Enhanced radiation sensitivity, decreased DNA damage repair, and differentiation defects in airway stem cells derived from patients with chronic obstructive pulmonary disease.

Radiation therapy (RT) is a common treatment for lung cancer. Still, it can lead to irreversible loss of pulmonary function and a significant reduction in quality of life for one-third of patients. Preexisting comorbidities, such as chronic obstructive pulmonary disease (COPD), are frequent in patients with lung cancer and further increase the risk of complications. Because lung stem cells are crucial for the regeneration of lung tissue following injury, we hypothesized that airway stem cells from patients with COPD with lung cancer might contribute to increased radiation sensitivity. We used the air-liquid interface model, a three-dimensional (3D) culture system, to compare the radiation response of primary human airway stem cells from healthy and patients with COPD. We found that COPD-derived airway stem cells, compared to healthy airway stem cell cultures, exhibited disproportionate pathological mucociliary differentiation, aberrant cell cycle checkpoints, residual DNA damage, reduced survival of stem cells and self-renewal, and terminally differentiated cells post-irradiation, which could be reversed by blocking the Notch pathway using small-molecule γ-secretase inhibitors. Our findings shed light on the mechanisms underlying the increased radiation sensitivity of COPD and suggest that airway stem cells reflect part of the pathological remodeling seen in lung tissue from patients with lung cancer receiving thoracic RT.

Increased vascular smooth muscle cell senescence in aneurysmal Fibulin-4 mutant mice.

Aortic aneurysms are dilatations of the aorta that can rupture when left untreated. We used the aneurysmal Fibulin-4R/R mouse model to further unravel the underlying mechanisms of aneurysm formation. RNA sequencing of 3-month-old Fibulin-4R/R aortas revealed significant upregulation of senescence-associated secretory phenotype (SASP) factors and key senescence factors, indicating the involvement of senescence. Analysis of aorta histology and of vascular smooth muscle cells (VSMCs) in vitro confirmed the senescent phenotype of Fibulin-4R/R VSMCs by revealing increased SA-ß-gal, p21, and p16 staining, increased IL-6 secretion, increased presence of DNA damage foci and increased nuclei size. Additionally, we found that p21 luminescence was increased in the dilated aorta of Fibulin-4R/R|p21-luciferase mice. Our studies identify a cellular aging cascade in Fibulin-4 aneurysmal disease, by revealing that Fibulin-4R/R aortic VSMCs have a pronounced SASP and a senescent phenotype that may underlie aortic wall degeneration. Additionally, we demonstrated the therapeutic effect of JAK/STAT and TGF-ß pathway inhibition, as well as senolytic treatment on Fibulin-4R/R VSMCs in vitro. These findings can contribute to improved therapeutic options for aneurysmal disease aimed at reducing senescent cells.

Hybrid Molecular and Functional Micro-CT Imaging Reveals Increased Myocardial Apoptosis Preceding Cardiac Failure in Progeroid Ercc1 Mice.

PURPOSE: In this study, we explored the role of apoptosis as a potential biomarker for cardiac failure using functional micro-CT and fluorescence molecular tomography (FMT) imaging techniques in Ercc1 mutant mice. Ercc1 is involved in multiple DNA repair pathways, and its mutations contribute to accelerated aging phenotypes in both humans and mice, due to the accumulation of DNA lesions that impair vital DNA functions. We previously found that systemic mutations and cardiomyocyte-restricted deletion of Ercc1 in mice results in left ventricular (LV) dysfunction at older age. PROCEDURES AND RESULTS: Here we report that combined functional micro-CT and FMT imaging allowed us to detect apoptosis in systemic Ercc1 mutant mice prior to the development of overt LV dysfunction, suggesting its potential as an early indicator and contributing factor of cardiac impairment. The detection of apoptosis in vivo was feasible as early as 12 weeks of age, even when global LV function appeared normal, underscoring the potential of apoptosis as an early predictor of LV dysfunction, which subsequently manifested at 24 weeks. CONCLUSIONS: This study highlights the utility of combined functional micro-CT and FMT imaging in assessing cardiac function and detecting apoptosis, providing valuable insights into the potential of apoptosis as an early biomarker for cardiac failure.

Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz syndrome patients provides insights into genotype-phenotype relation.

RATIONALE: Pathogenic (P)/likely pathogenic (LP) SMAD3 variants cause Loeys-Dietz syndrome type 3 (LDS3), which is characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with osteoarthritis. OBJECTIVES: Investigate the impact of P/LP SMAD3 variants with functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs), to optimize interpretation of SMAD3 variants. METHODS: A retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and functional analyses on SMAD3 patient-derived VSMCs and SMAD3 patient-derived fibroblasts, differentiated into myofibroblasts. RESULTS: Individuals with dominant negative (DN) SMAD3 variant in the MH2 domain exhibited more major events (66.7% vs. 44.0%, P = 0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. The age at first major event was 35.0 years [IQR 29.0-47.0] in individuals with DN variants in MH2, compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (P = 0.065). Fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential in HI SMAD3 variant fibroblasts. HI SMAD3 variant VSMCs showed elevated SMA expression and altered expression of alternative MYH11 isoforms. DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix formation compared to control cell lines. CONCLUSION: Distinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and SMA and MYH11 expression. The differences between DN and HI SMAD3 variant fibroblasts and VSMCs potentially contribute to the differences in disease manifestation. Notably, myofibroblast differentiation seems a suitable alternative in vitro test system compared to VSMCs.

Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response.

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/- and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/- aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/- mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/- VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/- aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/- aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/- mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.

Pre-transplant kidney quality evaluation using photoacoustic imaging during normothermic machine perfusion.

Due to the shortage of kidneys donated for transplantation, surgeons are forced to use the organs with an elevated risk of poor function or even failure. Although the existing methods for pre-transplant quality evaluation have been validated over decades in population cohort studies across the world, new methods are needed as long as delayed graft function or failure in a kidney transplant occurs. In this study, we explored the potential of utilizing photoacoustic (PA) imaging during normothermic machine perfusion (NMP) as a means of evaluating kidney quality. We closely monitored twenty-two porcine kidneys using 3D PA imaging during a two-hour NMP session. Based on biochemical analyses of perfusate and produced urine, the kidneys were categorized into 'non-functional' and 'functional' groups. Our primary focus was to quantify oxygenation (sO2) within the kidney cortical layer of depths 2 mm, 4 mm, and 6 mm using two-wavelength PA imaging. Next, receiver operating characteristic (ROC) analysis was performed to determine an optimal cortical layer depth and time point for the quantification of sO2 to discriminate between functional and non-functional organs. Finally, for each depth, we assessed the correlation between sO2 and creatinine clearance (CrCl), oxygen consumption (VO2), and renal blood flow (RBF). We found that hypoxia of the renal cortex is associated with poor renal function. In addition, the determination of sO2 within the 2 mm depth of the renal cortex after 30 min of NMP effectively distinguishes between functional and non-functional kidneys. The non-functional kidneys can be detected with the sensitivity and specificity of 80% and 85% respectively, using the cut-off point of sO2 < 39%. Oxygenation significantly correlates with RBF and VO2 in all kidneys. In functional kidneys, sO2 correlated with CrCl, which is not the case for non-functional kidneys. We conclude that the presented technique has a high potential for supporting organ selection for kidney transplantation.

Multi-scale cellular imaging of DNA double strand break repair.

Live-cell and high-resolution fluorescence microscopy are powerful tools to study the organization and dynamics of DNA double-strand break repair foci and specific repair proteins in single cells. This requires specific induction of DNA double-strand breaks and fluorescent markers to follow the DNA lesions in living cells. In this review, where we focused on mammalian cell studies, we discuss different methods to induce DNA double-strand breaks, how to visualize and quantify repair foci in living cells., We describe different (live-cell) imaging modalities that can reveal details of the DNA double-strand break repair process across multiple time and spatial scales. In addition, recent developments are discussed in super-resolution imaging and single-molecule tracking, and how these technologies can be applied to elucidate details on structural compositions or dynamics of DNA double-strand break repair.

DNA repair in cardiomyocytes is critical for maintaining cardiac function in mice.

