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.

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.

Fibulin-4 deficiency differentially affects cytoskeleton structure and dynamics as well as TGFß signaling.

Fibulin-4 is an extracellular matrix (ECM) protein essential for elastogenesis and mutations in this protein lead to aneurysm formation. In this study, we isolated vascular smooth muscle cells (VSMCs) from mice with reduced fibulin-4 protein expression (Fibulin-4R/R) and from mice with a smooth muscle cell specific deletion of the Fibulin-4 gene (Fibulin-4f/-/SM22Cre+). We subsequently analyzed and compared the molecular consequences of reduced Fibulin-4 expression versus total ablation of Fibulin-4 expression with regard to effects on the SMC specific contractile machinery, cellular migration and TGFß signaling. Analysis of the cytoskeleton showed that while Fibulin-4f/-/SM22Cre+ VSMCs lack smooth muscle actin (SMA) fibers, Fibulin-4R/R VSMCs were able to form SMA fibers. Furthermore, Fibulin-4f/-/SM22Cre+ VSMCs showed a decreased pCofilin to Cofilin ratio, suggesting increased actin depolymerization, while Fibulin-4R/R VSMCs did not display this decrease. Yet, both Fibulin-4 mutant VSMCs showed decreased migration. We found increased activation of TGFß signaling in Fibulin-4R/R VSMCs. However, TGFß signaling was not increased in Fibulin-4f/-/SM22Cre+ VSMCs. From these results we conclude that both reduction and absence of Fibulin-4 leads to structural and functional impairment of the SMA cytoskeleton. However, while reduced levels of Fibulin-4 result in increased TGFß activation, complete absence of Fibulin-4 does not result in increased TGFß activation. Since both mouse models show thoracic aortic aneurysm formation, we conclude that not only hampered TGFß signaling, but also SMA cytoskeleton dynamics play an important role in aortic aneurysmal disease.

Decreased mitochondrial respiration in aneurysmal aortas of Fibulin-4 mutant mice is linked to PGC1A regulation.

Aim: Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation. Methods and results: In Fibulin-4R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4R/R mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4R/R aortas. Consistently, functional studies in Fibulin-4R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4R/R VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1M318R/+ VSMCs, a mouse model for Loeys-Dietz syndrome (LDS). Interestingly, also human fibroblasts from Marfan (FBN1) and LDS (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial Complexes I-V activities were unaltered in Fibulin-4R/R heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4R/R mice displayed increased reactive oxygen species (ROS) levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4R/R VSMCs. Increased TGFß reduced PGC1α levels, indicating involvement of TGFß signalling in PGC1α regulation. Activation of PGC1α restored the decreased oxygen consumption in Fibulin-4R/R VSMCs and improved their reduced growth potential, emphasizing the importance of this key regulator. Conclusion: Our data indicate altered mitochondrial function and metabolic dysregulation, leading to increased ROS levels and altered energy production, as a novel mechanism, which may contribute to thoracic aortic aneurysm formation.

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure. The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

Longitudinal assessment of bleomycin-induced lung fibrosis by Micro-CT correlates with histological evaluation in mice.

BACKGROUND: The intratracheal instillation of bleomycin in mice induces early damage to alveolar epithelial cells and development of inflammation followed by fibrotic tissue changes and represents the most widely used model of pulmonary fibrosis to investigate human IPF. Histopathology is the gold standard for assessing lung fibrosis in rodents, however it precludes repeated and longitudinal measurements of disease progression and does not provide information on spatial and temporal distribution of tissue damage. Here we investigated the use of the Micro-CT technique to allow the evaluation of disease onset and progression at different time-points in the mouse bleomycin model of lung fibrosis. Micro-CT was throughout coupled with histological analysis for the validation of the imaging results. METHODS: In bleomycin-instilled and control mice, airways and lung morphology changes were assessed and reconstructed at baseline, 7, 14 and 21 days post-treatment based on Micro-CT images. Ashcroft score, percentage of collagen content and percentage of alveolar air area were detected on lung slides processed by histology and subsequently compared with Micro-CT parameters. RESULTS: Extent (%) of fibrosis measured by Micro-CT correlated with Ashcroft score, the percentage of collagen content and the percentage of alveolar air area (r2 = 0.91; 0.77; 0.94, respectively). Distal airway radius also correlated with the Ashcroft score, the collagen content and alveolar air area percentage (r2 = 0.89; 0.78; 0.98, respectively). CONCLUSIONS: Micro-CT data were in good agreement with histological read-outs as micro-CT was able to quantify effectively and non-invasively disease progression longitudinally and to reduce the variability and number of animals used to assess the damage. This suggests that this technique is a powerful tool for understanding experimental pulmonary fibrosis and that its use could translate into a more efficient drug discovery process, also helping to fill the gap between preclinical setting and clinical practice.

