Biology subjects

Yanagisawa, H.

Publications and source records attributed to Yanagisawa, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Dermal bone grows by cellular invasion and intercalary biomineralization

Dermal bone architecture bears great evolutionary significance for jawed vertebrates. Its thickness growth is believed to rely on matrix apposition by superficial osteoblasts. To test this hypothesis we employ genetic lineage mapping, conditional gene ablation, intravital matrix labelling and molecular 3D analysis at single cell resolution in vivo. We uncover an invasive mechanism incompatible with apposition. The pervasive arrangement of two layers sandwiching a third spongy layer develops from a molecularly defined bi-layer via a previously unknown developmental module: Outer layer osteoblasts form rosette-like assemblies around single invading cells. The latter bear unique molecular signatures and form de novo sheets inside the spongy layer, communicating with the layer below. The transcription factor Hand2 organizes this module by orchestrating rosette formation, their molecular heterogeneity, cellular invasion and spongy layer thickness growth. Surprisingly, invading osteoblasts secrete new biomineral matrix inside the older matrix, which keeps expanding. Such intercalary biomineralization provides new perspectives for bone biology and the evolution of endochondral ossification.

developmental biology↗

bioRxiv2026

Dermal bone remodelling by invasive osteoblasts evolved in stem gnathostomes

The evolutionary origin of the human face can be traced to the earliest vertebrates with a dermal skeleton in which canonical skeletal Issues and cell types arose. To elucidate its developmental evolution we performed genetic mosaic labelling, 3D single cell analysis and fluorescent matrix birthdating in vivo. We discover a common lineage origin of endothelial cells and osteoblasts and de novo vasculogenesis (not angiogenesis) as the dominant mechanism. Inside the spongy (cancellous) layer, invasive OPN+/RUNX2+ osteoblasts establish two orthogonal collagen sca[ff]olds, remodel them by directional secretion of matrix metalloproteinases and employ controllers of hydroxyapatite crystal resorption previously considered to be osteoclast- specific. Such intercalary biomineralization, comprising cellular sheet and volumetric biomineral expansion, renders all layers malleable even during postnatal stages. We trace this mechanism to bone ontogenies of the earliest skeletonizing vertebrates and resolve cell/Issue homologies controversial for almost two centuries. Our new model also has implications for bone matrix bioengineering and cancer osteomimicry that might replay some of these ontogenetic processes.

evolutionary biology↗

bioRxiv2026

Depth-resolved analysis reveals target-dependent cryo-FIB damage profiles

Cryogenic focused ion beam (cryo-FIB) milling enables cellular cryo-electron tomography but can compromise high-resolution structural information near milled surfaces. Here we establish recombinant human apoferritin (apoF) as a reporter of depth-dependent structural preservation in cellular lamellae. ApoF yielded a 2.05 [A] in situ reconstruction. Apparent FIB damage was most pronounced within the first 15 nm for xenon, 30 nm for gallium, 45 nm for argon and remained detectable at least 75 nm from oxygen-milled surfaces under the workflows tested. Endogenous 70S ribosomes in the same xenon-milled tomograms showed a broader 30-45 nm profile, whereas {beta}-galactosidase showed a similar narrow xenon-associated profile in a different specimen. Xenon-milled lamellae 125-150 nm thick yielded the highest resolution among the thickness groups examined, with lower resolution in the thinner and thickest groups. These findings show structural preservation is constrained by both surface-associated damage and lamella thickness, with the apparent extent of damage varying by molecular reporter. Previous ribosome-based surface-exclusion estimates may therefore be overly conservative for some molecular targets.

molecular biology↗

bioRxiv2026

Immunomodulatory mechanisms of submicron phosphatidylserine-exposing polymeric particles (PSPs)

