Biology subjects

Sengle, G.

Publications and source records attributed to Sengle, G..

12 recordsLinked to original sources

bioRxiv2026

A cell death-independent role for MLKL in TGF-β receptor recycling in Marfan syndrome

Marfan syndrome (MFS) causes thoracic aortic aneurysm through mutations in the fibrillin-1 gene and subsequent dysregulated TGF-{beta} signalling. Mixed lineage kinase domain-like protein (MLKL) executes necroptosis after RIPK3-dependent phosphorylation, but also functions in endosomal trafficking. Here we show that MLKL phosphorylation sustains TGF-{beta} receptor recycling and aortic wall integrity without causing cell death. Phosphorylation-dead MLKLS345A/S347A (MlklAA) mice developed aortic root dilation with disorganised collagen architecture and a secretory-pathway proteomic signature. Crossing them with Fbn1C1039G/+ MFS mice aggravated aortic dilation, disrupted collagen fibril ultrastructure and prolyl/lysyl hydroxylation, and caused lethal rupture under angiotensin II. Complete MLKL deficiency reproduced this phenotype. Phosphorylated MLKL was increased in MFS aortas, yet cleaved caspase-3, TUNEL positivity, smooth muscle cell content and leukocyte infiltration were unchanged. In smooth muscle cells, MLKL depletion dysregulated collagen-modifying enzymes, altered TGF-{beta}-driven matrix remodelling and impaired RAB11-dependent TGF{beta}R1 recycling. These findings suggest that phosphorylation-dependent, non-lytic functions of MLKL may contribute to the maintenance of thoracic aortic integrity in MFS.

cell biology↗

bioRxiv2025

Olfactory receptor 2 drives abdominal aortic aneurysm by promoting CX3CR1-mediated monocyte recruitment

BackgroundAbdominal aortic aneurysms (AAA) are characterized by an intricate interplay of extracellular matrix degradation and inflammation. Macrophages are centrally involved in these processes. The mechanisms underlying macrophage activation in AAA remain incompletely understood. Vascular macrophages have been shown to express olfactory receptor 2 (Olfr2), a G-protein coupled receptor involved in mediating the sense of smelling and regulating inflammatory activity in macrophages. Whether Olfr2 plays a role in modulating macrophage responses in AAA formation remains unknown. Methods & ResultsIn silico micro-array analysis showed increased expression of the human Olfr2 orthologue OR6A2 in AAA tissue compared to healthy aorta. Flow cytometric analysis revealed increased expression of OR6A2 on classical, non-classical, and intermediate monocytes of patients with a large AAA (> 5cm) in comparison to patients with smaller AAA (< 5cm). Up to 30% of vascular macrophages expressed OR6A2 in human AAA and Olfr2 in mouse AAA tissue. Olfr2 expression peaked on major histocompatibility complex II-high (MHCIIhigh) and C-C chemokine receptor type 2-low (CCR2low) aortic monocytes and macrophages on day 7 following experimental AAA initiation and decreased to baseline expression on day 28. Olfr2 gene (Olfr2-/-) deficiency protected mice from AAA formation, which was accompanied by lowered ECM degradation, reduced macrophage infiltration and increased smooth muscle cell content. Conversely, treatment with the Olfr2 agonist octanal exacerbated AAA formation and inflammation, while the antagonist citral reduced AAA formation in comparison to vehicle treated mice. Bulk transcriptome analysis of aortic tissue revealed reduced inflammatory gene expression in Olfr2-/- mice at day 7 following AAA initiation. Spectral flow cytometry resolved 20 aortic immune cell populations, which were largely reduced in quantity by Olfr2-deficiency at day 7 and day 28 post experimental AAA formation, while circulating leukocyte counts and monocyte subset distribution were not altered between Olfr2+/+ and Olfr2-/- mice. Circulating Ly6Chigh-monocytes exhibited reduced expression of the CX3C motif chemokine receptor 1 (CX3CR1) and CCR2 during AAA formation. Transcriptional analysis of monocytes confirmed downregulation of pathways associated with cell adhesion, motility and migration. In vitro, Olfr2-/- monocytes showed impaired migration towards the CX3CR1 ligand CX3CL1. Competitive transfer of Olfr2+/+ and Olfr2-/- monocytes confirmed reduced migratory capacity of Olfr2-/- monocytes into the developing AAA. ConclusionWe demonstrate a critical relevance for Olfr2 in the modulation of the inflammatory response underlying AAA, which is mediated by enhanced monocyte recruitment.

