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Sex differences in Vascular Aging and Atherosclerosis (ID 1413)
Clonal hematopeisis as an age-related risk for atherosclerosis (ID 1412)
O072 - Clonal Hematopoiesis and Trained Immunity in Individuals with Obesity (ID 1366)
Abstract
Background and Aims
Obesity is a risk factor for atherosclerotic cardiovascular disease (CVD) and is associated with low-grade inflammation. Recently, clonal hematopoiesis emerged as a novel risk factor for CVD. Innate immune memory (trained immunity) also contributes to atherosclerosis. We hypothesize that clonal hematopoiesis is a driver of inflammation and atherosclerosis in patients with obesity and that the presence of clonal hematopoiesis predisposes to trained immunity.Methods
In a cross-sectional study, we recruited 302 individuals with obesity (BMI>27) and median age 67. We performed extensive clinical and immunological profiling of these subjects, and identified clonal hematopoiesis driver mutations (CHDMs). We measured circulating inflammatory markers and leukocyte phenotype and cytokine production capacity upon ex vivo stimulation. Approximately 5 years after the initial data point we performed a follow-up study on a subgroup of patients with CHDM in DNMT3A (n=15), in TET2 (n=6), or without CDHDM (n=15) and performed trained immunity assays and single-cell RNA sequencing(scRNAseq).Results
We identified 110 CHDMs in 85 patients. CHDM did not increase the presence of atherosclerotic plaques. PBMCs from subjects with CHDMs had lower cytokine production capacity upon ex vivo stimulation. Training capacity of monocytes did not differ among individuals with or without CHDMs. However, macrophages from individuals with TET2 mutations showed a higher cytokine production capacity at baseline. scRNAseq analyses are currently being performed.Conclusions
CHDMs do not stimulate the presence of carotid plaques in obese individuals. CHDMs have distinctive effects on monocyte and macrophage function and does not affect the potential for trained immunity. We will investigate the differential transcriptomic profile of these samples with or without LPS stimulation.O073 - SINGLE-CELL RNA SEQUENCING IDENTIFIES MFSD2B IN MEGAKARYOCYTE PROGENITORS AS A REGULATOR OF VASCULAR SENESCENCE (ID 941)
Abstract
Background and Aims
Accumulation of senescent endothelial cells (ECs) advances with age and contributes to the development of atherosclerosis. Megakaryocyte progenitors (MkPs) orchestrate platelet function, which contributes to atherosclerosis. However, diversification and function of MkPs, which regulate EC senescence upon aging have not been explored.
Methods
Here, we defined the role and trajectory of MkP subpopulations in bone-marrow from old (>24-moths) and young (3-months) wild-type mice, using single-cell RNA-sequencing. We further identified Mfsd2b in rare MkP subpopulations as a functional regulator of S1P export, which thereby regulates a potential link between S1P homeostasis and EC senescence upon aging.
Results
Single-cell transcriptomic profiles of mouse MkPs delineate two distinct clusters of Mfsd2b-expressing cells enriched in bone-marrow from old mice. Our data show that Mfsd2b upregulation leads to higher circulating S1P-18:0 (measured by LC-MS/MS), which is positively correlated with vascular dysfunction in old mice. In line with this, we observed an increase in senescence markers SA-β-galactosidase, γ-H2A.X (telomere shortening), and p19 and p21 (cell-cycle arrest), accompanied by increased pro-inflammatory cytokines IL1β and IL6, as markers of SASP, in young aortic endothelial cells co-cultured with old MkPs. Employing RNAi approach, Mfsd2b-knockout in old MkPs rescues EC senescence by reducing numbers of SA-β-galactosidase-positive cells and downregulating cell-cycle arrest and SASP markers, leading to increases in EC migration and tube formation.
Conclusions
Our findings gain a deeper understanding of both the heterogeneity of bone-marrow MkPs upon aging and a complex crosstalk of MkP-Mfsd2b to endothelial senescence. We suggest Mfsd2b in specific MkP subpopulations as a potential target for personalized vascular regeneration.
O074 - HUMAN AND MURINE SINGLE-CELL RNA-SEQUENCING REVEAL FIBROBLAST HETEROGENEITY IN HEALTHY AND DISEASED VASCULATURE AND DIFFERENTIAL REGULATION BY AGEING AND SERUM CHOLESTEROL (ID 745)
Abstract
Background and Aims
As current markers are unable to distinguish adventitial fibroblasts from other vascular cells, we investigated their transcriptome to reveal cell type markers, heterogeneity, and regulation by cardiovascular disease (CVD) risk factors.
Methods
Single-cell RNA sequencing analysis of ~3700 CD45-/ICAM2-/PDGFRβ+ fibroblasts from healthy murine adventitia was followed by validation and regulatory studies in healthy and atherosclerotic tissue of mice and humans.
