Biology subjects

Kelm, M.

Publications and source records attributed to Kelm, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Vitamin K2 Limits Ferroptosis-Associated Lipid Peroxidation and Attenuates Aortic Valve Stenosis

BackgroundCalcific aortic valve stenosis (AS) is the most common valvular heart disease in the aging population and lacks effective pharmacological therapy. Oxidative stress is a key feature of valvular remodeling, yet the mechanisms linking oxidative injury to calcification remain unclear. Ferroptosis, a lipid peroxidation-driven form of regulated cell death, has emerged as a key mediator of oxidative tissue injury and may contribute to cardiovascular disease. Vitamin K was recently identified as a suppressor of ferroptosis and cardiovascular calcification, but whether ferroptosis links vitamin K status to disease progression in AS remains unknown. Methods and ResultsWe investigated the role of lipid peroxidation and ferroptosis in AS and their modulation by vitamin K2 using a translational approach. In human stenotic aortic valves, lipid peroxidation was markedly increased and localized to calcified regions, consistent with a ferroptosis-associated microenvironment. In primary human valvular interstitial cells (VICs), pro-calcific conditions induced lipid peroxidation and a pro-ferroptotic state. Pharmacological induction of ferroptosis enhanced VIC calcification, whereas its inhibition attenuated mineralization, supporting a causal role in osteogenic remodeling. Impaired vitamin K status was associated with increased valvular lipid peroxidation in AS patients. Conversely, vitamin K2 attenuated lipid peroxidation, preserved cell viability under ferroptotic stress, and partially normalized pro-ferroptotic and inflammatory transcriptional programs in VICs. In a murine model of AS, dietary vitamin K2 supplementation attenuated disease progression, reduced transvalvular gradients, and decreased valvular inflammation and lipid peroxidation-associated pathways. Finally, in a prospective cohort of patients with aortic sclerosis to moderate AS (n = 157), circulating undercarboxylated osteocalcin, a marker of impaired vitamin K status, was independently associated with accelerated disease progression. ConclusionsVitamin K2 counteracts ferroptosis-associated lipid peroxidation in AS and attenuates disease severity in vivo. Impaired vitamin K status is independently associated with accelerated progression in patients. These findings position vitamin K2 as a potential disease-modifying strategy and vitamin K status as a prognostic marker in AS. What Is New?O_LIFerroptotic lipid peroxidation is enriched in calcified regions of human stenotic aortic valves. Pharmacological induction of ferroptosis increases, and its inhibition reduces, calcification of human valvular interstitial cells, indicating a causal contribution to valvular mineralization. C_LIO_LIVitamin K2 suppresses ferroptotic lipid peroxidation, preserves cell viability under ferroptotic stress, and shifts pro-oxidative and pro-inflammatory transcriptional programs in human valvular interstitial cells toward a protective state. C_LIO_LIIn an in vivo model of aortic stenosis, dietary vitamin K2 attenuated hemodynamic progression, with lower peak transvalvular velocity and mean gradient, and reduced valvular inflammation and lipid peroxidation, without affecting coagulation C_LIO_LIIn a prospective cohort of 157 patients with aortic sclerosis to moderate stenosis, impaired vitamin K status, reflected by higher circulating undercarboxylated osteocalcin, was independently associated with accelerated disease progression and with higher rates of mortality. C_LI What Are the Clinical Implications?O_LIAortic stenosis currently has no medical therapy. Vitamin K2, an inexpensive, safe nutrient that does not interfere with anticoagulation, emerges as a candidate disease-modifying strategy that warrants testing in randomized trials. C_LIO_LICirculating undercarboxylated osteocalcin may serve as a biomarker to identify patients at risk of rapid progression and to enrich future vitamin K trials for those most likely to benefit. C_LIO_LITargeting valvular lipid peroxidation, through antioxidant repletion and/or inhibition of lipid-peroxidation enzymes, may be a mechanistically grounded approach to slow calcific aortic valve disease. C_LI