Macrophages are highly dynamic cells that maintain tissue homeostasis by regulating both initiation and resolution of inflammation. During efferocytosis, macrophages recognize the eat me signal, phosphatidylserine (PS), exposed at the surface of apoptotic cells, leading to the resolution of inflammation and acquisition of a pro-resolving phenotype. Inspired by this endogenous mechanism, PS-based biomaterials have demonstrated immunomodulatory potential. However, the molecular mechanisms underlying PS-mediated macrophage reprogramming remain poorly understood. Here, submicron PS-exposing polymeric particles (PSPs; [~]300 nm) were developed to improve the suitability of PSP formulations for future systemic administration while preserving their immunomodulatory activity. PSPs were efficiently internalized by macrophages through both actin- and dynamin-dependent pathways. PSP treatment significantly reduced IL-6 and IL-12p70 production in LPS-stimulated macrophages, whereas induction of the classical anti-inflammatory M2 marker CD206 was limited. Transcriptomic analysis revealed coordinated attenuation of inflammatory signaling pathways, including downregulation of Myd88, Nfkb1, Rel, and Irf8, together with activation of NRF2-associated antioxidant pathways characterized by increased expression of Nfe2l2, Hmox1, Prdx1, Gclm, and Gclc. Activation of antioxidant-associated genes together with reduced Irf8 expression suggests that PSP promotes inflammatory resolution through coordinated redox adaptation and selective attenuation of inflammatory signaling. Collectively, these findings provide mechanistic insight into PS-mediated macrophage reprogramming and support the future development of systemically administered therapies for chronic inflammatory diseases, including vascular inflammatory disorders. HighlightsO_LISubmicron PSPs retain immunomodulatory activity of apoptotic cell-mimicking biomaterials. C_LIO_LIPSPs are rapidly internalized through actin- and dynamin-dependent pathways. C_LIO_LIPSPs attenuate inflammatory signaling and selectively suppress IL-6 and IL-12p70 production. C_LIO_LIPSPs induce NRF2-associated antioxidant and glutathione responses. C_LIO_LITranscriptomics reveals an early redox-adaptive macrophage program. C_LI

bioengineering↗

bioRxiv2026

Osteopontin Upregulation Defines a Pre-Rupture State in Thoracic Aortic Aneurysms in Mice and Humans

BackgroundThoracic aortic aneurysm (TAA) is a life-threatening condition with an unpredictable lisk of rupture. Current clinical parameters have limited ability to accurately predict imminent rupture. Osteopontin (OPN) has been implicated in aortic aneurysm pathology, however, it role as a marker of imminent rupture remains. unclear. We investigated the dynamics of OPN expression dynamics in a mouse model with predictable rupture timing and validated our findings in human TAA. MethodsOne-month-old fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice were used as a TAA model; with wild-type (WT) mice served as controls. Angiotensin II (AngII) was administered to Fbn1mgR/mgR to induce acute aortic rupture. Single-section transcriptome analysis and immunofluorescence staining were performed on ascending aortic tissue at 24 and 72 hours after AngII infusion, with pre-treatment Fbn1mgR/mgR and WT mice serving as controls. To determine conservation in human disease, we reanalyzed publicly available single-cell RNA sequencing data from ascending thoracic aortic aneurysm (ATAA) patients. ResultsAngII infusion induced progressive mortality beginning at 24 hours, with approximately 60% survival at 72 hours and nearly no survival by 8 days in Fbn1mgR/mgR mice. At this pre-rupture time point, OPN showed prominent upregulation at both mRNA and protein levels in ascending aortic tissues compared to controls. Immunofluorescence staining revealed increased OPN expression in the aortic wall, particularly in regions exhibiting structural deterioration. Reanalysis of human ATAA single-cell data showed elevated OPN expression compared to controls, with enrichment in immune cell populations, especially macrophages. Within the macrophage compartment, subcluster analysis identified a stress-responsive subpopulation (MC1) that was markedly expanded and almost exclusively composed of ATAA-derived cells, representing the primary source of OPN upregulation. ConclusionsOPN upregulation represents a conserved molecular signature of the pre-rupture state in TAA across mice and humans. Our mode, which enables predictable rupture timing, allowed the capture of acute pre-rupture molecular changes, suggesting OPN as a potential biomarker for predicting imminent aortic rupture.

molecular biology↗

bioRxiv2026

The epidermal stem cell-supporting matricellular protein fibulin 7 modulates skin inflammatory response in a psoriasis model