Heart failure has reached epidemic proportions in a progressively ageing population. The molecular mechanisms underlying heart failure remain elusive, but evidence indicates that DNA damage is enhanced in failing hearts. Here, we tested the hypothesis that endogenous DNA repair in cardiomyocytes is critical for maintaining normal cardiac function, so that perturbed repair of spontaneous DNA damage drives early onset of heart failure. To increase the burden of spontaneous DNA damage, we knocked out the DNA repair endonucleases xeroderma pigmentosum complementation group G (XPG) and excision repair cross-complementation group 1 (ERCC1), either systemically or cardiomyocyte-restricted, and studied the effects on cardiac function and structure. Loss of DNA repair permitted normal heart development but subsequently caused progressive deterioration of cardiac function, resulting in overt congestive heart failure and premature death within 6 months. Cardiac biopsies revealed increased oxidative stress associated with increased fibrosis and apoptosis. Moreover, gene set enrichment analysis showed enrichment of pathways associated with impaired DNA repair and apoptosis, and identified TP53 as one of the top active upstream transcription regulators. In support of the observed cardiac phenotype in mutant mice, several genetic variants in the ERCC1 and XPG gene in human GWAS data were found to be associated with cardiac remodelling and dysfunction. In conclusion, unrepaired spontaneous DNA damage in differentiated cardiomyocytes drives early onset of cardiac failure. These observations implicate DNA damage as a potential novel therapeutic target and highlight systemic and cardiomyocyte-restricted DNA repair-deficient mouse mutants as bona fide models of heart failure.

Molecular Imaging of Aortic Aneurysm and Its Translational Power for Clinical Risk Assessment.

Aortic aneurysms (AAs) are dilations of the aorta, that are often fatal upon rupture. Diagnostic radiological techniques such as ultrasound (US), magnetic resonance imaging (MRI), and computed tomography (CT) are currently used in clinical practice for early diagnosis as well as clinical follow-up for preemptive surgery of AA and prevention of rupture. However, the contemporary imaging-based risk prediction of aneurysm enlargement or life-threatening aneurysm-rupture remains limited as these are restricted to visual parameters which fail to provide a personalized risk assessment. Therefore, new insights into early diagnostic approaches to detect AA and therefore to prevent aneurysm-rupture are crucial. Multiple new techniques are developed to obtain a more accurate understanding of the biological processes and pathological alterations at a (micro)structural and molecular level of aortic degeneration. Advanced anatomical imaging combined with molecular imaging, such as molecular MRI, or positron emission tomography (PET)/CT provides novel diagnostic approaches for in vivo visualization of targeted biomarkers. This will aid in the understanding of aortic aneurysm disease pathogenesis and insight into the pathways involved, and will thus facilitate early diagnostic analysis of aneurysmal disease. In this study, we reviewed these molecular imaging modalities and their association with aneurysm growth and/or rupture risk and their limitations. Furthermore, we outline recent pre-clinical and clinical developments in molecular imaging of AA and provide future perspectives based on the advancements made within the field. Within the vastness of pre-clinical markers that have been studied in mice, molecular imaging targets such as elastin/collagen, albumin, matrix metalloproteinases and immune cells demonstrate promising results regarding rupture risk assessment within the pre-clinical setting. Subsequently, these markers hold potential as a future diagnosticum of clinical AA assessment. However currently, clinical translation of molecular imaging is still at the onset. Future human trials are required to assess the effectivity of potentially viable molecular markers with various imaging modalities for clinical rupture risk assessment.

Genetic and clinical determinants of abdominal aortic diameter: genome-wide association studies, exome array data and Mendelian randomization study.