physiology↗

bioRxiv2026

Endothelial YAP/TAZ rewiring under cardiometabolic stress drives sex-divergent vascular remodeling in heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is widely linked to endothelial dysfunction, yet the molecular pathways translating cardiometabolic stress into microvascular remodeling remain poorly defined. Here, we identify endothelial YAP/TAZ signaling as a mechanistic regulator of sex-divergent vascular responses in HFpEF. Plasma proteomics from the UK Biobank revealed elevated circulating YAP1 levels associated with heart failure and increased mortality, particularly in male patients, where YAP1 coincided with increased levels of the endothelial activation marker ESM1. In a hypertensive cardiorenal mouse model, endothelial YAP/TAZ deletion preserved cardiac function, whereas endothelial TAZ gain-of-function aggravated disease. Under cardiometabolic stress (TNF and high glucose), endothelial cells exhibited sex-specific rewiring of YAP/TAZ-dependent transcriptional programs. Male endothelial cells showed increased extracellular YAP1 release, angiogenic instability with impaired extracellular matrix remodeling, whereas female cells adopted an immune-primed, stress-adaptive phenotype. Mechanistically, cardiometabolic stress uncoupled canonical YAP-TEAD transcription and engaged alternative cofactors, including VGLL3 and VGLL4, thereby reshaping the endothelial secretome and propagating sex-divergent microvascular remodeling. These findings identify endothelial YAP/TAZ rewiring as a molecular switch that converts cardiometabolic stress into sex-divergent microvascular remodeling in HFpEF and connect this process to circulating YAP1 and ESM1 in patients.

cell biology↗

bioRxiv2026

Kidney disease reprograms microbiome-host signaling to promote heart failure

BackgroundHeart failure is prevalent in chronic kidney disease (CKD) and linked to chronic inflammation. CKD-typical gut microbiome dysbiosis may stimulate inflammation, as bacterial aromatic metabolites are highly abundant and engage transcriptional programs through the aryl hydrocarbon receptor (AhR). Whether this axis drives cardiac remodeling and is therapeutically targetable remains unknown. MethodsWe used the subtotal nephrectomy model (STNx) and microbiome depletion by oral antibiotics. We investigated cardiac and renal function, AhR activity, metabolite profiles, and immunophenotypes by flow cytometry and transcriptomics. Candidate metabolite indoxyl sulfate (IxS) was tested in experimental HFpEF. In vivo and in vitro AhR inhibition (AhRi) was performed using a clinically tested compound. Mechanistic studies were performed in primary human and murine cardiac fibroblasts and T cells, as well as translational validation using UK Biobank data. ResultsMicrobiome depletion lowered bacterial metabolites and attenuated cardiac fibrosis and diastolic dysfunction in STNx, identifying AhR-driven expansion of interleukin-17A (IL-17A)-producing T helper cells (TH17) as key effector. Plasma IL-17A was stage-dependently elevated in CKD patients, particularly in HFpEF, and associated with all-cause mortality. Bacterial metabolite IxS promoted TH17 polarization and exacerbated cardiac dysfunction in HFpEF. AhRi using a small molecule inhibitor reduced TH17 abundance and attenuated cardiac fibrosis in STNx. Mechanistically, AhR and IL-17A signaling synergistically induced a conserved pro-fibrotic phenotype in human and murine cardiac fibroblasts, and AhR inhibition blocked ECM production in response to CKD patient serum. ConclusionA microbiome-AhR-IL-17A axis drives CKD-associated cardiac fibrosis. AhRi prevents remodeling, highlighting a potential therapeutic avenue to prevent cardiorenal multimorbidity.

immunology↗

bioRxiv2025

Butyrate Rescues Cardiac Metabolic Dysfunction in Hypertensive Heart Failure with Preserved Ejection Fraction

Diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF) is characterized by metabolic inflexibility. Unlike systolic heart failure, where ketone bodies support energy homeostasis, the failing heart in HFpEF lacks well-characterized alternative fuels to meet its high ATP demand. Here, we show that butyrate, a microbiota-derived short-chain fatty acid, serves as an ancillary energy source and improves diastolic function. Although cardiac power was preserved in rats with HFpEF, both experimental and human HFpEF hearts exhibited an impaired expression of proteins in mitochondrial electron transport chain and oxidative phosphorylation. Additionally, accumulation of 3-hydroxy-butyrate (BOH) in rat and also human HFpEF indicated that ketones do not rescue the cardiac energetic deficit. In HFpEF patients from the UK Biobank, higher BOH levels were associated with increased mortality, particularly those with hypertension. Applying 13C-butyrate to isolated perfused hearts with and without HFpEF resulted in isotope incorporation in butyryl-CoA and downstream TCA intermediates and thus proving its active metabolization. Butyrate was efficiently oxidized by cardiomyocytes and was overtaking BOH and amino acids in supporting respiration. Finaly, chronic butyrate supplementation improved survival, enhanced diastolic function, and reduced fibrosis and inflammation in HFpEF rats despite persistent hypertension. These findings identify butyrate as a compensatory fuel and a promising therapeutic candidate in energetically compromised HFpEF.

