Biology subjects

Sips, P.

Publications and source records attributed to Sips, P..

6 recordsLinked to original sources

bioRxiv2026

Integrated luminescence and phenotypic profiling for drug discovery in a zebrafish model of Marfan syndrome

BackgroundMarfan syndrome (MFS) is a life-threatening heritable connective tissue disorder caused by pathogenic variants in fibrillin-1, characterized by progressive cardiovascular disease. Current medical therapies slow disease progression but do not prevent major complications, underscoring the need for new treatment strategies and unbiased discovery approaches. MethodsWe used a zebrafish model of MFS lacking fibrillin-3 (fbn3-/-), which recapitulates key cardiovascular phenotypes including cardiac stress, valvular defects, arrhythmia, and aortic dilation. To enable sensitive, quantitative assessment of cardiac stress, we generated a novel transgenic zebrafish reporter expressing secreted nanoluciferase under control of the stress-responsive nppb promoter. This reporter was combined with morphological phenotyping and bulbus arteriosus (BA) imaging. We evaluated standard MFS therapies, targeted modulators of TGF-{beta} signaling, and performed an unbiased high-throughput drug screen of over 1 500 clinically approved compounds across multiple developmental treatment windows. Resultsfbn3-/- larvae exhibited markedly elevated nppb activity that correlated with phenotypic severity and peaked during stages of highest mortality. The nanoluciferase reporter provided a [~]1 000-fold dynamic range, substantially outperforming Firefly luciferase-based assays. Pharmacological inhibition of TGF-{beta} signaling produced transient or deleterious effects, while {beta}-blockers, losartan, and allopurinol failed to consistently improve cardiac stress, pericardial edema, or BA dilation. The unbiased high-throughput drug screen identified a small number of primary and secondary hits; however, none demonstrated reproducible phenotypic rescue upon rigorous multi-dose, multi-time window validation. ConclusionsThis study establishes a sensitive zebrafish-based platform for early, quantitative assessment of cardiovascular stress in MFS. Our findings highlight the limited efficacy of current therapies, the context-dependent nature of TGF-{beta} modulation, and the biological complexity underlying MFS pathogenesis. Although no definitive therapeutic candidates were identified, this work lays a robust foundation for expanded unbiased discovery efforts aimed at identifying disease-modifying interventions for MFS.

pharmacology and toxicology↗

bioRxiv2025

Evaluating Variants of Uncertain Significance in Adult Knock-in Zebrafish: A Proof of Concept with a COL1A2 Variant

Genomic variants of uncertain significance (VUS) impede clinical decision-making. In this study, we use a knock-in strategy in zebrafish to evaluate the COL1A2 c.2123G>A VUS, identified in a 59-year-old female with recurrent fractures. Using prime editing, we obtained different zebrafish lines respectively harboring the VUS, a known pathogenic variant, or a known benign variant. Comprehensive skeletal phenotyping revealed no significant abnormalities in the zebrafish modeling the benign variant and the VUS, while zebrafish modeling the pathogenic variant showed scoliosis of the vertebral column, vertebral fusions, vertebral compressions, fractures, and increased mineralization of the notochord and intervertebral ligament. Our findings demonstrate for the first time, that COL1A2 variant modeling in zebrafish models informs functional validation and shows potential for elucidating associated pathogenic mechanisms. This approach can be extended to study VUS in other genes.

genetics↗

bioRxiv2024

A novel genetic mouse model of fatal aortic dissection reveals massive inflammatory cell infiltration in the thoracic aorta

BackgroundAortic dissection (AD) is the separation of medial layers of the aorta and is a major cause of death in patients with connective tissue disorders such as Marfan syndrome. However, molecular triggers instigating AD, its temporospatial progression, and how vascular cells in each vessel layer interact and participate in the pathological process remain incompletely understood. To unravel the underlying molecular mechanism of AD, we generated a spontaneous AD mouse model. MethodsWe incorporated a novel missense variant (p.G234D) in FBN1, the gene for fibrillin-1, identified in a non-syndromic familial AD patient into mice using CRISPR/Cas9 system. We performed histopathological analyses of the aortic lesions by histology, immunofluorescence staining, electron microscopy, synchrotron-based imaging and single-cell (sc)RNA-sequencing. Biochemical analysis was performed to examine the binding capacity of mutant human FBN1G234D protein to latent Tgf{beta} binding proteins (LTBPs), and signaling pathways in the mutant aortic wall were examined by western blot analysis. Results50% of the Fbn1G234D/G234D mutant mice died within 5 weeks of age from multiple intimomedial tears that expanded longitudinally and progressed to aortic rupture accompanied by massive immune cell infiltration. scRNA-sequencing, validated by immunostaining, revealed a significant increase in MHC class II-positive pro-inflammatory macrophages and monocytes at the site of intima tears with upregulation of MMP2/9 and marked disruption of elastic lamina. Subendothelial matrices, such as type IV collagen and laminin, expanded into the medial layer, where fibronectin expression was highly upregulated. Fbn1G234D/G234D endothelial cells exhibited altered mechanosensing with loss of parallel alignment to blood flow and upregulation of VCAM-1 and ICAM-1, all of which likely contributed to the infiltration of immune cells. Biochemically, FBN1G234D lost the ability to bind to latent TGF{beta} binding protein (LTBP)-1, -2, and -4, resulting in the downregulation of TGF{beta} signaling in the aortic wall. ConclusionsWe show that dynamic interactions involving endothelial cells (ECs) and macrophages/monocytes in the intima, where the ECM microenvironment is altered with the reduced TGF{beta} signaling, contributes to the initiation of AD. Our novel AD mouse model provides a unique opportunity to identify target molecules involved in the intimomedial tears that can be utilized for development of therapeutic strategies.

