ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Aging , DNA Repair , Endonucleases , Extracellular Matrix , Muscle, Smooth, Vascular , Phenotype , Animals , Mice , Aging/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/deficiency , Endonucleases/metabolism , Endonucleases/deficiency , Endonucleases/genetics , Extracellular Matrix/metabolism , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/metabolismABSTRACT
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.
Subject(s)
Fibroblasts , Genetic Association Studies , Loeys-Dietz Syndrome , Muscle, Smooth, Vascular , Smad3 Protein , Humans , Smad3 Protein/genetics , Smad3 Protein/metabolism , Loeys-Dietz Syndrome/genetics , Loeys-Dietz Syndrome/pathology , Male , Female , Fibroblasts/metabolism , Adult , Middle Aged , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Cell Differentiation/genetics , Cell Line , Myocytes, Smooth Muscle/metabolism , Retrospective Studies , Phenotype , Myofibroblasts/metabolism , Myofibroblasts/pathology , MutationABSTRACT
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.