Biology subjects

Winkels, H.

Publications and source records attributed to Winkels, H..

9 recordsLinked to original sources

bioRxiv2026

An Apoptotic Failsafe Program Involving PDGF-dependent PI 3-Kinase Activity Contributes to Plaque Composition in Atherosclerosis

Background: Growth and stability of atherosclerotic plaques are impacted by vascular smooth muscle cell (VSMC) apoptosis which is believed to be suppressed by growth factor dependent activation of phosphoinositide 3-kinase (PI3K). Here, we analyzed the effects of chronically elevated platelet-derived growth factor receptor {beta} (PDGFR{beta}) signaling on VSMC apoptosis and aortic plaque composition during atherogenesis. Methods: We assessed atherosclerosis in low-density lipoprotein receptor deficient mice, lacking low-density lipoprotein receptor-related protein 1 in VSMCs (LDLR-/-smLRP1-/- mice), in that smLRP1 deficiency resulted in PDGFRB overexpression. Apoptosis and downstream signaling was investigated in cultured aortic VSMCs. Results: Chronically elevated PDGFR{beta} signaling in LDLR-/-smLRP1-/- mice unexpectedly increased VSMC apoptosis in atherosclerotic plaques. In vitro, PDGF exerted transient anti-apoptotic effects on VSMCs, whereas long-term stimulation promoted cell death. Mutation of the PI3K binding site in the PDGFR{beta} (PDGFR{beta}-F2/F2) reduced VSMC apoptosis in plaques as well as in cultured VSMCs. Further analyses revealed that PI3K isoform p110 mediates VSMC survival, whereas p110{delta} and p110{beta} rather promote apoptosis. We identified a pro-apoptotic PDGFR{beta}-driven signaling pathway in VSMCs involving PI3K-dependent induction of c-Myc that in turn suppresses the expression of anti-apoptotic Bcl-2. PDGF-dependent downregulation of Bcl-2 increases the susceptibility of VSMCs to apoptotic agonists like H2O2 or Fas ligand. Consistently, Bcl-2 expression was elevated in plaques from PDGFR{beta}-F2/F2 mice. Conclusions: Our study expands the understanding of the role of growth factors in regulating apoptosis and argues against their role as pure survival factors. This has direct implications in atherogenesis, as chronically elevated PDGF does not mediate VSMC survival in atherosclerotic plaques, but rather promotes cell death in the VSMC-rich fibrous cap, thus promoting plaque instability and rupture.

biochemistry↗

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↗

bioRxiv2026

Thymus Involution across the Human Menstrual Cycle

Structured AbstractO_ST_ABSImportanceC_ST_ABSThymic involution in humans has traditionally been conceptualized as a linear, age-associated process. Animal studies suggest transient hormone-dependent fluctuations in thymus size during reproductive transitions, including across the estrous cycle, but translational evidence in humans remains limited. ObjectiveTo determine whether thymus size changes across the menstrual cycle in healthy women and whether these changes are associated with fluctuations in estradiol and progesterone levels. DesignObservational repeated-measures study using quantitative computed tomography (qCT). Data were collected as part of an imaging study examining menstrual cycle-related physiological variation. Participants underwent assessments during menses and the early luteal phase within the same menstrual cycle. Mixed-effects models with robust variance estimation evaluated associations between cycle phase, hormonal contraception, reproductive hormone levels, and thymus size. Thymus segmentation was independently conducted by 2 blinded raters. SettingSingle-center university-based imaging study conducted at the University of Iowa. ParticipantsThirty-one non-smoking women with regular menstrual cycles were included, of whom n = 16 used oral hormonal birth control and n = 15 did not use hormonal contraception. Exclusion criteria included pregnancy, breastfeeding, postmenopausal status, diabetes, body mass index greater than 30 kg/m2, hysterectomy, or use of long-term noncyclic hormonal contraception. Participants tracked menstrual cycles using temperature monitoring and ovulation kits before completing visits during menses and the early luteal phase. All participants provided informed consent before study participation. Main Outcomes and MeasuresPrimary outcome was thymus size measured in mm3 using ultra-low-dose qCT imaging. Secondary outcomes included serum estradiol and progesterone levels. ResultsEstradiol levels increased from menses to the early luteal phase independent of hormonal contraceptive status. Progesterone levels were significantly lower among women using hormonal birth control. Thymus size differed significantly by hormonal contraceptive group, with larger thymus volumes observed among women not using hormonal birth control. Among women not using hormonal contraception, 66.7% demonstrated thymic involution from menses to the early luteal phase, compared with 37.5% of women using hormonal contraception. Estradiol and progesterone significantly interacted in predicting thymus size. Conclusions and RelevanceThese findings provide first-in-human evidence suggesting that thymus involution demonstrates short-term dynamics across the menstrual cycle and may be influenced by reproductive hormones and hormonal contraception. Key PointsO_ST_ABSQuestionC_ST_ABSDoes thymus size change dynamically across the human menstrual cycle in association with fluctuations in reproductive hormones and hormonal contraception use? FindingsIn this repeated-measures quantitative computed tomography study of n = 31 healthy women, thymus size demonstrated within-person variation across the menstrual cycle. Greater thymic involution was associated with larger increases in estradiol, whereas increases in thymus size were associated with larger increases in progesterone; thymus size also differed significantly by hormonal contraceptive status. MeaningThese findings suggest that thymic involution in humans may demonstrate short-term hormone-dependent dynamics beyond age-related atrophy, with potential implications for sex differences in immune regulation and inflammatory disease.

