Insight in the (Phospho)proteome of Vascular Smooth Muscle Cells Derived From Patients With Abdominal Aortic Aneurysm Reveals Novel Disease Mechanisms.

BACKGROUND: Abdominal aortic aneurysm (AAA) is characterized by weakening and dilatation of the aortic wall in the abdomen. The aim of this study was to gain insight into cell-specific mechanisms involved in AAA pathophysiology by analyzing the (phospho)proteome of vascular smooth muscle cells derived from patients with AAA compared with those of healthy donors. METHODS: A (phospho)proteomics analysis based on tandem mass spectrometry was performed on vascular smooth muscle cells derived from patients with AAA (n=24) and healthy, control individuals (C-SMC, n=8). Following protein identification and quantification using MaxQuant, integrative inferred kinase activity analysis was used to calculate kinase activity scores. RESULTS: Expression differences between vascular smooth muscle cells derived from patients with AAA and healthy, control individuals were predominantly found in proteins involved in ECM (extracellular matrix) remodeling (THSD4 [thrombospondin type-1 domain-containing protein 4] and ADAMTS1 [A disintegrin and metalloproteinase with thrombospondin motifs 1]), energy metabolism (GYS1 [glycogen synthase 1] and PCK2 [phosphoenolpyruvate carboxykinase 2, mitochondrial]), and contractility (CACNA2D1 [calcium voltage-dependent channel subunit α-2/δ-1] and TPM1 [tropomyosin α-1 chain]). Phosphorylation patterns on proteins related to actin cytoskeleton organization dominated the phosphoproteome of vascular smooth muscle cells derived from patients with AAA . Besides, phosphorylation changes on proteins related to energy metabolism (GYS1), contractility (PARVA [α-parvin], PPP1R12A [protein phosphatase 1 regulatory subunit 12A], and CALD1 [caldesmon 1]), and intracellular communication (GJA1 [gap junction α-1 protein]) were seen. Kinase activity of NUAK1 (NUAK family SNF1-like kinase 1), FYN (tyrosine-protein kinase Fyn), MAPK7 (mitogen-activated protein kinase 7), and STK10 (serine/threonine kinase 10) was different in vascular smooth muscle cells derived from patients with AAA compared with those from healthy, control individuals. CONCLUSIONS: This study revealed changes in expression and phosphorylation levels of proteins involved in various processes responsible for AAA progression and development (eg, energy metabolism, ECM remodeling, actin cytoskeleton organization, contractility, intracellular communication, and cell adhesion). These newly identified proteins, phosphosites, and related kinases provide further insight into the underlying mechanism of vascular smooth muscle cell dysfunction within the aneurysmal wall. Our omics data thereby offer the opportunity to study the relevance, either as drug target or biomarker, of these proteins in AAA development.

Metformin Improves the Function of Abdominal Aortic Aneurysm Patient Derived Aortic Smooth Muscle Cells.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor. Paradoxically, a decreased risk of abdominal aortic aneurysm (AAA) presence and growth rate is described among patients with T2DM, associated with metformin use. This study aimed to investigate the effect of metformin on AAA patient derived aortic smooth muscle cell (SMC) function. METHODS: Aortic biopsies were obtained from patients with AAA (n = 21) and controls (n = 17) during surgery. The SMCs of non-pathological aortic controls, non-diabetic patients with AAA, and diabetic patients with AAA were cultured from explants and treated with or without metformin. The SMC contractility was measured upon ionomycin stimulation, as well as metabolic activity, proliferation, and migration. Then, mRNA and protein expression of markers for contraction, metabolic activity, proliferation, and inflammation were measured. RESULTS: The mRNA expression of KLF4 and GYS1, genes involved in metabolic activity, differed between the SMCs from non-diabetic and diabetic patients with AAA before metformin stimulation (p < .041). However, the effect of metformin on the various SMC functions was similar between non-diabetic and diabetic patients with AAA. Upon stimulation, metformin increased the contractility of AAA patient SMCs (p = .001). The mRNA expression of smoothelin, a marker for the contractile phenotype, increased in the SMCs of patients with AAA after treatment with metformin (p = .006). An increase in metabolic activity (p < .001) and a decrease in proliferation (p < .001) and migration were found in the SMCs of controls and patients with AAA with metformin. Increased mRNA expression of PPARγ, a nuclear receptor involved in mitochondrial biogenesis (p < .009), and a decrease in gene expression of Ki-67, a marker for proliferation (p < .005), were observed. Gene expression of inflammation markers MCP-1 and IL-6, and protein expression of NF-κB p65 decreased after treatment with metformin in patients with AAA. CONCLUSION: This study found that metformin increases contractility and metabolic activity, and reduces proliferation, migration, and inflammation in aortic SMCs in vitro.

Molecular phenotyping and functional assessment of smooth muscle-like cells with pathogenic variants in aneurysm genes ACTA2, MYH11, SMAD3 and FBN1.

