Olga Moiseeva

Notch-dependent EMT is attenuated in patients with aortic aneurysm and bicuspid aortic valve.

Bicuspid aortic valve is the most common congenital heart malformation and the reasons for the aortopathies associated with bicuspid aortic valve remain unclear. NOTCH1 mutations are associated with bicuspid aortic valve and have been found in individuals with various left ventricular outflow tract abnormalities. Notch is a key signaling during cardiac valve formation that promotes the endothelial-to-mesenchymal transition. We address the role of Notch signaling in human aortic endothelial cells from patients with bicuspid aortic valve and aortic aneurysm. Aortic endothelial cells were isolated from tissue fragments of bicuspid aortic valve-associated thoracic aortic aneurysm patients and from healthy donors. Endothelial-to-mesenchymal transition was induced by activation of Notch signaling. Effectiveness of the transition was estimated by loss of endothelial and gain of mesenchymal markers by immunocytochemistry and qPCR. We show that aortic endothelial cells from the patients with aortic aneurysm and bicuspid aortic valve have down regulated Notch signaling and fail to activate Notch-dependent endothelial-to-mesenchymal transition in response to its stimulation by different Notch ligands. Our findings support the idea that bicuspid aortic valve and associated aortic aneurysm is associated with dysregulation of the entire Notch signaling pathway independently on the specific gene mutation.

Variants in the NOTCH1 Gene in Patients with Aortic Coarctation

BACKGROUND AND OBJECTIVE Malformations of the left ventricular outflow tract are one of the most common forms of congenital heart disorders. Recently, it has been shown that mutations in the NOTCH1 gene can lead to bicuspid aortic valve, aortic aneurysm, and hypoplastic left heart syndrome. The aim of our study was to estimate the frequency of NOTCH1 gene mutations/substitutions in patients with aortic coarctation, isolated or combined with bicuspid aortic valve. DESIGN AND PATIENTS The study included 51 children with coarctation. Detailed family history was obtained for every study subject, and echocardiographic data were obtained for the relatives when available. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Control DNA was obtained from 200 healthy donors. RESULTS In more than half of the cases, coarctation was combined with bicuspid aortic valve, and in approximately half of the cases, it was combined with hypoplasia of the aortic arch or descending aorta. Familial history of congenital heart disease was observed in 34.3% of the cases. In total, 29 variants of the NOTCH1 gene were identified in the patient group and in the control subjects. Four of those variants led to amino acid exchange, of which only one, R1279H, was identified in both the patient group and in the controls. This variant was significantly overrepresented in the patients with aortic coarctation compared with those in the control group (P < .05). We conclude that the R1279H substitution in the NOTCH1 gene is significantly overrepresented in patients with aortic coarctation and, therefore, may represent a disease-susceptibility allele.

Phenotypic and Functional Changes of Endothelial and Smooth Muscle Cells in Thoracic Aortic Aneurysms

Thoracic aortic aneurysm develops as a result of complex series of events that alter the cellular structure and the composition of the extracellular matrix of the aortic wall. The purpose of the present work was to study the cellular functions of endothelial and smooth muscle cells from the patients with aneurysms of the thoracic aorta. We studied endothelial and smooth muscle cells from aneurysms in patients with bicuspid aortic valve and with tricuspid aortic valve. The expression of key markers of endothelial (CD31, vWF, and VE-cadherin) and smooth muscle (SMA, SM22α, calponin, and vimentin) cells as well extracellular matrix and MMP activity was studied as well as and apoptosis and cell proliferation. Expression of functional markers of endothelial and smooth muscle cells was reduced in patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients. We show for the first time that aortic endothelial cell phenotype is changed in the thoracic aortic aneurysms compared to normal aortic wall. In conclusion both endothelial and smooth muscle cells from aneurysms of the ascending aorta have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis.