Study of potential health effects of electromagnetic fields of telephony and Wi-Fi, using chicken embryo development as animal model.

The objective of this study is to investigate possible biological effects of radiofrequency electromagnetic fields (RF-EMF) as used in modern wireless telecommunication in a well-controlled experimental environment using chicken embryo development as animal model. Chicken eggs were incubated under continuous experimental exposure to GSM (1.8 GHz), DECT (1.88 GHz), UMTS (2.1 GHz), and WLAN (5.6 GHz) radiation, with the appropriate modulation protocol, using a homogeneous field distribution at a field strength of approximately 3 V/m, representing the maximum field level in a normal living environment. Radiation-shielded exposure units/egg incubators were operating in parallel for exposed and control eggs in a climatized homogeneous environment, using 450 eggs per treatment in three successive rounds per treatment. Dosimetry of the exposure (field characteristics and specific absorption rate) were studied. Biological parameters studied included embryo death during incubation, hatching percentage, and various morphological and histological parameters of embryos and chicks and their organs, and gene expression profiles of embryos on day 7 and day 18 of incubation by microarray and qPCR. No conclusive evidence was found for induced embryonic mortality or malformations by exposure to the used EMFs, or for effects on the other measured parameters. Estimated differences between treatment groups were always small and the effect of treatment was not significant. In a statistical model that ignored possible interaction between rounds and exposure units, some of the many pairwise comparisons of exposed versus control had P-values lower than 0.05, but were not significant after correction for multiple testing. Bioelectromagnetics. 38:186-203, 2017. © 2017 Wiley Periodicals, Inc.

AT1-receptor blockade, but not renin inhibition, reduces aneurysm growth and cardiac failure in fibulin-4 mice.

AIMS: Increasing evidence supports a role for the angiotensin II-AT1-receptor axis in aneurysm development. Here, we studied whether counteracting this axis via stimulation of AT2 receptors is beneficial. Such stimulation occurs naturally during AT1-receptor blockade with losartan, but not during renin inhibition with aliskiren. METHODS AND RESULTS: Aneurysmal homozygous fibulin-4 mice, displaying a four-fold reduced fibulin-4 expression, were treated with placebo, losartan, aliskiren, or the ß-blocker propranolol from day 35 to 100. Their phenotype includes cystic media degeneration, aortic regurgitation, left ventricular dilation, reduced ejection fraction, and fractional shortening. Although losartan and aliskiren reduced hemodynamic stress and increased renin similarly, only losartan increased survival. Propranolol had no effect. No drug rescued elastic fiber fragmentation in established aneurysms, although losartan did reduce aneurysm size. Losartan also increased ejection fraction, decreased LV diameter, and reduced cardiac pSmad2 signaling. None of these effects were seen with aliskiren or propranolol. Longitudinal micro-CT measurements, a novel method in which each mouse serves as its own control, revealed that losartan reduced LV growth more than aneurysm growth, presumably because the heart profits both from the local (cardiac) effects of losartan and its effects on aortic root remodeling. CONCLUSION: Losartan, but not aliskiren or propranolol, improved survival in fibulin-4 mice. This most likely relates to its capacity to improve structure and function of both aorta and heart. The absence of this effect during aliskiren treatment, despite a similar degree of blood pressure reduction and renin-angiotensin system blockade, suggests that it might be because of AT2-receptor stimulation.

Extracellular matrix defects in aneurysmal Fibulin-4 mice predispose to lung emphysema.