Aortic aneurysms (AAs) are pathological dilatations of the aorta. Pathogenic variants in genes encoding for proteins of the contractile machinery of vascular smooth muscle cells (VSMCs), genes encoding proteins of the transforming growth factor beta signaling pathway and extracellular matrix (ECM) homeostasis play a role in the weakening of the aortic wall. These variants affect the functioning of VSMC, the predominant cell type in the aorta. Many variants have unknown clinical significance, with unknown consequences on VSMC function and AA development. Our goal was to develop functional assays that show the effects of pathogenic variants in aneurysm-related genes. We used a previously developed fibroblast transdifferentiation protocol to induce VSMC-like cells, which are used for all assays. We compared transdifferentiated VSMC-like cells of patients with a pathogenic variant in genes encoding for components of VSMC contraction (ACTA2, MYH11), transforming growth factor beta (TGFß) signaling (SMAD3) and a dominant negative (DN) and two haploinsufficient variants in the ECM elastic laminae (FBN1) to those of healthy controls. The transdifferentiation efficiency, structural integrity of the cytoskeleton, TGFß signaling profile, migration velocity and maximum contraction were measured. Transdifferentiation efficiency was strongly reduced in SMAD3 and FBN1 DN patients. ACTA2 and FBN1 DN cells showed a decrease in SMAD2 phosphorylation. Migration velocity was impaired for ACTA2 and MYH11 cells. ACTA2 cells showed reduced contractility. In conclusion, these assays for showing effects of pathogenic variants may be promising tools to help reclassification of variants of unknown clinical significance in AA-related genes.

The role of vascular smooth muscle cells in the development of aortic aneurysms and dissections.

BACKGROUND: Aortic aneurysms (AA) are pathological dilations of the aorta, associated with an overall mortality rate up to 90% in case of rupture. In addition to dilation, the aortic layers can separate by a tear within the layers, defined as aortic dissections (AD). Vascular smooth muscle cells (vSMC) are the predominant cell type within the aortic wall and dysregulation of vSMC functions contributes to AA and AD development and progression. However, since the exact underlying mechanism is poorly understood, finding potential therapeutic targets for AA and AD is challenging and surgery remains the only treatment option. METHODS: In this review, we summarize current knowledge about vSMC functions within the aortic wall and give an overview of how vSMC functions are altered in AA and AD pathogenesis, organized per anatomical location (abdominal or thoracic aorta). RESULTS: Important functions of vSMC in healthy or diseased conditions are apoptosis, phenotypic switch, extracellular matrix regeneration and degradation, proliferation and contractility. Stressors within the aortic wall, including inflammatory cell infiltration and (epi)genetic changes, modulate vSMC functions and cause disturbance of processes within vSMC, such as changes in TGF-ß signalling and regulatory RNA expression. CONCLUSION: This review underscores a central role of vSMC dysfunction in abdominal and thoracic AA and AD development and progression. Further research focused on vSMC dysfunction in the aortic wall is necessary to find potential targets for noninvasive AA and AD treatment options.

Patient-Specific 3-Dimensional Model of Smooth Muscle Cell and Extracellular Matrix Dysfunction for the Study of Aortic Aneurysms.

INTRODUCTION: Abdominal aortic aneurysms (AAAs) are associated with overall high mortality in case of rupture. Since the pathophysiology is unclear, no adequate pharmacological therapy exists. Smooth muscle cells (SMCs) dysfunction and extracellular matrix (ECM) degradation have been proposed as underlying causes. We investigated SMC spatial organization and SMC-ECM interactions in our novel 3-dimensional (3D) vascular model. We validated our model for future use by comparing it to existing 2-dimensional (2D) cell culture. Our model can be used for translational studies of SMC and their role in AAA pathophysiology. MATERIALS AND METHODS: SMC isolated from the medial layer of were the aortic wall of controls and AAA patients seeded on electrospun poly-lactide-co-glycolide scaffolds and cultured for 5 weeks, after which endothelial cells (EC) are added. Cell morphology, orientation, mechanical properties and ECM production were quantified for validation and comparison between controls and patients. RESULTS: We show that cultured SMC proliferate into multiple layers after 5 weeks in culture and produce ECM proteins, mimicking their behavior in the medial aortic layer. EC attach to multilayered SMC, mimicking layer interactions. The novel SMC model exhibits viscoelastic properties comparable to biological vessels; cytoskeletal organization increases during the 5 weeks in culture; increased cytoskeletal alignment and decreased ECM production indicate different organization of AAA patients' cells compared with control. CONCLUSION: We present a valuable preclinical model of AAA constructed with patient specific cells with applications in both translational research and therapeutic developments. We observed SMC spatial reorganization in a time course of 5 weeks in our robust, patient-specific model of SMC-EC organization and ECM production.

Inflammatory Gene Expression of Human Perivascular Adipose Tissue in Abdominal Aortic Aneurysms.