ECM composition and organization are greatly altered during inflammation but it is still elusive if ECM dynamics may protect tissue stem cells against aberrant inflammation. Fibulin 7 (encoded by Fbln7) is part of the basement membrane ECM where epidermal stem cells (EpSCs) reside. It supports the long-term potential of fast-cycling EpSCs and moderates aging-related inflammatory markers in keratinocytes. Here, we assessed fibulin 7s role during imiquimod (IMQ)-induced inflammation in 1-year-old mouse dorsal skin. We found that loss of Fbln7 aggravates epidermal inflammation, marked by increased epidermal thickness, proliferation, and phosphorylation of JNK (c-Jun N-terminal kinase). Fast-cycling EpSCs labeled with Slc1a3-creER-TdTomato demonstrated that IMQ-induced proliferation in Fbln7 KO mice is contributed by cell divisions in the suprabasal layers, a hallmark of inflammatory epidermal responses. EpSC transcriptomes further reveal IMQ-modulated genes that are more substantially affected in Fbln7 KO mice, including IL-17 pathway-related genes known in psoriasis pathogenesis. Mechanistically, fibulin 7 directly binds to IL-17A and decreases IL-17A-mediated p38 MAPK activation. In public human psoriasis datasets, FBLN7 is reduced in lesional skin compared with non-lesional or normal skin, and it is significantly correlated with common psoriasis-associated genes. Altogether, fibulin 7 is potentially beneficial to protect against skin inflammation.

cell biology↗

bioRxiv2026

Cryo-EM structure of pro-aggregant P301L/S320F double-mutant tau filaments formed in mouse brains following peripheral AAV delivery

Tauopathies are characterized by the accumulation of abnormally phosphorylated tau filaments, with disease-specific folds revealed by cryo-electron microscopy (cryo-EM). Here, we delivered a recombinant human tau carrying the P301L/S320F double mutation into AppNL-G-F/MAPT double knock-in mice using a blood-brain-barrier-permeable AAV (AAV-PHP.eB), enabling rapid induction of tau aggregation in vivo. The mutant tau was systemically administered via retro-orbital injection, providing a minimally invasive approach for widespread neuronal transduction. Phosphorylated tau aggregation, as well as seeding activity, were observed without the need for exogenous seeds. Our cryo-EM analysis resolved a novel filament fold incorporating residues 279-329, in which the P301L and S320F mutations introduced stabilizing interactions that reinforced filament assembly. This conformation was distinct from previously reported folds, including Alzheimer and Pick tau filaments. Moreover, the filaments adopted a more compact architecture than those observed in patient-derived samples or other model mice. This finding demonstrates that P301L/S320F double-mutant tau adopts a structurally unique, highly aggregation-prone fold, and that this system provides a rapid platform for modeling tau filament formation.

neuroscience↗

bioRxiv2026

Atomic resolution cryo-EM at 200 keV

Atomic resolution in cryo-electron microscopy was first demonstrated six years ago. This was accomplished using 300 kV electron microscopes equipped with new hardware that provided narrower energy spread, aberration correction, and energy filtering. Here, we report the achievement of 1.24 [A] atomic resolution on an upgraded 200 kV electron microscope featuring a cold field emission gun, a high-resolution objective lens polepiece, and an energy filter. These components transform the instrument into a cost-effective single particle cryo-EM platform with performance comparable to that of significantly more expensive 300 kV systems. The microscope can also be operated at 100 kV and by using a high-speed hybrid-pixel detector we were able to reach sub-2 [A] resolution.

molecular biology↗

bioRxiv2025

CCDC32 collaborates with the membrane to assemble the AP-2 clathrin adaptor complex

