Loss of Smooth Muscle α-Actin Leads to NF-κB-Dependent Increased Sensitivity to Angiotensin II in Smooth Muscle Cells and Aortic Enlargement.

RATIONALE: Mutations in ACTA2, encoding the smooth muscle isoform of α-actin, cause thoracic aortic aneurysms, acute aortic dissections, and occlusive vascular diseases. OBJECTIVE: We sought to identify the mechanism by which loss of smooth muscle α-actin causes aortic disease. METHODS AND RESULTS: Acta2-/- mice have an increased number of elastic lamellae in the ascending aorta and progressive aortic root dilation as assessed by echocardiography that can be attenuated by treatment with losartan, an angiotensin II (AngII) type 1 receptor blocker. AngII levels are not increased in Acta2-/- aortas or kidneys. Aortic tissue and explanted smooth muscle cells from Acta2-/- aortas show increased production of reactive oxygen species and increased basal nuclear factor κB signaling, leading to an increase in the expression of the AngII receptor type I a and activation of signaling at 100-fold lower levels of AngII in the mutant compared with wild-type cells. Furthermore, disruption of smooth muscle α-actin filaments in wild-type smooth muscle cells by various mechanisms activates nuclear factor κB signaling and increases expression of AngII receptor type I a. CONCLUSIONS: These findings reveal that disruption of smooth muscle α-actin filaments in smooth muscle cells increases reactive oxygen species levels, activates nuclear factor κB signaling, and increases AngII receptor type I a expression, thus potentiating AngII signaling in vascular smooth muscle cells without an increase in the exogenous levels of AngII.

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.

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-ß.

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-ß). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-ß 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-ß, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense 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 alpha-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.

Genetic variants promoting smooth muscle cell proliferation can result in diffuse and diverse vascular diseases: evidence for a hyperplastic vasculomyopathy.

Genetic predisposition to early onset of occlusive vascular diseases, including coronary artery disease, ischemic stroke, and Moyamoya disease, may represent varying presentations of a common underlying dysregulation of vascular smooth muscle cell proliferation. We discuss mutations in two genes, NF1 and ACTA2, which predispose affected individuals to diffuse and diverse vascular diseases. These patients show evidence of diffuse occlusive disease in multiple arterial beds or even develop seemingly diverse arterial pathologies, ranging from occlusions to arterial aneurysms. We also present the current evidence that both NF1 and ACTA2 mutations promote increased smooth muscle cell proliferation in vitro and in vivo, which leads us to propose that these diffuse and diverse vascular diseases are the outward signs of a more fundamental disease: a hyperplastic vasculomyopathy. We suggest that the concept of a hyperplastic vasculomyopathy offers a new approach not only to identifying mutated genes that lead to vascular diseases but also to counseling and possibly treating patients harboring such mutations. In other words, this framework may offer the opportunity to therapeutically target the inappropriate smooth muscle cell behavior that predisposes to a variety of vascular diseases throughout the arterial system.

Pathogenesis of thoracic and abdominal aortic aneurysms.

The major disease processes affecting the aorta are aortic aneurysms and dissections. Aneurysms are usually described in terms of their anatomic location, with thoracic aortic aneurysms (TAAs) involving the ascending and descending aorta in the thoracic cavity and abdominal aortic aneurysms (AAAs) involving the infrarenal abdominal aorta. Both thoracic and abdominal aortas are elastic arteries, and share similarities in their physical structures and cellular components. However, thoracic and abdominal aortas differ in their biochemical properties and the origin of their vascular smooth muscle cells (VSMCs). These similarities and differences between thoracic and abdominal aortas provide the basis for the various pathologic mechanisms observed in this disease. This review focuses on the comparison of the pathologic mechanisms involved in TAA and AAA.

Abnormal mechanosensing and cofilin activation promote the progression of ascending aortic aneurysms in mice.

