Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

Abstract

The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomeraseimmortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.thoracic aortic aneurysms | ACTA2 mutation | vascular disease | cell migration | human fibroblasts

Endosomal trafficking defects in patient cells with KIAA1109 biallelic variants

“…In addition, we observed qualitative and quantitative differences in the actin-based cytoskeleton with no observable differences in microtubule structure at steady state. Expression of mutant smooth muscle alpha actin ( ACTA2 ) has been shown to reduce the prominence of actin stress fibers in fibroblasts, 28 similar to what we observe in patient B fibroblasts upon staining with phalloidin ( Fig. 5 ).…”

Aortic Cellular Diversity and Quantitative Genome-Wide Association Study Trait Prioritization Through Single-Nuclear RNA Sequencing of the Aneurysmal Human Aorta

“…8,43,44 Histologically, aneurysms are characterized by disarray of VSMCs, including intracellular abnormalities such as loss of and dysfunction of actin stress fibers and focal adhesions (Figure 1A and 1B). 13,15,45 To better characterize the composition and cell-specific transcriptome differences between the aneurysmal and nonaneurysmal ascending aorta, we performed snRNA-seq on human aortic samples with particular focus on the VSMC population (Figure 1C).…”

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“…Expression analysis further suggested phenotypical changes in ANO1 knockdown smooth muscle cells from tail arteries toward a contractile phenotype (Kudryavtseva et al., 2013). We found upregulation of a potent co‐activator of smooth muscle contractile genes, myocardin‐related transcription factor A (Liu et al., 2017; Velasquez et al., 2013; Zhang et al., 2007) in the tail arteries from ANO1 heterozygous mice. This increase might be associated with upregulation of Rho‐related GTP‐binding protein RhoB that is involved in hypoxia‐induced pulmonary artery contraction and remodeling (Wojciak‐Stothard et al., 2012) although its role in the peripheral circulation is not elucidated.…”