in a 60 ml collection container pre- and post-dive. The samples were processed and stored at −80 °C until analyzed. Urine samples were spun down for 5 min at 3000 rpm in a tabletop centrifuge to remove any debris. The supernatant was then vortexed before use. Levels of angiogenic growth factors were assessed in undiluted urine. Samples were analyzed using a custom Bio-Plex assay (Bio-Rad, Hercules, CA) using manufacturers protocols. Samples were analyzed in duplicate. Analysis of experimental data was done using five-parametric curve fitting with reference to calibration curves prepared with analyte standards included in the kits.

      Creatinine levels

      Creatinine determination was performed with a Shimadzu Prominence high performance liquid chromatography system including a dual pump system, delivering a mobile phase consisting of 25 mM citric acid and 3% acetonitrile (pH 6.0) at a flow rate of 0.5 ml/min. After diluting urine 1:50 in mobile phase, separation was done on a kinetix 2.6μ XB-C18 100A (100 × 4.60 mm) column (Phenomenex) with UV absorbance detection at 250 nm. Quantification was based on peak height as compared to creatinine standards (Acros Organics).

      Statistical analysis

      All data was analyzed using JMP 11.2.0 (Cary, NC). Gene expression data was analyzed using a Wilcoxon Rank test with a p-value ≥0.05 considered significant with the assumption of equal variances. Paired pre-and post-dive serum and urine samples were analyzed using a paired t-test with a p-value ≥0.05 considered significant.

      Results

      Gene expression changes

      The data presented in Fig. 2 shows the relative quantitation for each gene as a ratio of change between the pre-dive compared tothe post-dive samples for patients with and without (control) radiation-induced cystitis. Nine samples were successfully analyzed for the cystitis group and 10 for the control patients. The box and whisker plots show that median change in gene expression for the control patients remained unchanged for the post-dive except for NOS2 which showed a 1.52-fold increase after the dive. The majority of genes also showed no change as a result of the dive in the cystitis patients except for NOS2 (0.82-fold) and TNFα (0.78-fold) which were reduced post dive. This resulted in a significant difference in NOS2 (p = 0.0178) and TNFα (p = 0.037) between the control and cystitis patients (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Changes in gene expression pre-and post-dive. The data is presented as the relative quantitation for each gene as a ratio of change of the post-divevalues compared to the pre-dive values for patients with (black) and without (grey) radiation-induced cystitis.

      Are there intrinsic differences in protein expression between radiation cystitis patients and controls?

      Several proteins were either not detectable in plasma or urine at the limit of sensitivity of the ELISA assays. Three proteins, IL-6, VEGF-A and TNFα produced reproducible data in the pre- and post-dive plasma and urine samples and were selected for comparative analysis.
      In the plasma samples when comparing patients with and without radiation cystitis, only VEGF-A showed a significant difference in the pre-dive samples (Fig. 3A). The concentration of VEGF-A in the cystitis patients was 40.3 ± 6.6 pg/ml compared to 22.8 ± 6.3 pg/ml in the non-cystitis patients (p = 0.0347). The values for IL-6 were 5.9 ± 1.6 pg/ml and 4.9 ± 1.7 pg/ml in non-cystitis versus cystitis patients and 2.1 ± 1.0 pg/ml and 1.3 ± 1.0 pg/ml for TNFα.
      Figure thumbnail gr3
      Fig. 3Protein expression in plasma (A) and urine (B) samples from cystitis and control patients prior to the HBO dive.
      In the urine samples, IL-6 was detectable in only 5 (45.5%) of the control patient samples whilst 8 of 11 (80%) samples were positive in the cystitis patients (Fig. 3B). The mean levels were 6.5 ± 50.7 pg/ml and 157 ± 53.2 pg.ml in controls versus cystitis patients, this resulted in a significant difference (p = 0.027). VEGF levels were detected in all 11 control samples and 9 of 10 cystitis patients and were generally higher in cystitis patients (135.5 ± 27.2 pg/ml) compared to controls (107.2 ± 25.9 pg/ml) but again this did not reach significance (p = 0.25). TNF-α was detectable in 10 of 11 control samples and 9 of 10 cystitis samples but the level of expression was uniformly low with means and S.E.M.of 2.8 ± 1.5 pg/ml and 3.2 ± 1.6 pg/ml in control versus cystitis patients.

      Does protein expression change differentially between cystitis and control patients after a HBO dive?

      Fig. 4 shows the correlation between the proteins before and after the HBO dive in both plasma and urine samples. In the plasma samples, levels of IL-6, VEGF-A and TNF-α were not significantly changed post-dive in both cystitis and non-cystitis patients.
      Figure thumbnail gr4
      Fig. 4Protein expression in plasma and urine samples pre- and post-dive specimens for control (gray symbol) and cystitis patients (black symbol). The dashed line in each graph represents the line of unity.
      All urine samples were analyzed for creatinine concentration which varied between patients with a mean and S.D. of 1359 ± 482 µg/ml in the control patients and 1859 ± 378 in the cystitis patients in the pre-dive samples compared to 1378 ± 317 and 2255 ± 571 and in the post-dive samples respectively. None of these differences was significant and there was no significant trend in change between pre- and post-dive samples. Analyzing the data as a function of creatinine did not change the significance of results compared to analysis using the protein concentration of each biomarker. When comparing the protein expression data in pre- and post-dive urine samples for VEGF and TNFα, there were no significant differences in the paired samples in either the controls or the cystitis patients. However, IL-6 showed a significant reduction in the cystitis patients post dive. The pre dive levels were 175.3 but were reduce to a mean of 117.7 after the dive (p = 0.0387).