NOTCH1 Mutations in Aortic Stenosis: Association with Osteoprotegerin/RANK/RANKL

Background. The NOTCH pathway is known to be important in the pathogenesis of calcific aortic valve disease, possibly through regulators of osteoprotegerin (OPG), receptor activator of nuclear factor κB (RANK), and its ligand (RANKL) system. The purpose of the present study was to search for possible associations between NOTCH1 gene mutations and circulating levels of OPG and soluble RANKL (sRANKL) in patients with aortic stenosis (AS). Methods. The study was performed on 61 patients with AS including 31 with bicuspid and 30 with tricuspid aortic valves. We applied a strategy of targeted mutation screening for 10 out of 34 exons of the NOTCH1 gene by direct sequencing. Serum OPG and sRANKL levels were assessed. Results. In total, 6 genetic variants of the NOTCH1 gene including two new mutations were identified in the study group. In an age- and arterial hypertension-adjusted multivariable regression analysis, the serum OPG levels and the OPG/sRANKL ratio were correlated with NOTCH1 missense variants. All studied missense variants in NOTCH1 gene were found in Ca(2+)-binding EGF motif of the NOTCH extracellular domain bound to Delta-like 4. Conclusion. Our results suggest that the OPG/RANKL/RANK system might be directly influenced by genetic variants of NOTCH1 in aortic valve calcification.

Mechanisms of Smooth Muscle Cell Differentiation Are Distinctly Altered in Thoracic Aortic Aneurysms Associated with Bicuspid or Tricuspid Aortic Valves

Cellular and molecular mechanisms of thoracic aortic aneurysm are not clear and therapeutic approaches are mostly absent. Thoracic aortic aneurysm is associated with defective differentiation of smooth muscle cells (SMC) of aortic wall. Bicuspid aortic valve (BAV) comparing to tricuspid aortic valve (TAV) significantly predisposes to a risk of thoracic aortic aneurysms. It has been suggested recently that BAV-associated aortopathies represent a separate pathology comparing to TAV-associated dilations. The only proven candidate gene that has been associated with BAV remains NOTCH1. In this study we tested the hypothesis that Notch-dependent and related TGF-β and BMP differentiation pathways are differently altered in aortic SMC of BAV- vs. TAV-associated aortic aneurysms. SMC were isolated from aortic tissues of the patients with BAV- or TAV-associated aortic aneurysms and from healthy donors used as controls. Gene expression was verified by qPCR and Western blotting. For TGF-β induced differentiation SMC were treated with the medium containing TGF-β1. To induce proosteogenic signaling we cultured SMC in the presence of specific osteogenic factors. Notch-dependent differentiation was induced via lentiviral transduction of SMC with activated Notch1 domain. MYOCD expression, a master gene of SMC differentiation, was down regulated in SMC of both BAV and TAV patients. Discriminant analysis of gene expression patterns included a set of contractile genes specific for SMC, Notch-related genes and proosteogenic genes and revealed that control cells form a separate cluster from both BAV and TAV group, while BAV- and TAV-derived SMC are partially distinct with some overlapping. In differentiation experiments TGF-β caused similar patterns of target gene expression for BAV- and TAV derived cells while the induction was higher in the diseased cells than in control ones. Osteogenic induction caused significant change in RUNX2 expression exclusively in BAV group. Notch activation induced significant ACTA2 expression also exclusively in BAV group. We show that Notch acts synergistically with proosteogenic factors to induce ACTA2 transcription and osteogenic differentiation. In conclusion we have found differences in responsiveness of SMC to Notch and to proosteogenic induction between BAV- and TAV-associated aortic aneurysms.