Results
Immunohistochemistry and flow cytometry validated platelet-derived growth factor receptor α and dipeptidase 1 as fibroblast markers across human and murine arteries. Pseudotime analysis predicted three fibroblast differentiation trajectories, validated in an independent dataset and on protein level. Gene ontology analysis supported divergent functional profiles related to vascular development, antigen presentation or collagen fibril organization. Fibroblast trajectories were significantly enriched for genes with known genome-wide associations to CVD. CVD risk factors aging and hypercholesterolemia differentially expanded murine trajectory 2 or 3 fibroblasts respectively, coinciding with increased adventitial collagen (1.2-fold, p<0.01). Immunohistochemistry, bulk, and single-cell transcriptomics of human carotid and aorta specimens showed trajectory presence in healthy and atherosclerotic arteries, and differential correlations to atherosclerotic plaque traits.
Conclusions
We provide adventitial fibroblast cell type markers and three transcriptionally divergent fibroblast differentiation trajectories. Regulation by CVD risk factors, presence in human atherosclerosis, and enrichment of genes associated with CVD, implicate their biological relevance in CVD.
O075 - IMPACT OF INNER MITOCHONDRIAL PROTEIN ON DIETARY LIPID METABOLISM AND ATHEROSCLEROSIS PROGRESSION (ID 965)
Abstract
Background and Aims
Mitochondria are central organelles involved in cellular metabolism, energy generation, calcium homeostasis, sterol, and bile acids (BAs) production. Mitochondria continuously undergo biogenesis, fusion, fission, and mitophagy, maintaining a continuous balance between all forms. On these premises, we test the impact of OPA1, an inner mitochondria membrane fusion protein, on mitochondrial tethering on lipid metabolism in the liver and the atherosclerotic plaque.
Methods
OPA1-liver KO (Opa1LKO) on HFD and Opa1LKO, OPA1 He male mice, OPA1 Tg on LDLR KO background were fed with Western-type diet (WTD) respectively for 12 weeks and 20 weeks. Inverse calorimetry, ITT, GTT, and LipidToleranceTest (LTT) were performed. Paraffin-embedded tissues were used for histological analysis. Tissues were used for OMICs analysis.
Results
Opa1LKO mice displayed altered systemic metabolism with reduced body weight and reduced circulating lipid levels. Opa1LKO mice showed altered bile acid production, as confirmed by RNAseq and proteomics data. Opa1LKO mice show an improved glycemic profile and reduced liver steatosis. Opa1 hepatocyte deficiency is associated with primary unconjugated bile acids accumulation with a significative reduction of primary conjugated bile acids and a consequent reduction in lipid uptake from diet. This is strongly affecting circulating cholesterol levels and atherosclerotic plaque development.
Conclusions
Hepatic Opa1 deficiency protects mice from HFD-induced metabolic dysfunction resulting in a reduction of lipid metabolism as a consequence of an alteration in bile acids production. OPA1 hepatic and systemic expression is significantly affecting lipoprotein metabolism and atherosclerotic plaque development.
O076 - REVERSING THE FUNCTIONAL- AND REGENERATIVE CAPACITY OF SENESCENT ENDOTHELIAL CELLS BY PHARMACOLOGICAL REPROGRAMMING (ID 366)
Abstract
Background and Aims
Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases contributing to the development of vascular dysfunction with a reduced angiogenic and regenerative potential. Targeting EC senescence and reversing the senescent phenotype might represent a promising therapeutic strategy to improve vascular function. Here we show a reversal of EC senescence following the application of a pharmacological reprogramming strategy by a timely restricted, non-genetic induction of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM).
Methods
Methods to characterize the effects of pharmacological reprogramming included the quantification of gene expression as well as the functional analysis of EC in vitro. In addition, the regenerative capacity of EC was evaluated in an ischemic hind-limb model in vivo.
Results
Application of a pharmacological cocktail of FDA approved substances to replicative senescent EC led to a timely-restricted and robust overexpression of OSKM associated with significantly enhanced functional properties as proliferation, migration and tube formation of the senescent EC compared to untreated control cells (P<0.05). Further, expression of senescent markers such as p16INK4A or p14ARF and expression of cytokines for example TNFa, Il1b and Il-6 was significantly reduced compared to untreated senescent cells. In vivo, a significantly improved blood flow was observed after hind limb ischemia in 22 months old C57BL/6 mice after 7 and 14 days.
Conclusions
In summary, we demonstrate that a short induction of OSKM via a pharmacological approach holds the potential to reverse senescence in EC in vitro and thus to enhance endothelial regenerative capacity in vivo.