BACKGROUND: In this study we set out to investigate the clinically observed relationship between chronic obstructive pulmonary disease (COPD) and aortic aneurysms. We tested the hypothesis that an inherited deficiency of connective tissue might play a role in the combined development of pulmonary emphysema and vascular disease. METHODS: We first determined the prevalence of chronic obstructive pulmonary disease in a clinical cohort of aortic aneurysms patients and arterial occlusive disease patients. Subsequently, we used a combined approach comprising pathological, functional, molecular imaging, immunological and gene expression analysis to reveal the sequence of events that culminates in pulmonary emphysema in aneurysmal Fibulin-4 deficient (Fibulin-4(R)) mice. RESULTS: Here we show that COPD is significantly more prevalent in aneurysm patients compared to arterial occlusive disease patients, independent of smoking, other clinical risk factors and inflammation. In addition, we demonstrate that aneurysmal Fibulin-4(R/R) mice display severe developmental lung emphysema, whereas Fibulin-4(+/R) mice acquire alveolar breakdown with age and upon infectious stress. This vicious circle is further exacerbated by the diminished antiprotease capacity of the lungs and ultimately results in the development of pulmonary emphysema. CONCLUSIONS: Our experimental data identify genetic susceptibility to extracellular matrix degradation and secondary inflammation as the common mechanisms in both COPD and aneurysm formation.

Absence of cardiovascular manifestations in a haploinsufficient Tgfbr1 mouse model.

Loeys-Dietz syndrome (LDS) is an autosomal dominant arterial aneurysm disease belonging to the spectrum of transforming growth factor ß (TGFß)-associated vasculopathies. In its most typical form it is characterized by the presence of hypertelorism, bifid uvula/cleft palate and aortic aneurysm and/or arterial tortuosity. LDS is caused by heterozygous loss of function mutations in the genes encoding TGFß receptor 1 and 2 (TGFBR1 and -2), which lead to a paradoxical increase in TGFß signaling. To address this apparent paradox and to gain more insight into the pathophysiology of aneurysmal disease, we characterized a new Tgfbr1 mouse model carrying a p.Y378* nonsense mutation. Study of the natural history in this model showed that homozygous mutant mice die during embryonic development due to defective vascularization. Heterozygous mutant mice aged 6 and 12 months were morphologically and (immuno)histochemically indistinguishable from wild-type mice. We show that the mutant allele is degraded by nonsense mediated mRNA decay, expected to result in haploinsufficiency of the mutant allele. Since this haploinsufficiency model does not result in cardiovascular malformations, it does not allow further study of the process of aneurysm formation. In addition to providing a comprehensive method for cardiovascular phenotyping in mice, the results of this study confirm that haploinsuffciency is not the underlying genetic mechanism in human LDS.

MicroRNA-29 in aortic dilation: implications for aneurysm formation.

RATIONALE: Aging represents a major risk factor for coronary artery disease and aortic aneurysm formation. MicroRNAs (miRs) have emerged as key regulators of biological processes, but their role in age-associated vascular pathologies is unknown. OBJECTIVE: We aim to identify miRs in the vasculature that are regulated by age and play a role in age-induced vascular pathologies. METHODS AND RESULTS: Expression profiling of aortic tissue of young versus old mice identified several age-associated miRs. Among the significantly regulated miRs, the increased expression of miR-29 family members was associated with a profound downregulation of numerous extracellular matrix (ECM) components in aortas of aged mice, suggesting that this miR family contributes to ECM loss, thereby sensitizing the aorta for aneurysm formation. Indeed, miR-29 expression was significantly induced in 2 experimental models for aortic dilation: angiotensin II-treated aged mice and genetically induced aneurysms in Fibulin-4(R/R) mice. More importantly, miR-29b levels were profoundly increased in biopsies of human thoracic aneurysms, obtained from patients with either bicuspid (n=79) or tricuspid aortic valves (n=30). Finally, LNA-modified antisense oligonucleotide-mediated silencing of miR-29 induced ECM expression and inhibited angiotensin II-induced dilation of the aorta in mice. CONCLUSION: In conclusion, miR-29-mediated downregulation of ECM proteins may sensitize the aorta to the formation of aneurysms in advanced age. Inhibition of miR-29 in vivo abrogates aortic dilation in mice, suggesting that miR-29 may represent a novel molecular target to augment matrix synthesis and maintain vascular wall structural integrity.