      Discussion

      In this novel pilot study, we investigated whether the physiological response of patients who have developed radiation-induced bladder cystitis differs to the general population when exposed to mild oxidative stress induced by HBO. It is well accepted that breathing greater than one atmosphere of oxygen will increase production of reactive oxygen species (ROS) [
      • Thom S.R.
      Oxidative stress is fundamental to hyperbaric oxygen therapy.
      ] and also reactive nitrogen species (RNS). These molecules are involved in signaling transduction cascades for a variety of growth factors, cytokines and hormones. Under normal conditions ROS act in conjunction with several redox systems involving glutathione, thioredoxin and pyridine nucleotides, and play central roles in coordinating cell signaling and also antioxidant protective pathways [
      • Valko M.
      • Leibfritz D.
      • Moncol J.
      • Cronin M.T.D.
      • Mazur M.
      • Telser J.
      Free radicals and antioxidants in normal physiological functions and human disease.
      ]. In this context oxidative stress is not synonymous with oxygen toxicity.
      However, exposure to radiation treatment produces excessive levels of ROS which have been shown to disrupt components of the electron transport chain in mitochondria, induce intracellular redox system imbalances and cause oxidative stress by reacting with biological molecules such as lipids, proteins, and DNA to cause lipid peroxidation, protein misfolding, and DNA strand breaks [
      • Kim W.
      • Lee S.
      • Seo D.
      • et al.
      Cellular Stress Responses in Radiotherapy.
      ]. Indeed, studies of exposure to radiation treatment have demonstrated a prolonged oxidative stress implying a persistent imbalance between ROS production and antioxidant defense systems [
      • Clutton S.M.
      • Townsend K.M.S.
      • Walker C.
      • Ansell J.D.
      • Wright E.G.
      Radiation-induced genomic instability and persisting oxidative stress in primary bone marrow cultures.
      ,
      • Rugo R.E.
      • Secretan M.B.
      • Schiestl R.H.
      X radiation causes a persistent induction of reactive oxygen species and a delayed reinduction of TP53 in normal human diploid fibroblasts.
      ,
      • Tulard A.
      • Hoffschir F.
      • de Boisferon F.H.
      • Luccioni C.
      • Bravard A.
      Persistent oxidative stress after ionizing radiation is involved in inherited radiosensitivity.
      ]. The pathophysiology of late radiation cystitis has still to be fully elucidated but endothelial cells appear to play an important role in this process. It has been demonstrated that submucosal vascularity is damaged by fibrosis of the vascular intima leading to vessel obstruction and submucosal/muscular fibrosis [
      • Helissey C.
      • Cavallero S.
      • Brossard C.
      • Dusaud M.
      • Chargari C.
      • Francois S.
      Chronic Inflammation and Radiation-Induced Cystitis: Molecular Background and Therapeutic Perspectives.
      ]. The consequence of these events is ensuing urothelial atrophy, hypoxia with diminished vascularization and ischemia of the bladder which leads to fibrosis and atrophy of the bladder tissue and the emergence of neovascularization in the form of telangiectasia that is susceptible to bleeding [
      • Helissey C.
      • Cavallero S.
      • Brossard C.
      • Dusaud M.
      • Chargari C.
      • Francois S.
      Chronic Inflammation and Radiation-Induced Cystitis: Molecular Background and Therapeutic Perspectives.
      ].
      The cellular response to HBO appears to be a combination of systemic events as well as local alterations. Regional angiogenic stimuli influence the efficiency of new blood vessel growth by angiogenesis in local endothelial cells and they stimulate the recruitment and differentiation of circulating stem/progenitor cells (SPCs) to form vessels de novo by vasculogenesis [
      • Thom S.R.
      Hyperbaric oxygen: its mechanisms and efficacy.
      ]. In radiation-induced cystitis, it is thought that HBO therapy facilitates better oxygen diffusion in tissues and disrupts the continuum between hypoxia and fibrosis through induction of primary neovascularization, secondary growth of healthy granulation tissue, and induces short-term vasoconstriction, which may help control active bleeding [
      • Helissey C.
      • Cavallero S.
      • Brossard C.
      • Dusaud M.
      • Chargari C.
      • Francois S.
      Chronic Inflammation and Radiation-Induced Cystitis: Molecular Background and Therapeutic Perspectives.
      ].
      In this study, patients were only exposed to a single dive in the chamber and, therefore, we were not studying the long-term benefits of HBO but assessing the physiological response of patients to this temporary low-level oxidative stress. We showed that gene expression in the control patients remained unchanged for the post-dive except for NOS2 which showed a 1.52-fold increase. In contrast, NOS2 levels were significantly reduced (0.82-fold) post-dive in the cystitis patients as was TNFα (0.78-fold); the other genes were unchanged. Experimental and clinical wound healing studies have established nitic oxide (NO) as a critical mediator of normal tissue repair [
      • Schwentker A.
      • Billiar T.R.
      Nitric oxide and wound repair.
      ] and its production is catalyzed by NOS2. Several studies have shown variable effects of HBO on NOS2 expression dependent on tissue type and disease context [