OBJECTIVE: Perivascular adipose tissue (PVAT) contributes to vascular homeostasis and is increasingly linked to vascular pathology. PVAT density and volume were associated with abdominal aortic aneurysm (AAA) presence and dimensions on imaging. However, mechanisms underlying the role of PVAT in AAA have not been clarified. This study aimed to explore differences in PVAT from AAA using gene expression and functional tests. METHODS: Human aortic PVAT and control subcutaneous adipose tissue were collected during open AAA surgery. Gene analyses and functional tests were performed. The control group consisted of healthy aorta from non-living renal transplant donors. Gene expression tests were performed to study genes potentially involved in various inflammatory processes and AAA related genes. Live PVAT and subcutaneous adipose tissue (SAT) from AAA were used for ex vivo co-culture with smooth muscle cells (SMCs) retrieved from non-pathological aortas. RESULTS: Adipose tissue was harvested from 27 AAA patients (n [gene expression] = 22, n [functional tests] = 5) and five control patients. An increased inflammatory gene expression of PTPRC (p = .008), CXCL8 (p = .033), LCK (p = .003), CCL5 (p = .004) and an increase in extracellular matrix breakdown marker MMP9 (p = .016) were found in AAA compared with controls. Also, there was a decreased anti-inflammatory gene expression of PPARG in AAA compared with controls (p = .040). SMC co-cultures from non-pathological aortas with PVAT from AAA showed increased MMP9 (p = .033) and SMTN (p = .008) expression and SAT increased SMTN expression in these SMC. CONCLUSION: The data revealed that PVAT from AAA shows an increased pro-inflammatory and matrix metallopeptidase gene expression and decreased anti-inflammatory gene expression. Furthermore, increased expression of genes involved in aneurysm formation was found in healthy SMC co-culture with PVAT of AAA patients. Therefore, PVAT from AAA might contribute to inflammation of the adjacent aortic wall and thereby plays a possible role in AAA pathophysiology. These proposed pathways of inflammatory induction could reveal new therapeutic targets in AAA treatment.

Mutations in PIH1D3 Cause X-Linked Primary Ciliary Dyskinesia with Outer and Inner Dynein Arm Defects.

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right body axis disturbance. Here we report maternally inherited and de novo mutations in PIH1D3 in four men affected with PCD. PIH1D3 is located on the X chromosome and is involved in the preassembly of both outer (ODA) and inner (IDA) dynein arms of cilia and sperm flagella. Loss-of-function mutations in PIH1D3 lead to absent ODAs and reduced to absent IDAs, causing ciliary and flagellar immotility. Further, PIH1D3 interacts and co-precipitates with cytoplasmic ODA/IDA assembly factors DNAAF2 and DNAAF4. This result has clinical and genetic counseling implications for genetically unsolved male case subjects with a classic PCD phenotype that lack additional phenotypes such as intellectual disability or retinitis pigmentosa.

Transdifferentiation of Human Dermal Fibroblasts to Smooth Muscle-Like Cells to Study the Effect of MYH11 and ACTA2 Mutations in Aortic Aneurysms.

Mutations in genes encoding proteins of the smooth muscle cell (SMC) contractile apparatus contribute to familial aortic aneurysms. To investigate the pathogenicity of these mutations, SMC are required. We demonstrate a novel method to generate SMC-like cells from human dermal fibroblasts by transdifferentiation to study the effect of variants in genes encoding proteins of the SMC contractile apparatus (ACTA2 and MYH11) in patients with aortic aneurysms. Dermal fibroblasts from seven healthy donors and cells from seven patients with MYH11 or ACTA2 variants were transdifferentiated into SMC-like cells within a 2-week duration using 5 ng/ml TGFß1 on a scaffold containing collagen and elastin. The induced SMC were comparable to primary human aortic SMC in mRNA expression of SMC markers which was confirmed on the protein level by immunofluorescence quantification analysis and Western blotting. In patients with MYH11 or ACTA2 variants, the effect of intronic variants on splicing was demonstrated on the mRNA level in the induced SMC, allowing classification into pathogenic or nonpathogenic variants. In conclusion, direct conversion of human dermal fibroblasts into SMC-like cells is a highly efficient method to investigate the pathogenicity of variants in proteins of the SMC contractile apparatus.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Osteogenic transdifferentiation of primary human fibroblasts to osteoblast-like cells with human platelet lysate.

Inherited bone disorders account for about 10% of documented Mendelian disorders and are associated with high financial burden. Their study requires osteoblasts which play a critical role in regulating the development and maintenance of bone tissue. However, bone tissue is not always available from patients. We developed a highly efficient platelet lysate-based approach to directly transdifferentiate skin-derived human fibroblasts to osteoblast-like cells. We extensively characterized our in vitro model by examining the expression of osteoblast-specific markers during the transdifferentiation process both at the mRNA and protein level. The transdifferentiated osteoblast-like cells showed significantly increased expression of a panel of osteogenic markers. Mineral deposition and ALP activity were also shown, confirming their osteogenic properties. RNA-seq analysis allowed the global study of changes in the transcriptome of the transdifferentiated cells. The transdifferentiated cells clustered separately from the primary fibroblasts with regard to the significantly upregulated genes indicating a distinct transcriptome profile; transdifferentiated osteoblasts also showed significant enrichment in gene expression related to skeletal development and bone mineralization. Our presented in vitro model may potentially contribute to the prospect of studying osteoblast-dependent disorders in patient-derived cells.