pathology↗

bioRxiv2024

Early mechanisms of aortic failure in a zebrafish model for thoracic aortic dissection and rupture

Thoracic aortic aneurysm and dissection (TAAD) associates with a high mortality rate. Despite the existence of different mouse models for TAAD, the underlying disease mechanisms remain elusive. Treatment options are limited and mainly consist of surgical repair at critical aortic diameters as current pharmacological interventions are unable to stop disease progression. In humans, loss of function (LOF) of SMAD3 and SMAD6 impairs vascular homeostasis, increasing the risk for TAAD. We developed a zebrafish model for thoracic aortic dissection/rupture by targeting both ohnologs of smad3 and smad6. At 10 days post fertilization, we found an increased diameter of the ventral aorta in smad3a-/-;smad3b-/- double knockout zebrafish, while smad6a-/-;smad6b-/- double knockout zebrafish have a reduced aortic diameter associated with early mortality. We discovered that a smad3a-/-;smad3b-/-;smad6a-/-;smad6b-/- quadruple knockout (qKO) zebrafish model is viable and survives to adulthood, although exposure to stress leads to sudden death. Histological analysis of the adult ventral aorta shows medial elastolysis, aortic dissections and ruptures at sites exposed to high biomechanical stress. RNA-sequencing of 5 days post fertilization qKO zebrafish indicates a profile of reduced negative regulation of proteolysis and upregulation of melanogenesis, a previously unaddressed pathway in this pathology. We confirm that pharmacological modulation of tyrosinase, the enzyme responsible for the production of melanin, influences aortic morphology. Overall, the qKO mutant, thus far the only known zebrafish model of thoracic aortic dissection and rupture, reveals novel SMAD3/6-dependent pathways that impact thoracic aortic homeostasis, in this way opening avenues for the development of novel treatments in TAAD.

developmental biology↗

bioRxiv2020

Loss of a subunit of vacuolar ATPase identifies unexpected biological signatures of reduced organelle acidification in vivo

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases has been associated with various human diseases including heritable connective tissue disorders, neurodegenerative diseases and cancer. Multiple studies have investigated the pleiotropic effects of reduced acidification in vitro, but the mechanisms elicited by impaired endo(lyso)somal acidification in vivo remain poorly understood. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, hypotonia and epidermal structural defects, reminiscent of the phenotypic manifestations in cutis laxa patients carrying a defect in the ATP6V1E1 gene. Mechanistically, we found that in vivo genetic depletion of atp6v1e1b leads to N-glycosylation defects and reduced maturation of endosomal and lysosomal vesicles, but retains the hypoxia-mediated response. In order to gain further insights into the processes affected by aberrant organelle acidification, we performed an untargeted analysis of the transcriptome and metabolome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid and fatty acid metabolism with profound defects in mitochondrial respiration. Taken together, our results identify new complex biological effects of reduced organelle acidification in vivo, which likely contribute to the multisystemic manifestations observed in disorders caused by v-ATPase deficiency.

molecular biology↗

bioRxiv2018

Zebrafish type I collagen mutants faithfully recapitulate human type I collagenopathies

The type I collagenopathies are a group of heterogeneous connective tissue disorders, that are caused by mutations in the genes encoding type I collagen and include specific forms of Osteogenesis Imperfecta (OI) and the Ehlers-Danlos syndrome (EDS). These disorders present with a broad disease spectrum and large clinical variability of which the underlying genetic basis is still poorly understood. In this study, we systematically analyzed skeletal phenotypes in a large set of zebrafish, with diverse mutations in the genes encoding type I collagen, representing different genetic forms of human OI, and the first zebrafish model of human EDS, which harbors characteristic defects in the soft connective tissues. Furthermore, we provide insight into how zebrafish and human type I collagen are compositionally and functionally related, which is relevant in the interpretation of human type I collagen related disease models. Our studies reveal a high degree of inter-genotype variability in phenotypic expressivity that closely correlates with associated OI severity. Further, we demonstrate the potential for select mutations to give rise to variable phenotypic penetrance, mirroring the clinical variability associated with human disease pathology. Therefore, our work suggests the potential for zebrafish to aid in identifying unknown genetic modifiers and mechanisms underlying the phenotypic variability in OI and related disorders. This will improve diagnostic strategies and enable the discovery of new targetable pathways for pharmacological intervention\n\nSIGNIFICANCE STATEMENTType I collagenopathies are a heterogenous group of connective tissue disorders, caused by genetic defects in type I collagen. Inherent to these disorders is a large clinical variability, of which the underlying molecular basis remains undefined. By systematically analyzing skeletal phenotypes in a large set of type I collagen zebrafish mutants we show that zebrafish models are able to both genocopy and phenocopy different forms of human type I collagenopathies, arguing for a similar pathogenetic basis. This study illustrates the potential of zebrafish as a tool to further dissect the molecular basis of phenotypic variability in human type I collagenopathies to improve diagnostic strategies as well as promote the discovery of new targetable pathways for pharmacological intervention of these disorders.

genetics↗