The contribution of individual lipoprotein species to the generation of the adrenal cholesterol pool used for the synthesis of anti-inflammatory glucocorticoid species remains unknown. Here we... Show moreThe contribution of individual lipoprotein species to the generation of the adrenal cholesterol pool used for the synthesis of anti-inflammatory glucocorticoid species remains unknown. Here we examined the impact of specific lowering of very low-density lipoprotein (VLDL) and low-density (LDL) levels on adrenal cholesterol and glucocorticoid homeostasis. Hereto, lethally-irradiated hypercholesterolemic apolipoprotein E (APOE) knockout mice received APOE-containing bone marrow from wild-type mice (n = 6) or APOE knockout control bone marrow (n = 10) and were subsequently fed a regular chow diet. Transplantation with wild-type bone marrow was associated with a 10-fold decrease in VLDL/LDL-cholesterol levels. No changes were observed in adrenal weights, adrenal cholesterol content, or basal plasma corticosterone levels. However, food deprivation-induced corticosterone secretion was 64% lower (P < 0.05) in wild-type bone marrow recipients as compared to APOE knockout bone marrow recipients, in the context of similar plasma adrenocorticotropic hormone (ACTH) levels. A parallel 19-29% decrease in adrenal relative mRNA expression levels of ACTH-responsive genes SR-BI (P < 0.01), STAR (P < 0.05), and CYP11A1 (P < 0.05) was detected. In support of relative glucocorticoid insufficiency, blood lymphocyte and eosinophil concentrations were respectively 2.4-fold (P < 0.01) and 8-fold (P < 0.001) higher in wild-type bone marrow recipients under food deprivation stress conditions. In conclusion, we have shown that a selective lowering of VLDL/LDL levels in APOE knockout mice through a transplantation with APOE-containing wild-type bone marrow is associated with a decreased maximal adrenal glucocorticoid output. Our studies provide experimental support for the hypothesis that, in vivo, VLDL/LDL serves as the primary source of cholesterol used for glucocorticoid synthesis during food deprivation stress. Show less
The immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B and T lymphocyte attenuator (BTLA) is a novel co-receptor that... Show moreThe immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B and T lymphocyte attenuator (BTLA) is a novel co-receptor that negatively regulates the activation of B and T cells, however, there have been no reports of BTLA and its function in atherosclerosis or cardiovascular disease (CVD). We aimed to assess the dominant BTLA expressing leukocyte in CVD patients and to investigate whether BTLA has a functional role in experimental atherosclerosis. Stimulation of the BTLA pathway in Ldlr-/-mice reduces initial lesion development and increases collagen content of established lesions, presumably by shifting the balance between atherogenic follicular B cells and atheroprotective B cells and directing CD4+ T cells towards regulatory T cells. We provide the first evidence that BTLA is a very promising target for the treatment of atherosclerosis.We show that BTLA is primarily expressed on B cells in CVD patients and follicular B2 cells in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We treated Ldlr-/- mice that were fed a Western-type diet (WTD) with PBS, an isotype antibody or an agonistic BTLA antibody (3C10) for 6 weeks. We report here that the agonistic BTLA antibody significantly attenuated atherosclerosis. This was associated with a strong reduction in follicular B2 cells, while regulatory B and T cells were increased. The BTLA antibody showed similar immunomodulating effects in a progression study in which Ldlr-/- mice were fed a WTD for 10 weeks before receiving antibody treatment. Most importantly, BTLA stimulation enhanced collagen content, a feature of stable lesions, in preexisting lesions. Show less
Mokry, M.; Depuydt, M.A.C.; Prange, K.H.M.; Slenders, L.; Elbersen, D.; Granneman, L.E.C.; ... ; Pasterkamp, G. 2019
Atherosclerotic plaque (AP) is a complex pathological formation, containing numerous interacting cell types. Our understanding of the cellular composition and dynamics of AP is mainly based on the... Show moreAtherosclerotic plaque (AP) is a complex pathological formation, containing numerous interacting cell types. Our understanding of the cellular composition and dynamics of AP is mainly based on the evaluation of a limited number of known markers and therefore may be incomplete and biased. Here we employ single cell transcriptomics (scRNAseq) to identify and define cell types in human plaques and to reconstruct their lineage trees and relationships.We have performed scRNAseq on atherosclerotic plaques obtained from 3 (discovery cohort) and 6 (validation cohort) carotid endarterectomy patients. We have employed the scRNAseq based on CEL-seq2/SORT-seq protocol coupled with fluorescence-activated cell sorting. This method allowed us to specifically select viable and nucleated single cells. Finally we have used RaceID and StemID algorithm to identify common and rare cell types and to perform the lineage tracing analysis.We have identified 15 different cell subtypes, among others endothelial cells (EC), macrophages and smooth muscle cells (SMC). Besides these major types we were able to detect less frequent cell populations - including ACKR1+ venular endothelial cells. Notably, we were able to find individual cells and cell clusters co-expressing both EC and SMC genes, such as SPARC, COL6A1, PECAM1 and CD34. These cells exhibited increased median transcriptome entropy and connectivity, relative to other EC and SMC clusters - altogether supporting their progenitor role and suggesting the plasticity of cell identity in AP.ScRNAseq enabled us to identify and define cell subtypes present in human AP. Our data suggest the cellular lineage plasticity in human AP. Show less
The immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B and T lymphocyte attenuator (BTLA) is a novel co-receptor that... Show moreThe immune system is strongly involved in atherosclerosis and immune regulation generally leads to attenuated atherosclerosis. B and T lymphocyte attenuator (BTLA) is a novel co-receptor that negatively regulates the activation of B and T cells, however, there have been no reports of BTLA and its function in atherosclerosis or cardiovascular disease (CVD). We aimed to assess the dominant BTLA expressing leukocyte in CVD patients and to investigate whether BTLA has a functional role in experimental atherosclerosis.\nStimulation of the BTLA pathway in Ldlr-/-mice reduces initial lesion development and increases collagen content of established lesions, presumably by shifting the balance between atherogenic follicular B cells and atheroprotective B cells and directing CD4+ T cells towards regulatory T cells. We provide the first evidence that BTLA is a very promising target for the treatment of atherosclerosis.\nWe show that BTLA is primarily expressed on B cells in CVD patients and follicular B2 cells in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. We treated Ldlr-/- mice that were fed a Western-type diet (WTD) with PBS, an isotype antibody or an agonistic BTLA antibody (3C10) for 6 weeks. We report here that the agonistic BTLA antibody significantly attenuated atherosclerosis. This was associated with a strong reduction in follicular B2 cells, while regulatory B and T cells were increased. The BTLA antibody showed similar immunomodulating effects in a progression study in which Ldlr-/- mice were fed a WTD for 10 weeks before receiving antibody treatment. Most importantly, BTLA stimulation enhanced collagen content, a feature of stable lesions, in preexisting lesions. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.A.C.; Wezel, A.; Smeets, H.; ... ; Slütter, B.A. 2019
CD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs.... Show moreCD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs. Theatherosclerotic lesion, however, is a complex microenvironment containing a plethora of inflammatory signals,which may affect CD8+T-cell activation. Here, we address how this environment affects the functionality ofCD8+T-cells. We compared the cytokine production of CD8+T-cells derived from spleens and en-zymatically digested aortas ofapoE−/−mice with advanced atherosclerosis byflow cytometry. Aortic CD8+T-cells produced decreased amounts of IFN-γand TNF-αcompared to their systemic counterparts. The observeddysfunctional phenotype of the lesion-derived CD8+T-cells was not associated with classical exhaustion mar-kers, but with increased expression of the ectonucleotidase CD39. Indeed, pharmacological inhibition of CD39 inapoE−/−mice partly restored cytokine production by CD8+T-cells. Using a bone-marrow transplantation ap-proach, we show that TCR signaling is required to induce CD39 expression on CD8+T-cells in atheroscleroticlesions. Importantly, analysis of human endarterectomy samples showed a strong microenvironment specificupregulation of CD39 on CD8+T-cells in the plaques of human patients compared to matched blood samples. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in thevessel wall induces an immune regulatory CD8+T-cell phenotype that is associated with decreased cytokineproduction through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosispatients. This provides a new understanding of immune regulation by CD8+T-cells in atherosclerosis. Show less
The presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to... Show moreThe presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to their characteristic Fcε-receptor, causes the release of their cytoplasmic granules. These granules are filled with neutral proteases such as tryptase, but also with histamine and pro-inflammatory mediators. Mast cells accumulate in high numbers within human atherosclerotic tissue, particularly in the shoulder region of the plaque. These findings are largely based on immunohistochemistry, which does not allow for the extensive characterization of these mast cells and of the local mast cell activation mechanisms. In this study, we thus aimed to develop a new flow-cytometry based methodology in order to analyze mast cells in human atherosclerosis. We enzymatically digested 22 human plaque samples, collected after femoral and carotid endarterectomy surgery, after which we prepared a single cell suspension for flow cytometry. We were able to identify a specific mast cell population expressing both CD117 and the FcεR, and observed that most of the intraplaque mast cells were activated based on their CD63 protein expression. Furthermore, most of the activated mast cells had IgE fragments bound on their surface, while another fraction showed IgE-independent activation. In conclusion, we are able to distinguish a clear mast cell population in human atherosclerotic plaques, and this study establishes a strong relationship between the presence of IgE and the activation of mast cells in advanced atherosclerosis. Our data pave the way for potential therapeutic intervention through targeting IgE-mediated actions in human atherosclerosis. Show less