biochemistry↗

bioRxiv2025

Blinatumomab-driven T-cell activation in αβ and γδ T-cell subsets: Insights from in vitro assays

Blinatumomab (BLN) is a bispecific T-cell engager that has revolutionized the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), significantly improving outcomes in both adults and children. By simultaneously binding to CD19 on B cells and CD3 on T cells, BLN triggers target cell-dependent T-cell activation, resulting in the cytolysis of CD19+ BCP-ALL cells. Despite the remarkable clinical advancements achieved with BLN, the immunological mechanisms underlying treatment response or failure remain poorly characterized. {gamma}{delta} T cells are attractive candidates for adoptive T-cell therapy due to potent cytotoxicity, capacity to present antigens, broad lysis of different tumor entities, and low alloreactivity. Because {gamma}{delta} T cells can also be redirected by BLN, we systematically studied BLN-driven effector functions of conventional {beta} and unconventional {gamma}{delta} T cells. We evaluated cytotoxicity and cytokine/effector release in freshly isolated and in vitro-expanded {beta} and {gamma}{delta} T cells from healthy adults against CD19 BCP-ALL lines (NALM-6, HAL-01), and profiled dynamic phenotypic alterations by multiparametric flow cytometry. CD19 targets were consistently reduced in the presence of BLN. Freshly isolated {beta}, especially CD8, displayed superior BLN-mediated cytotoxicity as compared to {gamma}{delta} T cells, with donor-dependent variability in {gamma}{delta} killing. Notably, zoledronate-expanded V{gamma}9V{delta}2 {gamma}{delta} T-cell lines achieved cytotoxicity comparable to PHA-expanded {beta} cells. However, {gamma}{delta} T-cell-killing benefited from higher BLN concentration when challenged with high tumor load. BLN induced CD3 down-modulation in {beta} T cells but not in {gamma}{delta} T cells, alongside higher soluble Fas ligand in {beta} cultures, consistent with stronger early activation, preceding activation-induced cell death. {gamma}{delta} T cells showed no such changes, suggesting reduced susceptibility to activation-induced cell death. Single-cell RNA and flow analyses corroborated these findings, showing robust activation/exhaustion programs in {beta} T cells and a stable effector-memory state with low checkpoint expression in {gamma}{delta} T cells. Together, these data reveal subset-specific BLN responses and support expanded V{gamma}9V{delta}2 {gamma}{delta} T cells as a rational adoptive partner to BLN -- particularly in settings of favorable antigen density/low tumor burden -- providing complementary cytotoxicity with potentially reduced inflammatory liability. These findings provide a framework for combining {gamma}{delta} T-cell-based therapies in BLN-treated patients for improving BLN efficacy in BCP-ALL patients.

immunology↗

bioRxiv2025

Large extracellular vesicles derived from red blood cells in coronary artery disease patients with anemia promote endothelial dysfunction