      • Gajendrareddy P.K.
      • Sen C.K.
      • Horan M.P.
      • Marucha P.T.
      Hyperbaric oxygen therapy ameliorates stress-impaired dermal wound healing.
      ,
      • Uusijärvi J.
      • Eriksson K.
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      • Schiffer T.
      • Lindholm P.
      • et al.
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      ,
      • Han G.
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      ]. However, the differential response of cystitis to control patients suggests imbalances in this pathway exist which merits further investigation. For instance, it has been shown that NO has dose-dependent effects in the pathogenesis of inflammation where it has an anti-inflammatory effect under normal physiological conditions but is considered as a pro-inflammatory in abnormal situations [
      • Sharma J.N.
      • Al-Omran A.
      • Parvathy S.S.
      Role of nitric oxide in inflammatory diseases.
      ]. Further studies will be required to assess NO and NOS2 levels in bladder tissue to assess the baseline level of these in cystitis and control patients.
      Interestingly, VEGF expression levels were not changed in either group of patients whereas synthesis of this most specific growth factor for neovascularization has been shown to be increased in response to hyperbaric oxygen wound healing studies [
      • Sheikh A.Y.
      • Gibson J.J.
      • Rollins M.D.
      • Hopf H.W.
      • Hussain Z.
      • Hunt T.K.
      Effect of hyperoxia on vascular endothelial growth factor levels in a wound model.
      ]. Again, the lack of effect may be due to the short-term exposure after a single HBO treatment. However, the plasma and urine samples yielded interesting results for this gene. Of note, VEGF protein expression was significantly higher in the plasma of cystitis patients compared to the controls and there was a trend for significant overexpression in urine. This has been observed in previous studies [
      • Zwaans B.M.M.
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      ,
      • Zwaans B.M.M.
      • Nicolai H.E.
      • Chancellor M.B.
      • Lamb L.E.
      Prostate cancer survivors with symptoms of radiation cystitis have elevated fibrotic and vascular proteins in urine.
      ] of prostate cancer patients who had developed cystitis and parallels data obtained in patients who developed radiation proctitis [
      • Trzcinski R.
      • Dziki A.
      • Brys M.
      • et al.
      Expression of vascular endothelial growth factor and its correlation with clinical symptoms and endoscopic findings in patients with chronic radiation proctitis.
      ]. Other studies have replicated this finding and suggested that anti-VEGF therapy may be a possible therapy to ameliorate abnormal angiogenesis in this patient population [
      • Karamanolis G.
      • Delladetsima I.
      • Kouloulias V.
      • et al.
      Increased expression of VEGF and CD31 in postradiation rectal tissue: implications for radiation proctitis.
      ,
      • Takeuchi H.
      • Kimura T.
      • Okamoto K.
      • Aoyagi E.
      • Miyamoto H.
      • Kaji M.
      • et al.
      A mechanism for abnormal angiogenesis in human radiation proctitis: analysis of expression profile for angiogenic factors.
      ]. In this context the findings from this present study are thought-provoking as HBO exposure decreased levels of VEGF in the cystitis patients but was without effect in the control population.
      The changes in urine and plasma levels after the dive were very modest with only urine levels of IL-6 showing a significant change. Indeed, the levels of IL-6 were highly elevated in the urine of some of the cystitis patients compared to the controls and were much than that found in plasma indicating the inflammation in the bladder. IL-6 has been reported to be elevated in the urine of interstitial cystitis patients [

      Lotz M, Villiger P, Hugli T, Koziol J, Zuraw BL. Interleukin-6 and interstitial cystitis. 1994;152:869–73.

      ] and is associated with bacterial infection. It was interesting that a single HBO dive was able to reduce IL-6 urine levels in cystitis patients but had no effect in control patients.
      The study does have limitations. The patient number was relatively small but this reflects that the study was designed to be a pilot study. Second, the original intention to have age-matched controls who had previously had radiation treatment for prostate cancer but who had never experienced cystitis proved impossible to recruit. The compromise was to study patients with other pathologies who were scheduled for HBO treatment as standard of care and therefore they can’t be considered as completely “normal” controls.
      In conclusion, this pilot study took the novel approach of studying the response of cystitis and control patients to an in vivo stimulus of HBO exposure to investigate whether there were differences in physiological responses that might shed further light on the pathology of the disease and indicate other treatment options. Further research is ongoing to study these biomarkers in patients prior to radiation treatment and to assess whether the acute exposure to HBO might be a physiological screening tool to identify patients susceptible to chronic radiation toxicity.

      Declaration of Competing Interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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