Sluis, R.J. van der; Depuydt, M.A.C.; Verwilligen, R.A.F.; Hoekstra, M.; Eck, M. van 2019
Apolipoprotein E (APOE) deficient mice exhibit unexplained hypercorticosteronemia. Given that APOE is also produced locally within the adrenals, we evaluated the effect of adrenal-specific APOE... Show moreApolipoprotein E (APOE) deficient mice exhibit unexplained hypercorticosteronemia. Given that APOE is also produced locally within the adrenals, we evaluated the effect of adrenal-specific APOE deficiency on the glucocorticoid function. Hereto, one adrenal containing or lacking APOE was transplanted into adrenalectomized wild-type mice. Adrenal APOE deficiency did not impact adrenal total cholesterol levels. Importantly, the ability of the two adrenal types to produce glucocorticoids was also not different as judged from the similar plasma corticosterone levels. Adrenal mRNA expression levels of HMG-CoA reductase and the LDL receptor were decreased by respectively 72% (p < 0.01) and 65% (p = 0.07), suggesting that cholesterol acquisition pathways were inhibited to possibly compensate the lack of APOE. In support, a parallel increase in the expression level of the cholesterol accumulation-associated ER stress marker CHOP was detected (+117%; p < 0.05). In conclusion, our studies show that elimination of adrenocortical APOE production does not impact glucocorticoid output in wild-type mice. Show less
The presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to... Show moreThe presence of mast cells in human atherosclerotic plaques has been associated with adverse cardiovascular events. Mast cell activation, through the classical antigen sensitized-IgE binding to their characteristic Fc epsilon-receptor, causes the release of their cytoplasmic granules. These granules are filled with neutral proteases such as tryptase, but also with histamine and pro-inflammatory mediators. Mast cells accumulate in high numbers within human atherosclerotic tissue, particularly in the shoulder region of the plaque. These findings are largely based on immunohistochemistry, which does not allow for the extensive characterization of these mast cells and of the local mast cell activation mechanisms. In this study, we thus aimed to develop a new flow-cytometry based methodology in order to analyze mast cells in human atherosclerosis. We enzymatically digested 22 human plaque samples, collected after femoral and carotid endarterectomy surgery, after which we prepared a single cell suspension for flow cytometry. We were able to identify a specific mast cell population expressing both CD117 and the Fc epsilon R, and observed that most of the intraplaque mast cells were activated based on their CD63 protein expression. Furthermore, most of the activated mast cells had IgE fragments bound on their surface, while another fraction showed IgE-independent activation. In conclusion, we are able to distinguish a clear mast cell population in human atherosclerotic plaques, and this study establishes a strong relationship between the presence of IgE and the activation of mast cells in advanced atherosclerosis. Our data pave the way for potential therapeutic intervention through targeting IgE-mediated actions in human atherosclerosis. Show less
Slütter, B.A.; Depuydt, M.A.C.; Duijn, J. van; Bot, I.; Wezel, A.; Koppejan, H.; ... ; Kuiper, J. 2018
CD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the... Show moreCD8+ T-cells are numerous in early and advanced atherosclerotic lesion, but their role in atherogenesis and plaque stability is still under debate. Recent work in murine models suggests the existence multiple CD8+ T-cell subsets that may differently impact the progression of disease. Here we aim to characterize the CD8+ T-cell population in human atherosclerotic lesions using a combination of histology, flow cytometry and mass cytometry (Cytof).Fresh carotid or femoral endarterectomy and matching blood samples were collect from a local hospital. Part of the sample was fixed for histology and the remainder was digested to obtain single cell suspensions. Cells were labelled with fluorescently labelled antibodies for flowcytometry and isotope labelled antibody for mass cytometry. Results were analyzed with Flowjo (flowcytometry and mass cytometry) and Cytosplore (mass cytometry) software.CD8+ T-cells were detected in every lesion (n=42) analyzed and on average constituted 23% of the total leukocyte. The percentage of CD8+ T-cells inversely correlated with the percentage of macrophages in the lesion, suggesting a role for CD8+ T-cells in preventing macrophage accumulation in the lesion. Mass cytometry revealed at least 13 phenotypically distinct CD8+ T-cells in the lesions, terminally differentiated CD27-/CD28- representing the largest CD8+ section.Human atherosclerotic lesions contain various CD8+ T-cell populations that may differentially affect disease progression and lesion stability. Although the overall effect of CD8+ T-cell presence in the lesion appear to be beneficial, identifying the protective subsets and expanding them may open new avenues for treatment. Show less