immunology↗

bioRxiv2022

Deficiency in hyaluronan synthase 3 attenuates ruptures in a murine model of abdominal aortic aneurysms by reduced aortic monocyte infiltration

Abdominal aortic aneurysms (AAA) are a common vascular disorder with a high mortality due to the prevalence of aortic ruptures. The underlying pathomechanisms are complex and involve immune cell infiltration and degradation of the vascular extracellular matrix (ECM). Hyaluronan (HA), synthesized at the plasma membrane by three HA synthase isoenzymes (HAS1-3), is not only a major constituent of the ECM but also known to directly affect the phenotype of vascular smooth muscle cells as well as immunological responses. Specifically, the HAS3 isoenzyme has been reported to play a major role in various inflammatory conditions. Therefore, the aim of the present study was to elucidate the role of HAS3-derived HA in the pathogenesis of abdominal aortic aneurysm. To this end, we used a murine model of Angiotensin II (AngII)-induced abdominal aortic aneurysms and dissections (AAAs/AADs) and could demonstrate that genetic depletion of Has3 improves survival in Apoe/Has3 double deficient (Apoe/Has3-DKO) mice via the reduced occurrence of aortic ruptures. Mechanistically, fewer elastica breaks were observed in Apoe/Has3-DKO mice compared to Apoe-KO littermates. This was associated with a decreased infiltration of myeloid immune cells into the vessel wall of Has3-deficient mice while in parallel elevated numbers of circulating leukocytes were detected. RNA seq analysis from aortic tissue pointed towards a disturbed endothelial-myeloid cell communication as a cause for the diminished recruitment of immune cells to the aortic wall. While endothelial cells were unaffected, upregulation of adhesion receptors as well as the HA receptor CD44, known to mediate leukocyte adhesion to the endothelium, was blunted in monocytes from Apoe/Has3-DKO mice in response to AngII treatment. These findings underline the pivotal detrimental role of monocytes HAS3-dependent pericellular HA matrix for an exaggerated immune cell recruitment to inflammatory foci giving here rise for an increased incidence of ruptured aortic aneurysms.

immunology↗

bioRxiv2022

PI 3-kinase isoform p110alpha controls smooth muscle cell functionality and protects against aortic aneurysm formation

BackgroundCatalytic class IA PI 3-kinase isoform p110 is a crucial regulator of cellular proliferation and survival in numerous cell types. While p110 is critically involved in pathogenic vascular remodeling, its physiological role for vascular integrity under stress conditions has not been studied. We report a protective function of smooth muscle p110 against abdominal aortic aneurysm (AAA) formation. Methods & ResultsIn mice lacking p110 in smooth muscle cells (sm-p110-/-), perfusion of the infrarenal aorta with porcine pancreatic elastase (PPE) yielded substantially enhanced AAA formation compared to wild type controls. This disease phenotype is partly attributable to a subtle preexisting vascular phenotype under basal conditions, as sm-p110-/- mice displayed a smaller media area, deranged aortic wall structure (detached smooth muscle cells, increased apoptotic cell death), and a diminished functional responsiveness of aortic rings to vasodilators. Furthermore, p110 is also implicated in regenerative processes during AAA development: Whereas wild type mice showed increased media hypertrophy, neointima formation and proliferation upon PPE intervention, these vascular remodeling processes were diminished in sm-p110-/- mice. Concomitantly, increased numbers of elastic fiber breaks and ECM degradation were detected in sm-p110-/- aorta. Mechanistically, we found that lack of p110 expression impaired smooth muscle cell proliferation, expression of contractile marker genes and production of elastin fibers. This phenotype largely depended on reduced phosphorylation and inactivation of FOXO1, as specific FOXO1 inhibition fully rescued proliferation of p110-/- smooth muscle cells, and knockdown of FOXO1 increased expression of calponin and elastin. ConclusionsSmooth muscle p110 protects against AAA disease by maintaining aortic wall homoeostasis and promoting SMC proliferation to compensate for cell loss during AAA development. Our findings have potential implications for current approaches aimed at p110 inhibition for cancer therapy and suggest new pharmacological strategies to activate p110 signaling in AAA disease.