Progressive dilation of the infrarenal aortic diameter is a consequence of the ageing process and is considered the main determinant of abdominal aortic aneurysm (AAA). We aimed to investigate the genetic and clinical determinants of abdominal aortic diameter (AAD). We conducted a meta-analysis of genome-wide association studies in 10 cohorts (n = 13 542) imputed to the 1000 Genome Project reference panel including 12 815 subjects in the discovery phase and 727 subjects [Partners Biobank cohort 1 (PBIO)] as replication. Maximum anterior-posterior diameter of the infrarenal aorta was used as AAD. We also included exome array data (n = 14 480) from seven epidemiologic studies. Single-variant and gene-based associations were done using SeqMeta package. A Mendelian randomization analysis was applied to investigate the causal effect of a number of clinical risk factors on AAD. In genome-wide association study (GWAS) on AAD, rs74448815 in the intronic region of LDLRAD4 reached genome-wide significance (beta = -0.02, SE = 0.004, P-value = 2.10 × 10-8). The association replicated in the PBIO1 cohort (P-value = 8.19 × 10-4). In exome-array single-variant analysis (P-value threshold = 9 × 10-7), the lowest P-value was found for rs239259 located in SLC22A20 (beta = 0.007, P-value = 1.2 × 10-5). In the gene-based analysis (P-value threshold = 1.85 × 10-6), PCSK5 showed an association with AAD (P-value = 8.03 × 10-7). Furthermore, in Mendelian randomization analyses, we found evidence for genetic association of pulse pressure (beta = -0.003, P-value = 0.02), triglycerides (beta = -0.16, P-value = 0.008) and height (beta = 0.03, P-value < 0.0001), known risk factors for AAA, consistent with a causal association with AAD. Our findings point to new biology as well as highlighting gene regions in mechanisms that have previously been implicated in the genetics of other vascular diseases.

Contraction pressure analysis using optical imaging in normal and MYBPC3-mutated hiPSC-derived cardiomyocytes grown on matrices with tunable stiffness.

Current in vivo disease models and analysis methods for cardiac drug development have been insufficient in providing accurate and reliable predictions of drug efficacy and safety. Here, we propose a custom optical flow-based analysis method to quantitatively measure recordings of contracting cardiomyocytes on polydimethylsiloxane (PDMS), compatible with medium-throughput systems. Movement of the PDMS was examined by covalently bound fluorescent beads on the PDMS surface, differences caused by increased substrate stiffness were compared, and cells were stimulated with ß-agonist. We further validated the system using cardiomyocytes treated with endothelin-1 and compared their contractions against control and cells incubated with receptor antagonist bosentan. After validation we examined two MYBPC3-mutant patient-derived cell lines. Recordings showed that higher substrate stiffness resulted in higher contractile pressure, while beating frequency remained similar to control. ß-agonist stimulation resulted in both higher beating frequency as well as higher pressure values during contraction and relaxation. Cells treated with endothelin-1 showed an increased beating frequency, but a lower contraction pressure. Cells treated with both endothelin-1 and bosentan remained at control level of beating frequency and pressure. Lastly, both MYBPC3-mutant lines showed a higher beating frequency and lower contraction pressure. Our validated method is capable of automatically quantifying contraction of hiPSC-derived cardiomyocytes on a PDMS substrate of known shear modulus, returning an absolute value. Our method could have major benefits in a medium-throughput setting.

In Vivo Renin Activity Imaging in the Kidney of Progeroid Ercc1 Mutant Mice.

Changes in the renin-angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin-angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/- mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/- mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/- mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/- compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging.

DNA Double Strand Break Repair Pathways in Response to Different Types of Ionizing Radiation.

The superior dose distribution of particle radiation compared to photon radiation makes it a promising therapy for the treatment of tumors. However, the cellular responses to particle therapy and especially the DNA damage response (DDR) is not well characterized. Compared to photons, particles are thought to induce more closely spaced DNA lesions instead of isolated lesions. How this different spatial configuration of the DNA damage directs DNA repair pathway usage, is subject of current investigations. In this review, we describe recent insights into induction of DNA damage by particle radiation and how this shapes DNA end processing and subsequent DNA repair mechanisms. Additionally, we give an overview of promising DDR targets to improve particle therapy.

A BRCA1 Coiled-Coil Domain Variant Disrupting PALB2 Interaction Promotes the Development of Mammary Tumors and Confers a Targetable Defect in Homologous Recombination Repair.