Background and purposeEndothelial dysfunction (ED) is a hallmark of cardiovascular disease (CVD). We recently showed that anemia is associated with worsening of endothelial function after acute myocardial infarction (AMI). Extracellular vesicles (EVs) are efficient communicators between cells and can functionally contribute to different CVD, including, AMI. However, their specific role of EVs in stable coronary artery disease (CAD)-associated with anemia, particularly their contribution to ED, has not yet been investigated systematically. Experimental approachRed blood cell-derived EVs (REVs) and plasma-derived EVs (PLEVs) from all blood cells and endothelium were isolated from patients with stable CAD. The isolated large REVs and PLEVs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting. Uptake assays were performed by co-incubating with fluorescently-labeled REVs and PLEVs with human umbilical vein endothelial cells (ECs). Nitric oxide (NO) consumption ability of REVs was analyzed using a chemiluminescence detector (CLD). After co-incubation of aortic rings explanted from wild-type (WT) mice with REVs and PLEVs from anemic and non-anemic CAD patients, endothelial function was assessed using a wire myograph system. To investigate differences in the content of REVs and PLEVs between anemic and non-anemic CAD patients, proteomic analysis was performed. Key resultsDLS analysis showed that both REVs and PLEVs were within the size distribution range of 100-1000 nm. NTA analysis revealed increased release of REVs in anemic patients compared to non-anemic patients. Co-incubation of labeled REVs and PLEVs with ECs demonstrated their uptake by ECs in vitro which was similar between anemic patients compared to non-anemic patients. REVs from anemic patients showed increased NO consumption compared to those from non-anemic patients. Aortic rings co-incubated with REVs from anemic patients showed attenuated endothelial NO-dependent relaxation responses compared to non-anemic patients. Proteomics analysis of REVs from anemic patients revealed numerous differentially expressed proteins, including decreased abundance of antioxidant proteins such as catalase 1 (CAT1), superoxide dismutase 1 (SOD1) and increased oxidative stress-promoting myeloperoxidase (MPO). Co-incubation of ECs with REVs from anemic patients demonstrated increased ROS production. ConclusionAnemia is associated with increased release of REVs and enhanced NO consumption, which promotes ED. This is further exacerbated by an altered redox balance and increased ROS production, implicating therapeutic importance in anemic patients with CAD. Graphical AbstractAnemia is associated with an increased release of RBC-derived large extracellular vesicles (REVs), which are taken up by endothelial cells (ECs). Anemic REVs show enhanced nitric oxide (NO) consumption, contributing to NO dysregulation in ECs. Additionally, REVs carry various redox enzymes, including the oxidative stress-promoting enzyme myeloperoxidase (MPO), as well as antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). An imbalance in these redox enzymes leads to increased oxidative stress and endothelial nitric oxide synthase (eNOS) uncoupling, resulting in impaired NO-mediated relaxation responses and subsequent endothelial dysfunction (ED). O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/642191v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19d4cccorg.highwire.dtl.DTLVardef@1d1b3forg.highwire.dtl.DTLVardef@e7f144org.highwire.dtl.DTLVardef@190b007_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Distinct effects of acute and chronic blood loss anemia on vascular function after acute myocardial infarction

BackgroundAnemia is frequently observed in patients with cardiovascular diseases (CVD). Anemia alone or in combination with other morbid conditions leads to poor prognosis in acute myocardial infarction (AMI). We recently showed that moderate blood loss anemia is associated with red blood cell (RBC) dysfunction and a compensatory increase in flow-mediated dilation (FMD) responses which are compromised in chronic blood loss anemia However, the effects of acute anemia (AA) and chronic anemia (CA) on endothelial function after AMI are unclear. In this study, we evaluated systemic endothelial function following AMI in established murine models of blood loss acute and chronic anemia. We hypothesize that both AA and CA aggravate systemic endothelial dysfunction (ED) after AMI. Methods and resultsAA or CA was induced in male C57BL/6J mice by repeated blood withdrawal for three consecutive days or six weeks, respectively. Separate groups of anemic and non-anemic mice underwent AMI via left anterior descending artery (LAD) ligation (45 min), followed by reperfusion. Endothelial function was assessed using both in vivo and in vitro methods 24 h post-AMI. Impaired flow-mediated dilation (FMD, in vivo) and endothelium-dependent relaxation (EDR) responses were observed in the aorta, femoral, and saphenous arteries of AA mice compared to their respective control groups 24 h post AMI. The aorta and saphenous arteries from CA mice showed significantly reduced vascular smooth muscle (VSM) contractile responses after AMI. Analysis of oxidative products of nitric oxide (NO) in plasma revealed reduced nitrite and nitrate levels in both AA and CA mice compared to controls 24 h post-AMI. Immunohistochemistry of aortic tissues from both anemic groups showed increased reactive oxygen species (ROS) product 4-Hydroxynonenal (4-HNE). Co-incubation of RBCs from anemic mice or anemic ST-elevation myocardial infarction (STEMI) patients with aortic rings from wild type mice demonstrated attenuated VSM contractile and EDR responses. Supplementation with the ROS scavenger N-acetyl cysteine (NAC) for four weeks improved both in vivo and ex vivo EDR in AA and CA mice 24 h post-AMI. ConclusionAfter AMI, both AA and CA are associated with severe ED, while VSM contractile responses specifically reduced in CA mice. These effects are accompanied by increased ROS and partly mediated by RBCs. Antioxidant supplementation with NAC is a potential therapeutic option to reverse the severe vascular dysfunction in anemia following AMI. Graphical AbstractDistinct effects of acute and chronic anemia on vascular function 24 h post-AMI. After acute myocardial infarction, acute and chronic anemia are associated with increased reactive oxygen species (ROS) and inflammation in endothelial cells (EC), leading to the inhibition of endothelial nitric oxide synthase (eNOS) and subsequent endothelial dysfunction by limiting NO bioavailability. Chronic anemia is additionally associated with decreased vascular smooth muscle cell (VSMC) function due to increased oxidative stress, leading to SMC dysfunction. After N-Acetyl-L-Cysteine (NAC) treatment, vascular function is improved in both anemic groups. O_FIG O_LINKSMALLFIG WIDTH=98 HEIGHT=200 SRC="FIGDIR/small/614629v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@136583aorg.highwire.dtl.DTLVardef@da8708org.highwire.dtl.DTLVardef@d73539org.highwire.dtl.DTLVardef@e7516a_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Investigation of hemodynamic bulk flow patterns caused by aortic stenosis using a combined 4D Flow MRI-CFD framework