cell biology↗

bioRxiv2022

Targeting of MMP-13 prevents aortic aneurysm formation in Marfan mice

Fibrillin-1 assembles into microfibrils that not only define the structural integrity and biomechanics of the aorta but also target and sequester growth factors within the extracellular microenvironment of aortic resident cells. To better understand how dominant negative effects on fibrillin microfibril stability manifest in growth factor driven aortic disease, we analyzed early events of aortic aneurysm formation within the first two weeks of postnatal life in the dominant negative Fbn1 GT8 Marfan mouse model. Echocardiography analysis of homozygous GT8 Fbn1 mice showed significant aortic root enlargement within the second week of postnatal life which correlated with the onset of fibrillin-1 fiber degradation, aberrantly increased BMP activity and upregulated transcript levels of the collagenase MMP-13. We also found the aortic collagen network structurally disturbed where the mutant GT8-fibrillin-1 was detected. Genetic ablation or pharmacological inhibition of MMP-13 in Fbn1 GT8 Marfan mice prevents aortic root dilatation implicating the relevance of this mechanism in aortic aneurysm formation in Marfan syndrome.

biochemistry↗

bioRxiv2022

Inhibition of myeloperoxidase prevents thoracic aortic aneurysm formation in Marfan mice

Marfan syndrome (MFS) is the most prevalent inherited connective tissue disorder, still remains uncurable, and is characterized by high mortality at early age driven by dissection and rupture of thoracic aortic aneurysms. MFS is caused by mutations in the fibrillin-1 gene and aberrant TGF{beta} signaling. Here we addressed whether myeloperoxidase (MPO), a leukocyte derived enzyme with potent matrix modulating properties also influences the aortic phenotype in MFS. MFS patients displayed increased circulating MPO levels compared to controls as well as marked aortic MPO deposition. In an MFS mouse model, MPO induced inflammatory endothelial activation and endothelial to mesenchymal transition which triggered aortic leukocyte recruitment. Moreover, MPO directly contributed to adverse extracellular matrix remodeling by promoting oxidative stress and nitration of proteins within the vascular wall. Genetic MPO deficiency and pharmacological MPO inhibition attenuated MFS-related aneurysm formation. We herein identify MPO as a critical mediator of MFS-related thoracic aortic aneurysm formation and - in the absence of any pharmacological treatment so far in this disease - a first anti-inflammatory target to modulate disease progression.

immunology↗

bioRxiv2022

Fibrillin microfibril structure identifies long-range effects of inherited pathogenic mutations affecting a key regulatory TGFβ-binding site

Genetic mutations in fibrillin microfibrils cause a range of serious inherited diseases such as Marfan syndrome (MFS) and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. In this study we reveal the detailed cryo-EM structure of native fibrillin microfibrils from mammalian tissue. The major bead region showed pseudo 8-fold symmetry and a buried protease resistant N-terminal core. Based on this structure, we show a WMS deletion mutant induces a rearrangement with long-range effects blocking interaction with latent TGF{beta}-binding protein (LTBP)-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. These results establish that in complex extracellular protein assemblies, such as in fibrillin, mutations may have long-range structural consequences to disrupt growth factor signalling and cause disease.

biochemistry↗

bioRxiv2022

Transglutaminase mediated asprosin oligomerization allows its tissue storage as fibers