Cells have evolved a variety of assembly chaperones to aid in the difficult process of forming macromolecular complexes in a crowded cytoplasm. Assembly of adaptor protein complex 2 (AP-2), the primary cargo adaptor in clathrin-mediated endocytosis, is regulated by the chaperones AAGAB and CCDC32, whose deletion causes loss of all AP-2 subunits in vivo. AAGAB and CCDC32 are thought to act sequentially to assemble the AP-2 tetramer from its constituent heterodimers. However, the molecular requirements and structural consequences of CCDC32 interaction with AP-2 are not yet understood. Here, using in vitro reconstitution and integrative structural analysis, we describe the molecular mechanism of CCDC32-mediated AP-2 assembly. First, CCDC32 interacts with the appendage domain of the AP-2 subunit, using the same binding site as canonical endocytic regulators in addition to a novel, yet highly conserved pocket on . CCDC32 contains cargo sorting motifs normally found in trans-membrane cargo and binds to AP-2 heterodimers using canonical cargo-binding sites. Additionally, two amphipathic helices in CCDC32 bind to the /{sigma}2 heterodimer. Surprisingly, in solution, we find that CCDC32 prevents complex assembly and actively disassembles AP-2 tetramers. Inhibition requires the amphipathic helices of CCDC32, which also mediate binding to PIP2-containing membranes. The presence of PIP2-containing membrane stabilizes the final stages of assembly. We propose that the membrane acts as a molecular switch to release inhibitory interactions, allowing for full complex assembly to proceed. Using cryo-EM, we visualize an assembly intermediate that mimics the conformation of AP-2 found in vesicles, with CCDC32 bound at both cargo binding sites and both membrane-binding sites, suggesting that assembly leads to deposition of active complexes on the plasma membrane.

molecular biology↗

bioRxiv2025

Integrin-binding matricellular protein fibulin-5 maintains epidermal stem cell heterogeneity during skin aging

The extracellular matrix (ECM) is crucial in building the extracellular environment and translating extracellular information into biochemical signals that sustain organ functions. Fibulin-5 (Fbln5) is a multifunctional ECM protein essential for forming elastic fibers and regulating cellular functions by binding to integrins. While fibulin-5 expression decreases with age in human skin, the functional implications of this decrease, particularly in epidermal stem cell regulation, have remained largely unexplored. Here, we find that the knocking out of Fbln5 in mice results in early impairments of epidermal stem cell properties, similar to the chronological aging of the skin. The Fbln5 deficiency suppresses the expression of integrins and other cell junction proteins, leading to the inactivation of YAP signaling in epidermal stem cells. This reduction in YAP signaling results in the downregulation of the fast-cycling epidermal stem cell marker SLC1A3 in human skin and primary keratinocytes. These results suggest that, in addition to its known function in elastic fiber formation, fibulin-5 also plays a critical role in regulating the balance of epidermal stem cell populations during skin aging and coordinating the crosstalk between the extracellular environment and intracellular signaling.

developmental biology↗

bioRxiv2025

Spatiotemporal dynamics of CD73 in mouse retina under physiological conditions

Adenosine is essential to energy metabolism and neuromodulation in the central nervous system. The retina is a highly energy-demanding neural tissue, and dysregulation of adenosine signaling causes retinal diseases. However, the dynamics of ecto-5-nucleotidase (CD73), a key enzyme for extracellular adenosine generation, remain elusive. Here, we investigate its spatiotemporal profile from development to adulthood using two transgenic mouse lines. We found that CD73 is transiently expressed in the early astrocyte lineage (Embryonic day 16.5 to Postnatal day [P] 3), becomes prominent in the rod-photoreceptor lineage by P3, and appears in the inner nuclear layer from P7 onward. CD73 deletion delays rod-response recovery and shortens the implicit time in scotopic ERG under dim light. These findings indicate that light and other physiological cues influence CD73 expression, which has significant consequences for retinal function, thereby providing a foundation for exploring disease-related alterations in CD73 and designing therapies that restore adenosine homeostasis.

developmental biology↗

bioRxiv2025

The Hitchdock domain in Kinesin-2 Tail Enables Adaptor Assembly and Cargo Binding