Aortic stenosis (AS) leads to alterations of supra-valvular flow patterns. These patterns might lead to, inter alia, increased damage of red blood cell (RBC) membranes. We investigated these patient specific patterns of a severe AS patient and their reversal in healthy flow through a 4D Flow MRI-based CFD methodology. Computational models of subject-specific aortic geometries were created using in-vivo medical imaging data. Temporally and spatially resolved boundary conditions derived from 4D Flow MRI were implemented for an AS patient and a healthy subject. After validation of the in-silico results with in-vivo data, a healthy inflow profile was set for the AS patient in the CFD model. Pathological versus healthy flow fields were compared regarding their blood flow characteristics, i.e. shear stresses on RBCs and helicity. The accuracy of the 4D Flow MRI-based CFD model was proven with excellent agreement between in-vivo and in-silico velocity fields and R{superscript 2} = 0.9. A pathological high shear stress region in the bulk flow was present during late systole with an increase of 125 % compared to both healthy flow. The physiological bihelical structure with predominantly right-handed helices vanished for the pathological state. Instead, a left-handed helix appeared, accompanied by an overall increase in turbulent kinetic energy in areas of accumulated left-handed helicity. The validated 4D Flow MRI-based CFD model identified marked differences between AS and healthy flow. It suggests that altered turbulent and helical structures in the bulk flow are the cause for increased, potentially damaging forces acting upon RBCs in AS. Author SummaryAortic stenosis (AS) is a condition that alters the flow of blood through the aorta, potentially increasing damage to red blood cells (RBCs). It is therefore crucial to understand the precise nature of these changes, especially in the bulk region where the RBCs are mainly circulating. To investigate this, we developed a computational model based on patient-specific 4D Flow MRI data to simulate blood flow in a patient with severe AS and a healthy individual. Our high-fidelity models show high agreement with real-world data. We then imposed synthetically generated healthy flow conditions in the AS patient to eliminate age- and geometry-related uncertainties. Our results revealed significantly higher shear stresses and changes in helical flow patterns in the AS patient compared to healthy flow, suggesting that these alterations could increase the risk of RBC membrane damage. These findings underscore the importance of deepening our understanding about the interplay between bulk flow alterations and their impact on RBCs, as previous studies were mostly focused on near wall regions. This methodology could also be extended to other cardiovascular conditions, aiding in the development of patient-specific treatment strategies.

bioengineering↗

bioRxiv2024

Novel fluorescence-based methods to determine infarct and scar size in murine models of reperfused myocardial infarction

BackgroundDetermination of infarct area and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in mice and other animal models. MethodsFor both, early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male C57BL/6J mice and hearts were extracted 24 hours, 7-, 21-, or 28-days following MI and stained with 2,3,5-Triphenyltetrazolium chloride (TTC) and Evans Blue, Hoechst, phalloidin, Sirius Red, Massons or Gomoris Trichrome or Wheat Germ Agglutinin (WGA). ResultsFluorescent staining combining Hoechst and phalloidin constitutes an alternative for TTC and Evans Blue, enabling a clear visualization of infarct area, area at risk, as well as remote area unaffected by MI. Infarct size early after reperfusion determined with TTC staining correlates strongly with that demarcated by phalloidin while combination of Hoechst and phalloidin staining can emulate classical TTC/Evans Blue staining 24 h post-MI. Moreover, WGA is equally accurate as the classical Sirius Red, Massons and Gomoris Trichrome stainings in identifying scar size in later phases (>7d) post-MI. Finally, we demonstrate feasibility of combining conventional fluorescence staining by localizing CD45+ leukocytes to specific regions of the infarcted myocardium. ConclusionWe established staining procedure is not inferior to classical TTC staining while providing substantial benefits including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/604384v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@17ccc83org.highwire.dtl.DTLVardef@56d433org.highwire.dtl.DTLVardef@857f37org.highwire.dtl.DTLVardef@63297d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