The BRCA1 tumor suppressor gene encodes a multidomain protein for which several functions have been described. These include a key role in homologous recombination repair (HRR) of DNA double-strand breaks, which is shared with two other high-risk hereditary breast cancer suppressors, BRCA2 and PALB2. Although both BRCA1 and BRCA2 interact with PALB2, BRCA1 missense variants affecting its PALB2-interacting coiled-coil domain are considered variants of uncertain clinical significance (VUS). Using genetically engineered mice, we show here that a BRCA1 coiled-coil domain VUS, Brca1 p.L1363P, disrupts the interaction with PALB2 and leads to embryonic lethality. Brca1 p.L1363P led to a similar acceleration in the development of Trp53-deficient mammary tumors as Brca1 loss, but the tumors showed distinct histopathologic features, with more stable DNA copy number profiles in Brca1 p.L1363P tumors. Nevertheless, Brca1 p.L1363P mammary tumors were HRR incompetent and responsive to cisplatin and PARP inhibition. Overall, these results provide the first direct evidence that a BRCA1 missense variant outside of the RING and BRCT domains increases the risk of breast cancer. SIGNIFICANCE: These findings reveal the importance of a patient-derived BRCA1 coiled-coil domain sequence variant in embryonic development, mammary tumor suppression, and therapy response.See related commentary by Mishra et al., p. 6080.

Molecular phenotyping and functional assessment of smooth muscle-like cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.

BRCA2 binding through a cryptic repeated motif to HSF2BP oligomers does not impact meiotic recombination.

BRCA2 and its interactors are required for meiotic homologous recombination (HR) and fertility. Loss of HSF2BP, a BRCA2 interactor, disrupts HR during spermatogenesis. We test the model postulating that HSF2BP localizes BRCA2 to meiotic HR sites, by solving the crystal structure of the BRCA2 fragment in complex with dimeric armadillo domain (ARM) of HSF2BP and disrupting this interaction in a mouse model. This reveals a repeated 23 amino acid motif in BRCA2, each binding the same conserved surface of one ARM domain. In the complex, two BRCA2 fragments hold together two ARM dimers, through a large interface responsible for the nanomolar affinity - the strongest interaction involving BRCA2 measured so far. Deleting exon 12, encoding the first repeat, from mBrca2 disrupts BRCA2 binding to HSF2BP, but does not phenocopy HSF2BP loss. Thus, results herein suggest that the high-affinity oligomerization-inducing BRCA2-HSF2BP interaction is not required for RAD51 and DMC1 recombinase localization in meiotic HR.

Transforming Growth Factor-ß and the Renin-Angiotensin System in Syndromic Thoracic Aortic Aneurysms: Implications for Treatment.

Thoracic aortic aneurysms (TAAs) are permanent pathological dilatations of the thoracic aorta, which can lead to life-threatening complications, such as aortic dissection and rupture. TAAs frequently occur in a syndromic form in individuals with an underlying genetic predisposition, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). Increasing evidence supports an important role for transforming growth factor-ß (TGF-ß) and the renin-angiotensin system (RAS) in TAA pathology. Eventually, most patients with syndromic TAAs require surgical intervention, as the ability of present medical treatment to attenuate aneurysm growth is limited. Therefore, more effective medical treatment options are urgently needed. Numerous clinical trials investigated the therapeutic potential of angiotensin receptor blockers (ARBs) and ß-blockers in patients suffering from syndromic TAAs. This review highlights the contribution of TGF-ß signaling, RAS, and impaired mechanosensing abilities of aortic VSMCs in TAA formation. Furthermore, it critically discusses the most recent clinical evidence regarding the possible therapeutic benefit of ARBs and ß-blockers in syndromic TAA patients and provides future research perspectives and therapeutic implications.

Multi-Omics Profiling in Marfan Syndrome: Further Insights into the Molecular Mechanisms Involved in Aortic Disease.