physiology↗

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↗

bioRxiv2023

Pentose Phosphate Pathway Inhibition activates Macrophages towards phagocytic Lymphoma Cell Clearance

Macrophages in the B-cell lymphoma microenvironment represent a functional node in progression and therapeutic response. We assessed metabolic regulation of macrophages in the context of therapeutic antibody-mediated phagocytosis. Pentose phosphate pathway (PPP) inhibition by specific compounds and shRNA targeting induced increased phagocytic lymphoma cell clearance. Moreover, macrophages provided decreased support for survival of lymphoma cells. PPP inhibition induced metabolic activation, cytoskeletal re-modelling and pro-inflammatory polarization of macrophages. A link between PPP and immune regulation was identified as mechanism of macrophage repolarization. Inhibition of the PPP causes suppression of glycogen synthesis and subsequent modulation of the immune modulatory UDPG-Stat1-Irg1-Itaconate axis. PPP inhibition rewired macrophage maturation and activation in vivo. Addition of the PPP inhibitor S3 to antibody therapy achieved significantly prolonged overall survival in an aggressive B-cell lymphoma mouse model. We hypothesize the PPP as key regulator and targetable modulator of macrophage activity in lymphoma to improve efficacy of immunotherapies. HighlightsO_LIMacrophage-mediated lymphoma cell phagocytosis is increased by pentose phosphate pathway (PPP) inhibition as an immune regulatory switch for macrophage function and polarization C_LIO_LIPPP inhibition is linked to decreased glycogen synthesis and subsequent modulation of the UDPG-Stat1-Irg1-Itaconate axis C_LIO_LIPPP inhibition is tolerable in vivo and facilitates therapeutic targeting of B-cell lymphoma C_LI

cancer biology↗

bioRxiv2022

Vascular ultrasound for in vivo assessment of arterial pathologies in a murine model of atherosclerosis and aortic aneurysm

BackgroundVascular diseases like atherosclerosis or aortic aneurysms are common pathologies in the western world, promoting various, potentially fatal conditions. Hence, a plethora of animal models have been developed to investigate underlying mechanisms and potential therapeutics. Here we evaluate high resolution (HR) ultrasound in mouse models of atherosclerosis and abdominal aortic aneurysm (AAA) for noninvasive monitoring of morphological and functional vascular changes in vivo. MethodsEight-week-old ApoE-/- mice were used for disease models. For induction of atherosclerosis, mice were fed a western diet over 12 weeks. To trigger AAA development, osmotic minipumps were implanted, permanently releasing Angiotensin II continuously for 28 days. All animals were on C57Bl6/J background. HR vascular ultrasound of the carotid artery or the abdominal aorta was performed, respectively. Images obtained were analyzed by a speckle tracking algorithm (VevoVasc software) and were correlated with histological analyses by Picro Sirius Red staining and automated collagen quantification. ResultsArterial wall distensibility and global radial strain (GRS) as measures of arterial wall elasticity were reduced in the carotids of atherosclerotic mice as well as in the aortas of AAA mice. Pulse wave velocity (PWV) was elevated in both disease models. Intima-media thickness (IMT) was significantly increased in the atherosclerosis model. Matching those findings, area of the tunica media was enlarged in ApoE-/- mice fed a western diet, and in Angiotensin II treated mice as measured by automated image analysis, depicting higher collagen depositions in diseased arteries. Simple regression analysis revealed a strong correlation of media collagen content and area in AAA with IMT and GRS, respectively. In atherosclerosis, media collagen content significantly correlated with PWV and GRS, whereas wall distensibility was associated with the size of media area. ConclusionVascular imaging using latest generation HR ultrasound devices is suitable to trace changes of arterial wall properties in murine models of atherosclerosis and AAA. Obtained results not only correlate with histological findings but deliver information on functional parameters which may be used as early disease and risk markers in a longitudinal experimental approach.