Smooth muscle cells (SMCs) and the extracellular matrix (ECM) are intimately associated in the aortic wall. Fbln4(SMKO) mice with an SMC-specific deletion of the Fbln4 gene, which encodes the vascular ECM component fibulin-4, develop ascending aortic aneurysms that have increased abundance of angiotensin-converting enzyme (ACE); inhibiting angiotensin II signaling within the first month of life prevents aneurysm development. We used comparative proteomics analysis of Fbln4(SMKO) aortas from postnatal day (P) 1 to P30 mice to identify key molecules involved in aneurysm initiation and expansion. At P14, the actin depolymerizing factor cofilin was dephosphorylated and thus activated, and at P7, the abundance of slingshot-1 (SSH1) phosphatase, an activator of cofilin, was increased, leading to actin cytoskeletal remodeling. Also, by P7, biomechanical changes and underdeveloped elastic lamina-SMC connections were evident, and the abundance of early growth response 1 (Egr1), a mechanosensitive transcription factor that stimulates ACE expression, was increased, which was before the increases in ACE abundance and cofilin activation. Postnatal deletion of Fbln4 in SMCs at P7 prevented cofilin activation and aneurysm formation, suggesting that these processes required disruption of elastic lamina-SMC connections. Phosphoinositide 3-kinase (PI3K) is involved in the angiotensin II-mediated activation of SSH1, and administration of PI3K inhibitors from P7 to P30 decreased SSH1 abundance and prevented aneurysms. These results suggest that aneurysm formation arises from abnormal mechanosensing of SMCs resulting from the loss of elastic lamina-SMC connections and from increased SSH1 and cofilin activity, which may be potential therapeutic targets for treating ascending aortic aneurysms.

Fibulin-4 and fibulin-5 in elastogenesis and beyond: Insights from mouse and human studies.

The fibulin family of extracellular matrix/matricellular proteins is composed of long fibulins (fibulin-1, -2, -6) and short fibulins (fibulin-3, -4, -5, -7) and is involved in protein-protein interaction with the components of basement membrane and extracellular matrix proteins. Fibulin-1, -2, -3, -4, and -5 bind the monomeric form of elastin (tropoelastin) in vitro and fibulin-2, -3, -4, and -5 are shown to be involved in various aspects of elastic fiber development in vivo. In particular, fibulin-4 and -5 are critical molecules for elastic fiber assembly and play a non-redundant role during elastic fiber formation. Despite manifestation of systemic elastic fiber defects in all elastogenic tissues, fibulin-5 null (Fbln5(-/-)) mice have a normal lifespan. In contrast, fibulin-4 null (Fbln4(-/-)) mice die during the perinatal period due to rupture of aortic aneurysms, indicating differential functions of fibulin-4 and fibulin-5 in normal development. In this review, we will update biochemical characterization of fibulin-4 and fibulin-5 and discuss their roles in elastogenesis and outside of elastogenesis based on knowledge obtained from loss-of-function studies in mouse and in human patients with FBLN4 or FBLN5 mutations. Finally, we will evaluate therapeutic options for matrix-related diseases.

Angiotensin-converting enzyme-induced activation of local angiotensin signaling is required for ascending aortic aneurysms in fibulin-4-deficient mice.

Aortic aneurysms are life-threatening and often associated with defects in connective tissues and mutations in smooth muscle cell (SMC) contractile proteins. Despite recent advances in understanding altered signaling in aneurysms of Marfan syndrome, the underlying mechanisms and options for pharmacological treatment for other forms of aneurysms are still under investigation. We previously showed in mice that deficiency in the fibulin-4 gene in vascular SMCs (Fbln4(SMKO)) leads to loss of the SMC contractile phenotype, hyperproliferation, and ascending aortic aneurysms. We report that abnormal up-regulation of angiotensin-converting enzyme (ACE) in SMCs and subsequent activation of angiotensin II (AngII) signaling are involved in the onset of aortic aneurysms in Fbln4(SMKO) mice. In this model, aneurysm formation was completely prevented by inhibition of the AngII pathway with losartan or captopril within a narrow therapeutic window during the first month of life, even though the altered mechanical properties of blood vessel walls were not reversed by the pharmacological treatment. The therapeutic effects of losartan in Fbln4(SMKO) mice do not require the AngII receptor type 2 (Agtr2) but likely require both type 1a (Agtr1a) and 1b (Agtr1b) receptors. The results indicate that fibulin-4 is a vascular matrix component required for regulation of local angiotensin signaling and development and maintenance of the SMC phenotype.