High-resolution respirometry reveals altered mammalian tissue ketone body oxidation in different cardiometabolic diseases

Background and aimsReduced mitochondrial function has been implicated in metabolic disorders like type 2 diabetes (T2D), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD), which are tightly linked to insulin resistance and impaired metabolic flexibility. However, the contribution of the ketone bodies (KBs) {beta}-hydroxybutyrate (HBA) and acetoacetate (ACA) as substrates for mitochondrial oxidative phosphorylation (OXPHOS) in these insulin resistant states remains unclear. MethodsTargeted high-resolution respirometry protocols were applied to detect the differential contribution of HBA and ACA to OXPHOS capacity in heart, skeletal muscle, kidney, and liver of distinct human and mouse cohorts with T2D, obesity, and MASLD. ResultsIn humans with T2D, KB-driven mitochondrial OXPHOS capacity was [~]30% lower in the heart (p<0.05) and skeletal muscle (p<0.05) compared to non-diabetic controls. The relative contribution of KB to maximal OXPHOS capacity in T2D was also lower in both the heart ([~]25%, p<0.05) and skeletal muscle ([~]50%, p<0.05). Similarly, in kidney cortex from high-fat diet-induced obese mice, both the absolute and relative contribution of KB to OXPHOS capacity was [~]15% lower (p<0.05). Finally, hepatic HBA-driven mitochondrial OXPHOS capacity was 29% lower (p<0.05) in obese humans with MASLD compared to humans without MASLD. ConclusionsMitochondrial KB-driven OXPHOS capacity is impaired in insulin resistant states in various organs in absolute and relative terms, likely reflecting impaired mitochondrial metabolic flexibility. Our data suggest that KB respirometry can provide a sensitive readout of impaired mitochondrial function in diabetes, obesity, and MASLD.

cell biology↗

bioRxiv2024

Platelets induce cell apoptosis of cardiac cells via FasL after acute myocardial infarction

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players of hemostasis and play a crucial role in vessel occlusion, inflammation and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3 positive resident cardiac cells while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction of caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR suggesting that platelet induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and BcL-2 expression suggesting that platelets modulate the intrinsic and the extrinsic pathway of apoptosis leading to reduced infarct size after myocardial I/R injury. Therefore, platelet induced cardiac damage needs to be taken into account while optimizing antithrombotic/antiplatelet strategies for patients with AMI.

molecular biology↗

bioRxiv2024

Crosstalk between thrombospondin-1 and CD36 modulates platelet-RBC interaction limiting thrombosis and abdominal aneurysm formation

Red blood cells (RBCs) contribute to hemostasis and thrombosis by interaction with platelets via the FasL-FasR pathway to induce procoagulant activity and thrombin formation. Here, we identified a novel mechanism of platelet-RBC interaction via the CD36-thrombospondin-1 (TSP-1) signaling pathway, which is important in thrombus formation and the recruitment of RBCs to collagen-adherent platelets. Platelet-released TSP-1 can bind to CD36 at the RBC membrane to enhance procoagulant activity and to increase the activation of integrin IIb{beta}3, which represents an additional ligand for erythroid FasR, suggesting that both mechanisms of platelet-RBC interaction act in concert to propagate thrombus formation. In patients with abdominal aortic aneurysm (AAA), enhanced procoagulant activity of RBCs and platelets is accompanied by elevated exposure of TSP-1 and FasL at the platelet surface and accumulation of TSP-1 in the aortic wall and the intraluminal thrombus, suggesting that platelet-RBC interaction plays an important role in AAA pathology. TSP-1-deficient mice are protected against aortic diameter expansion in an experimental model of AAA, highlighting the crucial role of the CD36-TSP-1 axis in AAA. Thus, interfering with platelet-RBC interaction may be a promising therapeutic approach to reduce pro-coagulant activity and preserve AAA patients from surgery or rupture.

pathology↗

bioRxiv2023

Inhibition of proline-rich-tyrosine kinase 2 restores cardioprotection by remote ischemic preconditioning in type 2 diabetes mellitus