Asprosin, the C-terminal furin cleavage product of profibrillin-1, was reported to act as a hormone that circulates at nanomolar levels and is recruited to the liver where it induces G protein-coupled activation of the cAMP-PKA pathway and stimulates rapid glucose release into the circulation. Although derived upon C-terminal cleavage of fibrillin-1, a multidomain extracellular matrix glycoprotein with a ubiquitous distribution in connective tissues, little is known about the mechanisms controlling the bioavailability of asprosin in tissues. In the current view, asprosin is mainly produced by white adipose tissue from where it is released into the blood in monomeric form. Here, by employing newly generated specific asprosin antibodies we monitored the distribution pattern of asprosin in human and murine connective tissues such as placenta, and muscle. Thereby we detected the presence of asprosin positive extracellular fibers. Further, by screening established cell lines for asprosin synthesis we found that most cells derived from musculoskeletal tissues render asprosin into an oligomerized form. Our analyses show that asprosin already multimerizes intracellularly, but that stable multimerization via covalent bonds is facilitated by transglutaminase activity. Further, asprosin fiber formation requires an intact fibrillin-1 fiber network for proper linear deposition. Our data suggest a new extracellular storage mechanism of asprosin in an oligomerized form which may regulate its cellular bioavailability in tissues.

biochemistry↗

bioRxiv2021

LTBP1 promotes fibrillin incorporation into the extracellular matrix

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillinmicrofibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGF{beta} growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGF{beta}-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

cell biology↗

bioRxiv2021

Vertebrate hemicentin-1 interacts physically and genetically with nidogen-2

Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mouse and zebrafish hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 in basement membrane integrity. We show that HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately ten-fold lower affinity and in a competitive manner. Furthermore, immunofluorescence and immunogold labelling revealed that HMCN1/Hmcn1 is localized close to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as the loss of Laminin-5 (Lama5) or Hmcn1. Finally, combined partial loss-of-function studies indicated that nid2a genetically interacts with both hmcn1 and lama5. Together, these findings suggest that despite their mutually exclusive physical binding, hemicentins, nidogens, and laminins tightly cooperate and support each other during formation, maintenance, and function of basement membranes to confer tissue linkage.

developmental biology↗

bioRxiv2021

Lack of evidence for a role of anthrax toxin receptors as surface receptors for collagen VI and for its cleaved off C5 domain (endotrophin)

The widely expressed microfibril-forming collagen VI is subject to proteolytic cleavage and it has been proposed that the cleaved off C-terminal Kunitz domain (C5) of the 3 chain is an adipokine important for tumor progression and fibrosis. Under the name "endotrophin" the C5 fragment has also been shown to be a potent biomarker for fibro-inflammatory diseases. However, the biochemical mechanisms behind endotrophin activity have not been investigated. In earlier studies, the anthrax toxin receptor 1 was found to bind to C5, but this potential interaction has not been further studied. Given the proposed physiological role of endotrophin we aimed to determine how the endotrophin signal is transmitted to the recipient cells. Surprisingly, we could not detect any interaction between endotrophin and anthrax toxin receptor 1 or its close relative, anthrax toxin receptor 2. Moreover, we could not detect binding of fully assembled collagen VI to either anthrax toxin receptor. We also performed similar experiments with the collagen VI surface receptor NG2 (CSPG4). We could confirm that NG2 is a collagen VI receptor that binds to assembled collagen VI, but not to the cleaved C5/endotrophin. A cellular receptor for C5/endotrophin therefore still remains elusive.

cell biology↗

bioRxiv2020

LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro

ObjectiveSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin Ds beneficial effects are mediated by up-regulating LL-37, a well-known antimicrobial peptide with antiviral effects. MethodsRecombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37s ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2. ResultsWe could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/SStrep KD = 410 nM, LL-37/SHis KD = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/SStrep KD = 370 nM, hACE2/SHis KD = 370 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 290 nM. Further, inhibition of the binding of SStrep (IC50 = 740 nM), S1e (IC50 = 170 nM), and RBD (IC50 = 130 nM) to hACE2 by LL-37 was demonstrated. ConclusionsWe have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.

immunology↗