Impaired vascular contractility and aortic wall degeneration in fibulin-4 deficient mice: effect of angiotensin II type 1 (AT1) receptor blockade.

Medial degeneration is a key feature of aneurysm disease and aortic dissection. In a murine aneurysm model we investigated the structural and functional characteristics of aortic wall degeneration in adult fibulin-4 deficient mice and the potential therapeutic role of the angiotensin (Ang) II type 1 (AT(1)) receptor antagonist losartan in preventing aortic media degeneration. Adult mice with 2-fold (heterozygous Fibulin-4(+/R)) and 4-fold (homozygous Fibulin-4(R/R)) reduced expression of fibulin-4 displayed the histological features of cystic media degeneration as found in patients with aneurysm or dissection, including elastin fiber fragmentation, loss of smooth muscle cells, and deposition of ground substance in the extracellular matrix of the aortic media. The aortic contractile capacity, determined by isometric force measurements, was diminished, and was associated with dysregulation of contractile genes as shown by aortic transcriptome analysis. These structural and functional alterations were accompanied by upregulation of TGF-ß signaling in aortas from fibulin-4 deficient mice, as identified by genome-scaled network analysis as well as by immunohistochemical staining for phosphorylated Smad2, an intracellular mediator of TGF-ß. Tissue levels of Ang II, a regulator of TGF-ß signaling, were increased. Prenatal treatment with the AT(1) receptor antagonist losartan, which blunts TGF-ß signaling, prevented elastic fiber fragmentation in the aortic media of newborn Fibulin-4(R/R) mice. Postnatal losartan treatment reduced haemodynamic stress and improved lifespan of homozygous knockdown fibulin-4 animals, but did not affect aortic vessel wall structure. In conclusion, the AT(1) receptor blocker losartan can prevent aortic media degeneration in a non-Marfan syndrome aneurysm mouse model. In established aortic aneurysms, losartan does not affect aortic architecture, but does improve survival. These findings may extend the potential therapeutic application of inhibitors of the renin-angiotensin system to the preventive treatment of aneurysm disease.

Multimodality imaging reveals a gradual increase in matrix metalloproteinase activity at aneurysmal lesions in live fibulin-4 mice.

BACKGROUND: We imaged the protease activity of matrix metalloproteinases (MMPs) upregulated during aneurysm formation, using protease-activatable near-infrared fluorescence probes. We tested whether these protease-activatable sensors can directly report the in vivo activity of the key biomarkers in aneurysm, using our genetically modified fibulin-4 mouse models for aneurysm formation. Mice homozygous for the fibulin-4 reduced-expression allele (fibulin-4(R/R)) show dilatation of the ascending aorta and a tortuous, stiffened aorta resulting from disorganized elastic fiber networks. Strikingly, even a moderate reduction in expression of fibulin-4 in the heterozygous fibulin-4(+/R) mice occasionally results in modest aneurysm formation. METHODS AND RESULTS: Aorta transcriptome and protein expression analysis of fibulin-4(+/R) and fibulin-4(R/R) animals identified excessive transforming growth factor-ß signaling as the critical event in the pathogenesis of aneurysm formation. To determine whether a perturbed elastin lamellar structure arose from induction of transforming growth factor-ß-regulated MMPs, we performed gelatin zymography and used a protease-activatable near-infrared fluorescence probe to monitor and quantify MMP upregulation in animals, using various in vivo optical imaging modules and coregistration of the fluorescence signal with CT images of the same animals. Gelatin zymography demonstrated a significant increase in the presence of the active form of MMP-9 in the aortic arch of fibulin-4(R/R) mice. In vivo analysis of MMP upregulation using the near-infrared fluorescence probe and subsequent isosurface concentration mapping from reconstructed tomographic images from fibulin-4(+/R) and fibulin-4(R/R) mice revealed a graded increase in activation of MMPs within the aneurysmal lesions. CONCLUSIONS: We aimed to develop molecular imaging procedures for faster, earlier, and easier recognition of aortic aneurysms. We show that in vivo coregistration of MMP activity by noninvasive tomographic imaging methods allows the detection of increased MMP activity, even before the aneurysm has actually formed.