physiology↗

bioRxiv2022

Using TCR and BCR sequencing to unravel the role of T and B cells in abdominal aortic aneurysm

BackgroundAbdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease, and the pathogenesis is still poorly understood. Recent evidence suggests that AAA displays characteristics of an autoimmune disease and it gained increasing prominence that specific antigen-driven T cells in the aortic tissue may contribute to the initial immune response. Single-cell RNA T- and B cell receptor (TCR and BCR) sequencing is a powerful tool to investigate TCR and BCR clonality and thus to further test this hypothesis. However, difficulties such as very limited numbers of isolated cells must be considered during implementation and data analysis making biological interpretation of the data challenging. Here, we perform a representative analysis of scRNA TCR and BCR sequencing data of experimental murine AAA and show a reliable and streamlined bioinformatic processing pipeline highlighting opportunities and limitations of this approach. MethodsWe performed single-cell RNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aortic segment of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham operated mice at day 3 and 28 as well as non-operated mice served as controls. ResultsComparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed no differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified multiple clones in 11 of 16 AAA samples and in 1 of 8 control samples. Comparison of the immune receptor repertoires indicated that only few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and TRBV12-2+TRBV13-2. ConclusionIn summary, we found no clonal expansion of TCRs or BCRs in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive amount of T and B cells to prevent undersampling and to enable detection of potential rare clones. Using this current scSeq-based approach we did not identify clonal enrichment of T or B cells in experimental AAA.

immunology↗

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

Inhibition of MLKL impairs abdominal aortic aneurysm development by attenuating smooth muscle cell necroptosis

BackgroundReceptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) dependent cell death has been identified as a crucial mediator of abdominal aortic aneurysm (AAA) development. RIPK3 mediates phosphorylation of Mixed lineage kinase domain like pseudokinase (MLKL) thereby inducing its oligomerization and translocation to the cell membrane. Given the dual role of RIPKs being involved in necroptosis as well as in apoptosis induction, the specific role of MLKL-induced necroptotic cell death in AAA remains unclear. MethodsWe monitored elastase-perfusion (PPE) induced progression of AAA in C57BL/6N (WT), RIPK1 kinase-inactive (Ripk1D138N/D138N), MLKL knockout (Mlkl-/-) and MLKL phospho-deficient (MlklAA) mice by ultrasound measurements, histological analyses and bulk mRNA sequencing to assess structural and molecular aortic changes. Bone marrow transplantations in WT and MlklAA mice were utilized to dissect the role of MLKL in smooth muscle cells (SMCs) and myeloid cells in AAA development. MLKL expressing human SMCs were generated to investigate necroptosis-induced proinflammatory cytokine secretion and subsequent polymorphonuclear neutrophil (PMN) migration and activation in vitro. ResultsUltrasound analysis showed that ~70% of the WT animals developed PPE induced-AAA with significant aortic structural alterations and enhanced myeloid cell infiltration. In contrast, Ripk1D138N/D138N, MlklAA, and Mlkl-/- mice were protected from AAA. This protection was associated with reduced adverse extracellular matrix (ECM) remodeling and leukocyte infiltration. MLKL deficiency was associated with a significant downregulation of genes involved in fibrinolysis, anti-inflammatory response, immune response and complement activation in aortic tissue in AAA. Bone marrow transplantation studies showed the lack of MLKL in SMCs to be the main driver of AAA protection. Proinflammatory cytokine secretion was elevated in necroptosis induced SMCs and resulted in a significant accumulation and activation of PMN. ConclusionsOverall, these findings indicate that MLKL-induced necroptotic SMC death and subsequent proinflammatory leukocyte activation play a causative role in AAA development and suggest that pharmacological inhibition of MLKL may represent a promising treatment strategy for AAA disease.

cell biology↗