Thoracic aortic aneurysm is a potentially life-threatening disease with a strong genetic contribution. Despite identification of multiple genes involved in aneurysm formation, little is known about the specific underlying mechanisms that drive the pathological changes in the aortic wall. The aim of our study was to unravel the molecular mechanisms underlying aneurysm formation in Marfan syndrome (MFS). We collected aortic wall samples from FBN1 variant-positive MFS patients (n = 6) and healthy donor hearts (n = 5). Messenger RNA (mRNA) expression levels were measured by RNA sequencing and compared between MFS patients and controls, and between haploinsufficient (HI) and dominant negative (DN) FBN1 variants. Immunohistochemical staining, proteomics and cellular respiration experiments were used to confirm our findings. FBN1 mRNA expression levels were highly variable in MFS patients and did not significantly differ from controls. Moreover, we did not identify a distinctive TGF-ß gene expression signature in MFS patients. On the contrary, differential gene and protein expression analysis, as well as vascular smooth muscle cell respiration measurements, pointed toward inflammation and mitochondrial dysfunction. Our findings confirm that inflammatory and mitochondrial pathways play important roles in the pathophysiological processes underlying MFS-related aortic disease, providing new therapeutic options.

Pre-Operative Fasting Provides Long Term Protection Against Chronic Renal Damage Induced by Ischaemia Reperfusion Injury in Wild Type and Aneurysm Prone Fibulin-4 Mice.

OBJECTIVE: Renal ischaemia reperfusion injury (IRI) is inevitable during open repair of pararenal aortic aneurysms. Pre-operative fasting potently increases resistance against IRI. The effect of fasting on IRI was examined in a hypomorphic Fibulin-4 mouse model (Fibulin-4+/R), which is predisposed to develop aortic aneurysms. METHODS: Wild type (WT) and Fibulin-4+/R mice were either fed ad libitum (AL) or fasted for two days before renal IRI induction by temporary clamping of the renal artery and vein of both kidneys. Six hours, 48 h, and seven days post-operatively, serum urea levels, renal histology, and mRNA expression levels of inflammatory and injury genes were determined to assess kidney function and damage. Additionally, matrix metalloproteinase activity in the kidney was assessed six months after IRI. RESULTS: Two days of fasting improved survival the first week after renal IRI in WT mice compared with AL fed mice. Short term AL fed Fibulin-4+/R mice showed improved survival and kidney function compared with AL fed WT mice, which could not be further enhanced by fasting. Both fasted WT and Fibulin-4+/R mice showed improved survival, kidney function and morphology compared with AL fed mice six months after renal IRI. Fibulin-4+/R kidneys of fasted mice showed reduced apoptosis together with increased matrix metalloprotease activity levels compared with AL fed Fibulin-4+/R mice, indicative of increased matrix remodelling. CONCLUSION: Fibulin-4+/R mice are naturally protected against the short-term, but not long-term, consequences of renal IRI. Pre-operative fasting protects against renal IRI and prevents (long-term) deterioration of kidney function and morphology in both WT and Fibulin-4+/R mice. These data suggest that pre-operative fasting may decrease renal damage in patients undergoing open abdominal aneurysm repair.

Comparison of High- and Low-LET Radiation-Induced DNA Double-Strand Break Processing in Living Cells.

High-linear-energy-transfer (LET) radiation is more lethal than similar doses of low-LET radiation types, probably a result of the condensed energy deposition pattern of high-LET radiation. Here, we compare high-LET α-particle to low-LET X-ray irradiation and monitor double-strand break (DSB) processing. Live-cell microscopy was used to monitor DNA double-strand breaks (DSBs), marked by p53-binding protein 1 (53BP1). In addition, the accumulation of the endogenous 53BP1 and replication protein A (RPA) DSB processing proteins was analyzed by immunofluorescence. In contrast to α-particle-induced 53BP1 foci, X-ray-induced foci were resolved quickly and more dynamically as they showed an increase in 53BP1 protein accumulation and size. In addition, the number of individual 53BP1 and RPA foci was higher after X-ray irradiation, while focus intensity was higher after α-particle irradiation. Interestingly, 53BP1 foci induced by α-particles contained multiple RPA foci, suggesting multiple individual resection events, which was not observed after X-ray irradiation. We conclude that high-LET α-particles cause closely interspaced DSBs leading to high local concentrations of repair proteins. Our results point toward a change in DNA damage processing toward DNA end-resection and homologous recombination, possibly due to the depletion of soluble protein in the nucleoplasm. The combination of closely interspaced DSBs and perturbed DNA damage processing could be an explanation for the increased relative biological effectiveness (RBE) of high-LET α-particles compared to X-ray irradiation.