BackgroundEndothelial function and cardioprotection through remote ischemic preconditioning (rIPC) are severely impaired in type 2 diabetes mellitus (T2DM). Proline-rich tyrosine kinase 2 (Pyk2), a downstream target of the insulin receptor, reduces endothelial nitric oxide synthase (eNOS) activity. Therapeutic options to rescue cardioprotection in T2DM and improve outcomes after acute myocardial infarction (AMI) are lacking. We hypothesized that vascular endothelium contributes to rIPC, and that inhibition of Pyk2 restores cardioprotection in T2DM through modulation of eNOS, thus limiting infarct size. MethodsNew Zealand Obese (NZO) mice were used as a polygenic model of T2DM. Effects of Pyk2-inhibition on endothelial function, remote ischemic preconditioning (rIPC), and infarct size (IS) after ischemia/reperfusion (I/R) were compared in NZO, eNOS KO, and C57Bl/6 (Bl6) mice. Plasma derived from mice and individuals with or without T2DM at baseline and after rIPC was transferred to isolated hearts and aortic rings to assess the effects of Pyk2-inhibition on remote tissue protection. ResultsTransfer experiments with plasma drawn from non-diabetic humans and mice exposed to rIPC demonstrate that endothelium-dependent signals for remote tissue protection are conveyed by plasma. Key features reflecting the glucometabolic spectrum in T2DM were detected in NZO mice, including hyperinsulinemia, insulin resistance, obesity, and impaired glucose tolerance. Similar to T2DM patients, these mice also revealed endothelial dysfunction with decreased flow-mediated dilation (FMD), reduced circulating nitrite levels, elevated arterial blood pressure, and larger infarct size after I/R. Pyk2 increased the phosphorylation of eNOS on its inhibitory site (Tyr656). Cardioprotective effects by rIPC were lost in NZO mice. Inhibition of Pyk2 restored endothelial function and rescued endothelium-dependent cardioprotection after rIPC displayed by lower IS and improved LV function post I/R. ConclusionEndothelial function contributing to remote tissue protection is severely impaired in diabetes mellitus. Proline-rich tyrosine kinase 2 is a novel target to rescue cardioprotection through endothelium-dependent remote ischemic preconditioning, advocating its role in limiting infarct size in diabetes mellitus. Clinical perspective What is new?O_LIVascular endothelium contributes to remote tissue protection in ischemic preconditioning, which is severely impaired in diabetes C_LIO_LIProline-rich tyrosine kinase 2 reduces eNOS-activity, causes endothelial dysfunction, and impairs cardioprotection through ischemic preconditioning C_LIO_LIInhibition of proline-rich tyrosine kinase 2 restores eNOS activity, endothelial function, and cardioprotective effects of remote ischemic preconditioning limiting infarct size in an experimental model of diabetes. C_LI What are the clinical implications?O_LIProper endothelial function is cirtical to maintain cardiovascular health. Endothelial dysfunction contributes to impaired remote tissue protection in diabetes. C_LIO_LIThese data demonstrate for the first time that endothelium-dependent cardioprotection in myocardial ischemia/reperfusion through remote ischemic preconditioning can be restored in diabetes. C_LIO_LIProline-rich tyrosine kinase 2 is a novel target to restore endothelium-dependent remote cardioprotection to improve the outcome of diabetic patients with acute myocardial infarction. C_LI

physiology↗

bioRxiv2023

Inhibition of CD40-TRAF6 signaling protects against aneurysm development and progression

ObjectiveInflammation is a critical process during the progressive development and complication of abdominal aortic aneurysm. The co-stimulatory dyad CD40-CD40L is a major driver of inflammation and modulates immune responses. This study evaluates the potential of a small molecule inhibitor, which blocks the interaction between CD40 and tumor necrosis factor (TNF) receptor-associated factor (TRAF)-6, referred to as TRAF-STOP, in the early and later phase during AAA progression. Methods and resultsAAAs were induced in C57BL/6J mice by infrarenal aortic porcine pancreatic elastase infusion for 7, 14 or 28 days. Inhibition of CD40 signaling by TRAF-STOP resulted in less severe AAA formation and reduced the incidence of AAA development. TRAF-STOP treatment attenuated aortic structural remodeling, characterized by a reduced elastic fiber degradation, lowered expression of matrix metalloproteinase (MMP)-2 and MMP9, as well as preserved collagen type IV content in aneurysmal tissue. Furthermore, this is accompanied by the reduction of key pro-inflammatory genes such as TNF. ConclusionPharmacological inhibition of CD40-TRAF6 signaling protects from adverse aortic structural remodeling during the early phase of AAA progression representing a translational strategy to limit progression of human AAA disease.

pharmacology and toxicology↗

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

Quantitative assessment of angioplasty induced vascular inflammation with 19F cardiovascular magnetic resonance imaging