Functional Properties of Smooth Muscle Cells in Ascending Aortic Aneurysm

Thoracic aortic aneurysm (TAA) develops as a result of complex sequential events that dynamically alter the structure and composition of the aortic vascular extracellular matrix (ECM). The main cellular elements that alter the composition of aortic wall are smooth muscle cells (SMCs). The purpose of the present work was to study alterations of smooth muscle cell functions derived from the patients with TAA and from healthy donors. Since it is believed that TAA associates with bicuspid aortic valve (BAV) and with tricuspid aortic valve (TAV) differed in their pathogenesis, we have compared SMCs and tissue samples from BAV and TAV patients and healthy donors. The comparison was done by several parameters: SMC growth, migration and apoptotic dynamics, metalloproteinase MMP2 and MMP9 activity (zymography), and elastin, collagen, and fibrillin content (Western blot) in both tissue samples and cultured SMCs. Proliferation of BAV and TAV SMCs was decreased and migration ability in scratch tests was increased in TAV-derived SMCs compared to donor cells. BAV-cells migration ability was not changed compared to donor SMCs. Elastin content was decreased in TAA SMCs, whereas the content of fibrillin and collagen was not altered. At the same time, the elastin and collagen protein level was significantly higher in tissue samples of TAA patients than in donorderived samples. SMC proliferation and migration is differently affected in TAV and BAV-associated TAA that supports the idea on different nature of these two TAA groups. Our data also show that SMC functional properties are altered in TAA patients and these alterations could play a significant role in the disease pathogenesis.

Asymmetric Dimethylarginine in Patients with Ascending Aortic Aneurysms

BACKGROUND Ascending thoracic aortic aneurysm (aTAA) is a heterogeneous group of disorders that involve impaired endothelial function. The nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) serves as an endothelial dysfunction marker. Thus, we investigated ADMA levels in patients with aTAA. METHODS Eighty-six patients with aTAA and 18 healthy individuals were enrolled. All patients underwent echocardiography. Plasma ADMA levels were measured using high-performance liquid chromatography. RESULTS ADMA levels were higher in aTAA patients than in control patients (p = 0.034). According to the multivariable regression model, higher ADMA levels were associated with ascending aortic diameter (p = 0.017), smoking (p = 0.016), and log-transformed estimated glomerular filtration rate (eGFR, p = 0.005). CONCLUSION This pilot study demonstrates an association of ADMA with ascending aortic dilatation; however, further studies are needed to investigate whether increased ADMA levels underlie aTAA development.

Notch, BMP and WNT/β-catenin network is impaired in endothelial cells of the patients with thoracic aortic aneurysm

Cellular and molecular mechanisms of thoracic aortic aneurysm are still not clear and therapeutic approaches are mostly absent. The role of endothelial cells in aortic wall integrity is emerging from recent studies. Although Notch pathway ensures endothelial development and integrity, and NOTCH1 mutations have been associated with thoracic aortic aneurysms, the role of this pathway in aneurysm remains elusive. The purpose of the present work was to study functions of Notch genes in endothelial cells of patients with sporadic thoracic aortic aneurysm. Aortic endothelial cells were isolated from aortic tissue of patients with thoracic aortic aneurysm and healthy donors. Gene expression of Notch and related BMP and WNT/β-catenin pathways was estimated by qPCR; WNT/β-catenin signaling was studied by TCF-luciferase reporter. To study the stress-response the cells were subjected to laminar shear stress and the expression of corresponding genes was estimated by qPCR. Analyses of mRNA expression of Notch genes, Notch target genes and Notch related pathways showed that endothelial cells of aneurysm patients have dysregulated Notch/BMP/WNT pathways compared to donor cells. Activity of Wnt pathway was significantly elevated in endothelial cells of the patients. Cells from patients had attenuated activation of DLL4, SNAIL1, DKK1 and BMP2 in response to shear stress. In conclusion endothelial cells of the patients with thoracic aortic aneurysm have dysregulated Notch, BMP and WNT/β-catenin related signaling. Shear stress-response and cross-talk between Notch and Wnt pathways that normally ensures aortic integrity and resistance of endothelial cells to stress is impaired in aneurysmal patients.

Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves

AIMS Calcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups. METHODS AND RESULTS We used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. CONCLUSION Early signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.