Acute thoracic aortic dissections, a life-threatening complication of thoracic aortic aneurysms, can be prevented if at-risk individuals are identified. Up to 25% of individuals with thoracic aorti...
Background: Thoracic aortic aneurysms and/or dissections (TAD) are associated with pathogenic variants in genes that involved in either smooth muscle cell (SMC) contraction or extracellular matrix (ECM). The inability to maintain homeostasis between SMCs and ECM is pivotal in TAD pathogenesis. Focal adhesions (FA) act as mechanosensors in cells, transferring mechanical signals from the ECM to the intracellular contractile filaments and regulating downstream Rho/ROCK signaling. However, the landscape of SMC-ECM interactions, has not been fully explored at the early stages of TAD. Methods: C57BL/6J wild-type mice aged 21 days postnatal (P21) were administered the ECM cross-link blocker, BAPN, for up to four weeks to induce TAD. Proximal aortas were harvested for single-cell RNA sequencing at P35. Immunofluorescence and immunoblots were used to validate findings. Results: Single-cell RNA sequencing classified SMCs into 5 clusters marked by Rgs5 (SMC Rgs5 + ), high levels of Palld (SMC Palld + ), Tnnt2 (SMC Tnnt2 + , marker of second heart field-derived cells), Atf3 (SMC Atf3+ cluster), or no clear marker (SMC NOS ). The analysis of how BAPN exposure affects different cell types, based on the Jensen-Shannon divergence in the probability distribution between groups, demonstrated that SMCs, and specifically SMC Atf3 + and SMC NOS , are the most perturbed cell cluster by BAPN. Gene set enrichment analysis identified significantly dysregulated pathways of FA and Rho/ROCK signaling. The expression of Itga5 and Fn1 (fibronectin) were increased and Itga8 was decreased. An SMC-SMC interaction analysis found Fn1 interactions were the most upregulated interaction in 4 SMC clusters. When analyzing downstream pathways due to the changes in interaction, 80% of the top 25 interactions were related to integrins, including increased Fn1 interactions with Itga5-Itgb1 and Itgav-Itgb3. In SMC Rgs5 + cluster, the most affected interactions are related to TGF-β signaling. Conclusions: These findings suggest that, in the majority of aortic SMCs, mechanosensing through FAs, driven by fibronectin interactions with specific integrin receptor pairs, is activated with BAPN treatment, potentially contributing to the pathogenesis of TAD.
Cerebrovascular disease and risk of stroke in patients with ACTA2 R179H and R179L mutations Ellen S. Regalado 1 , John R. Østergaard 2 , Vijeya Ganesan 3 , Dong-chuan Guo 1 , and Dianna M. Milewicz 1 1 Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA; 2 Centre for Rare Diseases, Department of Paediatrics, Aarhus University Hospital, Skejby, Denmark; 3 Neurosciences Unit, Institute of Child Health, University College London, London, UK Mutations in the ACTA2 gene, which codes for the smooth muscle specific isoform of alpha actin, predispose to a variety of vascular diseases, including thoracic aortic aneurysm and dissection, early onset stroke, and coronary artery disease. Recently we reported a severe de novo ACTA2 p.R179H mutation that causes a syndromic smooth muscle dysfunction resulting in aortic aneurysms, patent ductus arteriosus, fixed dilated pupils, hypotonic bladder, malrotation and hypoperistalsis of the gut, and pulmonary hypertension. Additionally, childhood onset of cerebrovascular disease is a prominent feature of this syndrome. To understand the extent of cerebrovascular disease and risk of stroke in these patients, we examined the clinical data of 9 patients recruited worldwide (8 Caucasians, 1 Asian; female to male ratio, 5:4; 8 with p.R179H mutation, 1 with p.R179L mutation). These patients ranged in age from 1 to 27 years old at the time of diagnosis of the ACTA2 mutation. All of these patients demonstrated multiple areas of narrowing of the intracranial vessels and increased T2 signal intensity in the periventricular white matter. Eight patients had bilateral stenosis of the distal internal carotid arteries and ectasia of the internal carotid arteries involving the petrous, cavernous and clinoidal segments and one patient had occlusion of the basilar artery only. Of these 9 patients, 5 have presented with ischemic stroke or recurrent transient ischemic attacks (age range, 2-27 years old; female to male ratio, 4:1), 4 of whom demonstrated cerebral infarction on MR imaging (age range, 2-16 years old). Two patients underwent bilateral revascularization procedures at age 9 and 6 years; the 9 year old patient developed an acute ischemic stroke within a year of surgery whereas the 6 year old remains stroke free 10 years after surgery. These data demonstrates the severity of cerebrovascular disease presentation in this subset of patients with the ACTA2 mutation and underlines the need for early diagnosis and careful surveillance and management in order to prevent ischemic strokes.
Abstract Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.
Pathogenic variants in ACTA2 , which encodes smooth muscle α-actin ( α-SMA ), predispose to thoracic aortic disease. ACTA2 variants disrupting arginine 179 ( R179 ) also predispose to moyamoya-like ( MMD ) cerebrovascular disease, characterized by occlusion of the distal internal carotid arteries by neointimal cells that stain positive for smooth muscle cell ( SMC ) markers. Mice with conditional knockin of the Acta2 R179C mutation specifically in SMCs were generated ( Acta2 SMC-R179C/+ ). RNA sequencing and 2D gel electrophoresis confirmed presence of the mutation. Acta2 SMC-R179C/+ SMCs are not fully differentiated, maintain stem cell marker expression, and proliferate and migrate faster than wildtype ( WT ) SMCs. Seahorse analyses show increased glycolysis and decreased oxidative respiration ( OXPHOS ) in Acta2 SMC-R179C/+ SMCs, which have reduced mitochondrial mass and complex I activity, suggestive of deficient electron transport chain activity. Nicotinamide riboside ( NR ) augments complex I activity to boost OXPHOS without altering mitochondrial mass in Acta2 SMC-R179C/+ SMCs. NR also increases differentiation and reduces migration in Acta2 SMC-R179C/+ SMCs. With left carotid artery ligation ( LCAL ), 33% of Acta2 SMC-R179C/+ mice die within 4 days due to strokes, and 100% of surviving Acta2 SMC-R179C/+ mice at postop day 21 have occlusive lesions in the injured artery (8% in WT mice) containing α-SMA+ cells and fibrin deposits. Acta2 SMC-R179C/+ mice also exhibit markedly increased left carotid artery diameter and increased number, diameter, and tortuosity of left-sided collateral vessels compared to WT mice at postop day 21. NR treatment reduces deaths of LCAL-injured Acta2 SMC-R179C/+ mice to 8%, completely resolves occlusive lesion formation in 50% of mutant mice, and attenuates collateral formation and remodeling. These data suggest that immature SMCs with the ACTA2 R179C variant proliferate and migrate into the neointima causing MMD-like occlusive disease. Shifting metabolism to OXPHOS with NR drives differentiation and reduces migration, which diminishes arterial lesions in Acta2 SMC-R179C/+ mice. These data support the use of NR as a therapeutic to prevent MMD-like lesions and ischemic strokes in children with ACTA2 p.R179 variants.
Rationale: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. Objective: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. Methods and Results: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II–induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. Conclusions: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.
