Smooth Muscle Hyperplasia due to ACTA2/MYH11 Mutations: Identification of Novel Pathology and Pathways Leading to Aneurysms and Diverse Vascular Occlusive Diseases

Missense mutations in smooth muscle cell (SMC) specific ACTA2 (á-actin) and MYH11 (â-myosin heavy chain) cause diffuse and diverse vascular diseases, including thoracic aortic aneurysms and dissections (TAAD) and early onset coronary artery disease and stroke. The mechanism by which these mutations lead to dilatation of some arteries but occlusion of others is unknown. We hypothesized that the mutations act through two distinct mechanisms to cause varied vascular diseases: a loss of function, leading to decreased SMC contraction and aneurysms, and a gain of function, leading to increased SMC proliferation and occlusive disease. To test this hypothesis, ACTA2 mutant SMCs and myofibroblasts were assessed and found to not form á-actin filaments whereas control cells did, suggesting a dominant negative effect of ACTA2 mutations on filament formation. A loss of á-actin filaments would be predicted to cause decreased SMC contractility. Histological examination of vascular tissues from patients revealed SMC hyperplasia leading to arterial stenosis and occlusion, supporting a gain of function associated with the mutant gene. Furthermore, ACTA2 mutant SMCs and myofibroblasts proliferated more rapidly in static culture than control cells (p\u3c0.05). We also determined that Acta2-/- mice have ascending aortic aneurysms. Histological examination revealed aortic medial SMC hyperplasia, but minimal features of medial degeneration. Acta2-/- SMCs proliferated more rapidly in culture than wildtype (p\u3c0.05), and microarray analysis of Acta2-/- SMCs revealed increased expression of Actg2, 15 collagen genes, and multiple focal adhesion genes. Acta2-/- SMCs showed altered localization of vinculin and zyxin and increased phosphorylated focal adhesion kinase (FAK) in focal adhesions. A specific FAK inhibitor decreased Acta2-/- SMC proliferation to levels equal to wildtype SMCs (p\u3c0.05), suggesting that FAK activation leads to the increased proliferation. We have described a unique pathology associated with ACTA2 and MYH11 mutations, as well as an aneurysm phenotype in Acta2-/- mice. Additionally, we identified a novel pathogenic pathway for vascular occlusive disease due to loss of SMC contractile filaments, alterations in focal adhesions, and activation of FAK signaling in SMCs with ACTA2 mutations

Characterization of the inflammatory cells in ascending thoracic aortic aneurysms in patients with Marfan syndrome, familial thoracic aortic aneurysms, and sporadic aneurysms.

OBJECTIVE: This study sought to characterize the inflammatory infiltrate in ascending thoracic aortic aneurysm in patients with Marfan syndrome, familial thoracic aortic aneurysm, or nonfamilial thoracic aortic aneurysm. BACKGROUND: Thoracic aortic aneurysms are associated with a pathologic lesion termed medial degeneration, which is described as a noninflammatory lesion. Thoracic aortic aneurysms are a complication of Marfan syndrome and can be inherited in an autosomal dominant manner of familial thoracic aortic aneurysm. METHODS: Full aortic segments were collected from patients undergoing elective repair with Marfan syndrome (n = 5), familial thoracic aortic aneurysm (n = 6), and thoracic aortic aneurysms (n = 9), along with control aortas (n = 5). Immunohistochemistry staining was performed using antibodies directed against markers of lymphocytes and macrophages. Real-time polymerase chain reaction analysis was performed to quantify the expression level of the T-cell receptor beta-chain variable region gene. RESULTS: Immunohistochemistry of thoracic aortic aneurysm aortas demonstrated that the media and adventitia from Marfan syndrome, familial thoracic aortic aneurysm, and sporadic cases had increased numbers of T lymphocytes and macrophages when compared with control aortas. The number of T cells and macrophages in the aortic media of the aneurysm correlated inversely with the patient\u27s age at the time of prophylactic surgical repair of the aorta. T-cell receptor profiling indicated a similar clonal nature of the T cells in the aortic wall in a majority of aneurysms, whether the patient had Marfan syndrome, familial thoracic aortic aneurysm, or sporadic disease. CONCLUSION: These results indicate that the infiltration of inflammatory cells contributes to the pathogenesis of thoracic aortic aneurysms. Superantigen-driven stimulation of T lymphocytes in the aortic tissues of patients with thoracic aortic aneurysms may contribute to the initial immune response

Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-β

Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2−/− mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-β). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-β activation and Acta2−/− SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-β, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutation

Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2 mutations

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations

Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations

Mutations in Smooth Muscle Alpha-Actin (ACTA2) Cause Coronary Artery Disease, Stroke, and Moyamoya Disease, Along with Thoracic Aortic Disease

The vascular smooth muscle cell (SMC)-specific isoform of α-actin (ACTA2) is a major component of the contractile apparatus in SMCs located throughout the arterial system. Heterozygous ACTA2 mutations cause familial thoracic aortic aneurysms and dissections (TAAD), but only half of mutation carriers have aortic disease. Linkage analysis and association studies of individuals in 20 families with ACTA2 mutations indicate that mutation carriers can have a diversity of vascular diseases, including premature onset of coronary artery disease (CAD) and premature ischemic strokes (including Moyamoya disease [MMD]), as well as previously defined TAAD. Sequencing of DNA from patients with nonfamilial TAAD and from premature-onset CAD patients independently identified ACTA2 mutations in these patients and premature onset strokes in family members with ACTA2 mutations. Vascular pathology and analysis of explanted SMCs and myofibroblasts from patients harboring ACTA2 suggested that increased proliferation of SMCs contributed to occlusive diseases. These results indicate that heterozygous ACTA2 mutations predispose patients to a variety of diffuse and diverse vascular diseases, including TAAD, premature CAD, ischemic strokes, and MMD. These data demonstrate that diffuse vascular diseases resulting from either occluded or enlarged arteries can be caused by mutations in a single gene and have direct implications for clinical management and research on familial vascular diseases