Early macrophage rich vascular inflammation is a key feature in the pathophysiology of restenosis after angioplasty. 19F MRI with intravenously applied perfluorooctyl bromide-nanoemulsion (PFOB-NE) could offer ideal features for serial imaging of the inflammatory response after angioplasty. We aimed to non-invasively image monocyte/macrophage infiltration in response to angioplasty in pig carotid arteries using Fluorine-19 magnetic resonance imaging (19F MRI) to assess early inflammatory response to mechanical injury. Early macrophage rich vascular inflammation is a key feature in the pathophysiology of restenosis after angioplasty. 19F MRI with intravenously applied perfluorooctyl bromide-nanoemulsion (PFOB-NE) could offer ideal features for serial imaging of the inflammatory response after angioplasty. In eight minipigs, injury of the right carotid artery was induced by either balloon oversize angioplasty only (BA, n=4) or in combination with endothelial denudation (BA + ECDN, n=4). PFOB-NE was administered intravenously three days after injury followed by 1H and 19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using immunohistology. Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and adventitial infiltration of monocytes/macrophages. 19F signal could be detected in vivo in four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 {+/-} 4.8. Ex vivo analysis revealed a linear correlation of 19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was as about 400 cells/mm2. Therefore, 19F MRI enables quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This might open an avenue to non-invasively monitor inflammatory response to mechanical injury after angioplasty and thus to identify individuals with distinct patterns of vascular inflammation promoting restenosis. One Sentence Summary19F MRI enables radiation-free quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity.

immunology↗

bioRxiv2022

Platelet pannexin-1 channels modulate inflammation during abdominal aortic aneurysm formation

Abdominal aortic aneurysm (AAA) is a common disease and highly lethal if untreated. The progressive dilatation of the abdominal aorta is accompanied by degradation and remodeling of the vessel wall due to chronic inflammation. Pannexins represent anion-selective channels and play a crucial role in non-vesicular ATP release to amplify paracrine signaling in cells. Thus, pannexins are involved in many (patho-) physiological processes. Recently, Panx1 channels were identified to be significantly involved in AAA formation through endothelial derived Panx1 regulated inflammation and aortic remodeling. In platelets, Panx1 becomes activated following activation of glycoprotein (GP)VI. Since platelets play a role in cardiovascular diseases including AAA, we analyzed the contribution of platelet Panx1 in the progression of AAA. We detected enhanced Panx1 plasma levels in AAA patients. In experimental AAA using the pancreatic porcine elastase (PPE) mouse model, a major contribution of platelet Panx1 channels in platelet activation, pro-coagulant activity of platelets and platelet-mediated inflammation has been detected. In detail, platelets are important for the migration of neutrophils into the aortic wall induced by direct cell interaction and by activation of endothelial cells. Decreased platelet activation and inflammation did not affect ECM remodeling or wall thickness in platelet-specific Panx1 knock-out mice following PPE surgery. Thus, aortic diameter expansion at different time points after elastase infusion of the aortic wall was unaltered in platelet-specific Panx1 deficient mice suggesting that the modulation of inflammation alone does not affect AAA formation and progression. In conclusion, our data strongly supports the role of platelets in inflammatory responses in AAA via Panx1 channels and adds important knowledge about the significance of platelets in AAA pathology important for the establishment of an anti-platelet therapy for AAA patients.

cell biology↗

bioRxiv2022

Ageing impairs the neuro-vascular interface in the heart

Aging is a major risk factor for impaired cardiovascular health. The aging myocardium is characterized by electrophysiological dysfunctions such as a reduced heart rate variability. These alterations can be intrinsic within cardiomyocytes, but might be modulated by the cardiac autonomic nervous system, as well1. It is known that nerves align with vessels during development2, but the impact of aging on the cardiac neuro-vascular interface is unknown. Here, we report that aging reduces nerve density specifically in the left ventricle and dysregulates vascular-derived neuro-regulatory genes. Aging leads further to a down-regulation of miR-145 and de-repression of the neuro-repulsive factor Semaphorin-3A. miR-145 deletion increased Sema3a expression and reduced axon density, thus mimicking the observed aged heart phenotype. Removal of senescent cells, which accumulated with chronological age while nerve density declined, rescued from age-induced dennervation, reduced Sema3a expression and preserved heart rate variability. These data suggest that senescence-associated regulation of neuro-regulatory genes contributes to a declined nerve density of the aging heart and thereby to a reduced heart rate variability.

physiology↗