Intracellular transport relies on motor proteins like kinesins to deliver essential cargoes along microtubules, yet the mechanisms of cargo recognition remain unclear. Here, we present high-resolution cryo-electron microscopy structures of the heterotrimeric kinesin-2 complex (KIF3A/KIF3B/KAP3) bound to the adenomatous polyposis coli (APC) cargo. Our findings reveal a previously uncharacterized KIF3 tail motif, termed the "Hitchdock domain," which plays a pivotal role in mediating interactions with both the KAP3 adaptor and the APC cargo. In this domain, the KIF3A helical regions facilitate specific cargo binding, while the {beta}-hairpin region and KIF3B provide structural support. Mutagenesis and molecular dynamics simulations confirm the domains functional importance. Interestingly, the Hitchdock/KAP3 structure suggests a conserved structural basis for cargo recognition across molecular motors, including kinesin-1 and dynein, which utilize similar hook-like architectures, highlighting the potential universality of this mechanism. Furthermore, our findings provide insights into kinesin-2 cargo specificity and offer a molecular framework for understanding related diseases.

cell biology↗

bioRxiv2025

Repeatability of protein structural evolution following convergent gene fusions

Convergent evolution of proteins provides insights into repeatability of genetic adaptation. While local convergence of proteins at residue or domain level has been characterized, global structural convergence by inter-domain/molecular interactions remains largely unknown. Here we present structural convergent evolution on fusion enzymes of aldehyde dehydrogenases (ALDHs) and alcohol dehydrogenases (ADHs). We discovered BdhE (bifunctional dehydrogenase E), an enzyme clade that emerged independently from the previously known AdhE family through distinct gene fusion events. AdhE and BdhE showed shared enzymatic activities and non-overlapping phylogenetic distribution, suggesting common functions in different species. Cryo-electron microscopy revealed BdhEs form donut-like homotetramers, contrasting AdhEs helical homopolymers. Intriguingly, despite distinct quaternary structures and >70% unshared amino acids, both enzymes form resembled dimeric structure units by ALDH-ADH interactions via convergently elongated loop structures. These findings suggest convergent gene fusions recurrently led to substrate channeling evolution to enhance two-step reaction efficiency. Our study unveils structural convergence at inter-domain/molecular level, expanding our knowledges on patterns behind molecular evolution exploring protein structural universe.

evolutionary biology↗

bioRxiv2025

Defining epithelial stem cell heterogeneity through undulating structures of the skin and oral mucosa

Epithelial stem cells exhibit heterogeneity, with distinct stem cell populations occupying specific tissue regions. The human skin displays a characteristic undulating structure at the epidermal-dermal junction, which supports mechanical strength and influences the spatial organization of epithelial stem cells. Unlike human skin, mouse skin lacks these undulations, complicating studies into the effects of tissue architecture on stem cell distribution. Here, we leverage the mouse oral mucosa, which possesses an undulating structure similar to human skin, to characterize stem cell division dynamics and long-term fate in vivo. Using a combination of H2B-GFP pulse-chase analysis and lineage tracing with Dlx1-CreER and Slc1a3-CreER models, we demonstrate that slow-and fast-cycling stem cells localize to distinct anatomical regions relative to the undulating structure and maintain their respective compartments during tissue homeostasis. A three-dimensional culture model using micropatterned collagen scaffolds that recapitulate the undulating structures in vitro reveals that the mechanical environment generated by the undulating structures partially induces proliferative heterogeneity in epithelial stem cells. This study proposes tissue undulating surface structure as a common principle as a niche component that defines the localization of compartmentalized stem cell populations across different epithelial tissues. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/634195v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@b3046aorg.highwire.dtl.DTLVardef@e6cd8forg.highwire.dtl.DTLVardef@c169ccorg.highwire.dtl.DTLVardef@8f772_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Cryo-EM of PMEL Amyloids Reveals Pathogenic Mechanism of Pigment Dispersion Syndrome

PMEL amyloids provide a vital scaffold for melanin deposition in melanosomes, playing a central role in pigmentation. Despite their importance, the high-resolution structure of PMEL amyloids has remained elusive. Here, we determined near-atomic resolution structures of wild-type PMEL amyloids using cryo-electron microscopy, revealing two distinct polymorphic forms with unique structural features. We further examined the pathogenic G175S mutation linked to pigment dispersion syndrome (PDS). Structural analysis showed that the G175S mutation introduces an additional hydrogen bond, stabilizing a novel fibril conformation. In vitro assays demonstrated a fourfold increase in polymerization efficiency for the G175S mutant compared to the wild-type. This enhanced polymerization correlated with a [~]70% increase in secreted amyloids in G175S-expressing cells without detectable changes in melanosome morphology or number. These findings suggest that the G175S mutation promotes amyloidogenesis within melanosomes, increasing amyloid load and contributing to PDS pathophysiology. This study provides insights into the molecular basis of PMEL amyloid formation in both physiological and pathological contexts, offering new perspectives on their structural diversity and dysregulation in pigmentation disorders.