Genetic basis of thoracic aortic aneurysms and dissections: focus on smooth muscle cell contractile dysfunction.

Thoracic aortic aneurysms leading to type A dissections (TAAD) can be inherited in isolation or in association with genetic syndromes, such as Marfan syndrome and Loeys-Dietz syndrome. When TAAD occurs in the absence of syndromic features, it is inherited in an autosomal dominant manner with decreased penetrance and variable expression, the disease is referred to as familial TAAD. Familial TAAD exhibits significant clinical and genetic heterogeneity. The first genes identified to cause TAAD were FBN1, TGFBR2, and TGFBR1. The identification and characterization of these genes suggested that increased TGF-beta signaling plays a role in pathogenesis. The recent discovery that mutations in the vascular smooth muscle cell (SMC)-specific beta-myosin (MYH11) and alpha-actin (ACTA2) can also cause this disorder has focused attention on the importance of the maintenance of SMC contractile function in preserving aortic structure and preventing TAAD.

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's 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.

MYH11 mutations result in a distinct vascular pathology driven by insulin-like growth factor 1 and angiotensin II.

Non-syndromic thoracic aortic aneurysms and dissections (TAADs) are inherited in an autosomal dominant manner in approximately 20% of cases. Familial TAAD is genetically heterogeneous and four loci have been mapped for this disease to date, including a locus at 16p for TAAD associated with patent ductus arteriosus (PDA). The defective gene at the 16p locus has recently been identified as the smooth muscle cell (SMC)-specific myosin heavy chain gene (MYH11). On sequencing MYH11 in 93 families with TAAD alone and three families with TAAD/PDA, we identified novel mutations in two families with TAAD/PDA, but none in families with TAAD alone. Histopathological analysis of aortic sections from two individuals with MYH11 mutations revealed SMC disarray and focal hyperplasia of SMCs in the aortic media. SMC hyperplasia leading to significant lumen narrowing in some of the vessels of the adventitia was also observed. Insulin-like growth factor-1 (IGF-1) was upregulated in mutant aortas as well as explanted SMCs, but no increase in transforming growth factor-beta expression or downstream targets was observed. Enhanced expression of angiotensin-converting enzyme and markers of Angiotensin II (Ang II) vascular inflammation (macrophage inflammatory protein-1alpha and beta) were also found. These data suggest that MYH11 mutations are likely to be specific to the phenotype of TAAD/PDA and result in a distinct aortic and occlusive vascular pathology potentially driven by IGF-1 and Ang II.

Mutations in smooth muscle alpha-actin (ACTA2) lead to thoracic aortic aneurysms and dissections.

The major function of vascular smooth muscle cells (SMCs) is contraction to regulate blood pressure and flow. SMC contractile force requires cyclic interactions between SMC alpha-actin (encoded by ACTA2) and the beta-myosin heavy chain (encoded by MYH11). Here we show that missense mutations in ACTA2 are responsible for 14% of inherited ascending thoracic aortic aneurysms and dissections (TAAD). Structural analyses and immunofluorescence of actin filaments in SMCs derived from individuals heterozygous for ACTA2 mutations illustrate that these mutations interfere with actin filament assembly and are predicted to decrease SMC contraction. Aortic tissues from affected individuals showed aortic medial degeneration, focal areas of medial SMC hyperplasia and disarray, and stenotic arteries in the vasa vasorum due to medial SMC proliferation. These data, along with the previously reported MYH11 mutations causing familial TAAD, indicate the importance of SMC contraction in maintaining the structural integrity of the ascending aorta.