Biology subjects

van der Pluijm, I.

Publications and source records attributed to van der Pluijm, I..

3 recordsLinked to original sources

bioRxiv2025

Generation and characterization of Induced Pluripotent Stem Cell models with SMAD3 mutations to study the molecular mechanism of Loeys-Dietz Syndrome Type III aneurysm formation

Loeys-Dietz Syndrome type 3 (LDS3) is caused by pathogenic (P)/likely pathogenic (LP) variants in the SMAD3 gene and is characterized by aneurysm formation and arterial tortuosity, which can lead to life-threatening complications. There is an unmet need for suitable cell models to study LDS3 at a cellular and molecular level. Induced pluripotent stem (iPS) cells offer a promising approach because they can be genetically modified using CRISPR/Cas9 technology and differentiated into disease-relevant cell types. As it is difficult to obtain aortic vascular smooth muscle cells (VSMCs) from patients, iPS cells differentiated into VSMCs provide an ideal model to study cellular aneurysmal phenotypes. In this study, we generated iPS cell models carrying (P/LP) SMAD3 variants. These cell models were generated either by using CRISPR/Cas9 mediated introduction of indels and deletions to introduce SMAD3 variants, or by reprogramming of fibroblasts derived from SMAD3 patients. These iPS cell lines were characterized for SMAD3 expression by Western blotting and validated for pluripotency through immunofluorescence and qPCR. Moreover, the patient-derived iPS cell lines were shown to differentiate into smooth muscle cells (SMCs), which are relevant to study the molecular mechanisms underlying aneurysm formation in LDS3 patients. Our findings highlight the potential of these iPS-based models to investigate the pathophysiology of LDS3 and facilitate the development of therapeutic strategies for aortic aneurysms.

molecular biology↗

bioRxiv2023

Functional analysis of cell lines derived from SMAD3-related Loeys-Dietz Syndrome patients provides insights into genotype-phenotype relations

IntroductionPathogenic (P) and likely pathogenic (LP) variants in the SMAD3 gene cause Loeys-Dietz syndrome type 3 (LDS3), also known as aneurysms-osteoarthritis syndrome (AOS). The phenotype of LDS3 is highly variable and characterized by arterial aneurysms, dissections and tortuosity throughout the vascular system combined with skeletal, cutaneous and facial features. ObjectivesInvestigate the impact of P/LP SMAD3 variants through conducting functional tests on patient-derived fibroblasts and vascular smooth muscle cells (VSMCs).The resulting knowledge will optimize interpretation of SMAD3 variants. Material and methodsWe conducted a retrospective analysis on clinical data from individuals with a P/LP SMAD3 variant and utilized patient-derived VSMCs to investigate the functional impacts of dominant negative (DN) and haploinsufficient (HI) variants in SMAD3. Additionally, to broaden our cell model accessibility, we performed similar functional analyses on patient-derived fibroblasts carrying SMAD3 variants, differentiating them into myofibroblasts with the same variants. This enabled us to study the functional effects of DN and HI variants in SMAD3 across both patient-derived myofibroblasts and VSMCs. ResultsIndividuals with dominant negative (DN) variants in the MH2 protein interaction domain of SMAD3 exhibited a higher frequency of major events (66.7% vs. 44.0%, p=0.054), occurring at a younger age compared to those with haploinsufficient (HI) variants. Moreover, the age at the onset of the first major event was notably younger in individuals with DN variants in MH2, 35.0 years [IQR 29.0-47.0], compared to 46.0 years [IQR 40.0-54.0] in those with HI variants (p=0.065). In functional assays, fibroblasts carrying DN SMAD3 variants displayed reduced differentiation potential, contrasting with increased differentiation potential observed in fibroblasts with HI SMAD3 variants. Additionally, HI SMAD3 variant VSMCs showed elevated SMA expression, while exhibiting altered expression of alternative MYH11 isoforms. Conversely, DN SMAD3 variant myofibroblasts demonstrated reduced extracellular matrix (ECM) formation compared to control cell lines. These findings collectively indicate distinct functional consequences between DN and HI variants in SMAD3 across fibroblasts and VSMCs, potentially contributing to the observed differences in disease manifestation and age of onset of major events. ConclusionDistinguishing between P/LP HI and DN SMAD3 variants can be achieved by assessing differentiation potential, and evaluating SMA and MYH11 expression. Notably, myofibroblast differentiation seems to be a suitable alternative in vitro test system in comparison to VSMCs. Moreover, there is a notable trend of aortic events occurring at younger age in individuals with a DN SMAD3 variant in the MH2 domain, distinguishing them from those with a DN variant in the MH1 domain or a HI variant.

genetics↗

bioRxiv2019

SLC2A knockout mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicate GLUT10 in transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations do not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in rodents. Gulo;Slc2a10 knock-out mice show mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 knock-out mice do not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. TGF{beta} signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

genetics↗