cell biology↗

bioRxiv2024

Tension-induced suppression of allosteric conformational changes explains coordinated stepping of kinesin-1

The dimeric motor protein kinesin-1 walks along microtubules by alternating ATP hydrolysis and movement of its two motor domains ("head"). The detached head preferentially binds to the forward tubulin-binding site after ATP binds to the microtubule-bound head, but the mechanism preventing premature binding to the microtubule while the partner head awaits ATP remains unknown. Here, we examined the role of the neck linker, the segment connecting the two heads, in this mechanism. High-resolution structural analyses of the nucleotide-free head revealed a bulge just ahead of the neck linkers base that creates an asymmetric constraint on its mobility. While the neck linker can stretch freely backward, it must navigate around this bulge to extend forward. Based on this finding, we hypothesized that premature binding of the tethered head is suppressed by an intolerable increase in neck linker tension. Molecular dynamic simulations and single-molecule fluorescent assays supported this model. These findings demonstrate a tension-based regulation mechanism where off-pathway conformational transitions are thermodynamically suppressed through entropy loss associated with neck linker stretching, suggesting that neck linker tension influences the allosteric conformational transition rather than directly affecting the nucleotide state.

biophysics↗

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↗

bioRxiv2023

Novel Insights into the Aortic Mechanical Properties of Mice Modeling Hereditary Aortic Diseases

OBJECTIVEHereditary aortic diseases (hADs) increase the risk of aortic dissections and ruptures. Recently, we have established an objective approach to measure the rupture force of the murine aorta, thereby explaining the outcomes of clinical studies and assessing the added value of approved drugs in vascular Ehlers-Danlos syndrome (vEDS). Here, we applied our approach to six additional mouse hAD models. APPROACH AND RESULTWe used two mouse models of Marfan syndrome (MFS) as well as one smooth-muscle-cell-specific knockout (SMKO) of Efemp2 and three CRISPR/Cas9-engineered knock-in models (Ltbp1, Mfap4, and Timp1). One of the two MFS models was subjected to 4-week-long losartan treatment. Per mouse, three rings of the thoracic aorta were prepared, mounted on a tissue puller, and uniaxially stretched until rupture. The aortic rupture force of the SMKO and both MFS models was significantly lower compared with wild-type mice but in both MFS models higher than in mice modeling vEDS. In contrast, the Ltbp1, Mfap4, and Timp1 knock-in models presented no impaired aortic integrity. As expected, losartan treatment reduced aneurysm formation but surprisingly had no impact on the aortic rupture force of our MFS mice. CONCLUSIONSOur read-out system can characterize the aortic biomechanical integrity of mice modeling not only vEDS but also related hADs, allowing the aortic-rupture-force- focused comparison of mouse models. Furthermore, aneurysm progression alone may not be a sufficient read-out for aortic rupture, as antihypertensive drugs reducing aortic dilatation might not strengthen the weakened aortic wall. Our results may enable identification of improved medical therapies of hADs. HighlightsO_LIAssay to measure the aortic rupture force as read-out for the biomechanical integrity identified weakened murine thoracic aorta prior to micro- and macroscopic changes in Fbn1+/C1041G mice. C_LIO_LIDespite reducing aneurysm growth, losartan treatment did not have any impact on the aortic rupture force of Fbn1mgR/mgR mice modeling MFS. C_LIO_LISmooth-muscle-cell-knock-out of Efemp2 significantly impaired the rupture force of the murine ascending aorta. C_LIO_LIAortic rupture force measurements could clarify the causality of novel candidate gene(s)/variant(s) in mouse models of aortic diseases. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/553452v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@a29d79org.highwire.dtl.DTLVardef@10a272org.highwire.dtl.DTLVardef@19071forg.highwire.dtl.DTLVardef@122ab40_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