Single-Cell Analysis Uncovers Osteoblast Factor Growth Differentiation Factor 10 as Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation Associated with Plaque Rupture in Human Carotid Artery Disease.

(1) Background: Vascular smooth muscle cells (VSMCs) undergo a complex phenotypic switch in response to atherosclerosis environmental triggers, contributing to atherosclerosis disease progression. However, the complex heterogeneity of VSMCs and how VSMC dedifferentiation affects human carotid artery disease (CAD) risk has not been clearly established. (2) Method: A single-cell RNA sequencing analysis of CD45- cells derived from the atherosclerotic aorta of Apolipoprotein E-deficient (Apoe-/-) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the site-specific predisposition to atherosclerosis was performed. Growth Differentiation Factor 10 (GDF10) role in VSMCs phenotypic switch was investigated via flow cytometry, immunofluorescence in human atherosclerotic plaques. (3) Results: scRNAseq analysis revealed the transcriptomic profile of seven clusters, five of which showed disease-relevant gene signature of VSMC macrophagic calcific phenotype, VSMC mesenchymal chondrogenic phenotype, VSMC inflammatory and fibro-phenotype and VSMC inflammatory phenotype. Osteoblast factor GDF10 involved in ossification and osteoblast differentiation emerged as a hallmark of VSMCs undergoing phenotypic switch. Under hypercholesteremia, GDF10 triggered VSMC osteogenic switch in vitro. The abundance of GDF10 expressing osteogenic-like VSMCs cells was linked to the occurrence of carotid artery disease (CAD) events. (4) Conclusions: Taken together, these results provide evidence about GDF10-mediated VSMC osteogenic switch, with a likely detrimental role in atherosclerotic plaque stability.

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis.

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1ß secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1ß in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.

Anti-Apolipoprotein A-1 IgG Influences Neutrophil Extracellular Trap Content at Distinct Regions of Human Carotid Plaques.

BACKGROUND: Neutrophils accumulate in atherosclerotic plaques. Neutrophil extracellular traps (NET) were recently identified in experimental atherosclerosis and in complex human lesions. However, not much is known about the NET marker citrullinated histone-3 (H3Cit) expression and functionality in human carotid plaques. Moreover, the association between the proatherosclerotic autoantibody anti-apolipoprotein A-1 (anti-ApoA-1 IgG) and NET has never been investigated. METHODS: Atherosclerotic plaques have been obtained from 36 patients with severe carotid stenosis that underwent carotid endarterectomy for severe carotid stenosis. Samples were sectioned into upstream and downstream regions from the same artery segment. Plaque composition and expression of NET markers neutrophil elastase (NE) and H3Cit were quantified by immunohistochemistry. H3Cit expression and function was evaluated by immunofluorescence and confocal analysis in a subset of patients. RESULTS: Pathological features of vulnerable phenotypes were exacerbated in plaques developed at downstream regions, including higher accumulation of neutrophils and enhanced expression of NE and H3Cit, as compared to plaques from upstream regions. The H3Cit signal was also more intense in downstream regions, with significant extracellular distribution in spaces outside of neutrophils. The percentage of H3Cit colocalization with CD66b (neutrophils) was markedly lower in downstream portions of carotid plaques, confirming the extrusion of NET in this region. In agreement, the maximum distance of the H3Cit signal from neutrophils, extrapolated from vortex distance calculation in all possible directions, was also higher in downstream plaques. The serum anti-ApoA-1index positively correlated with the expression of H3Cit in downstream segments of plaques. Expression of the H3Cit signal outside of neutrophils and H3Cit maximal distance from CD66b-positive cells increased in plaques from serum positive anti-ApoA-1 patients compared with serum negative patients. CONCLUSION: NET elements are differentially expressed in upstream versus downstream regions of human carotid plaques and may be influenced by circulating levels of anti-ApoA-1 IgG. These findings could warrant the investigation of NET elements as potential markers of vulnerability.

Combined Genetic Deletion of IL (Interleukin)-4, IL-5, IL-9, and IL-13 Does Not Affect Ischemic Brain Injury in Mice.

Background and Purpose- After ischemic injury, microglia and infiltrated macrophages may acquire different polarization phenotypes promoting inflammation and injury (M1) or repair and protection (M2). There is evidence that immunomodulation, via type 2 helper T-cells (Th2) cytokines, exerts neuroprotection after ischemia. We investigated the consequences of simultaneous genetic deletion of Th2 cytokines (IL [interleukin]-4, IL-5, IL-9, IL-13) on the histopathologic outcome, microglia and infiltrated macrophages markers, and ischemic microenvironment at different time points after ischemic injury in mice subjected to permanent occlusion of the middle cerebral artery. Methods- Wild-type and Th2 cytokine-deficient mice (4KO) were subjected to permanent occlusion of the middle cerebral artery by electrocoagulation and followed up to 5 weeks after permanent occlusion of the middle cerebral artery. Neuropathologic outcome was assessed at 24 hours (n=6), 7 days (n=6), and 5 weeks (n=6-7) by examination of the ischemic lesion, neuronal count, microglia and infiltrated macrophages markers, brain atrophy, collagen deposition, and GFAP (glial fibrillary acidic protein) immunohistochemistry. Selected gene expression was investigated at 7 days (n=6). Results- 4KO mice showed no difference in lesion and neuronal count 7 days and up to 5 weeks after permanent occlusion of the middle cerebral artery compared with wild type. Ischemic 4KO mice had lower CD16/32 expression at 24 hours, lower CD11b and CD16/32 expression at 7 days than wild type. They had higher CD206 expression at 24 hours, higher CD206 and arginase1 at 7 days, and increased mRNA for CXCL9 (chemokine [C-X-C motif] ligand 9) compared with wild type. Additional histopathologic analysis, including brain atrophy, gliotic scar, and collagenous scar confirmed no difference between genotypes at 5 weeks. Conclusions- This study casts light on the proposed neuroprotective function of Th2 cytokines, showing that combined IL-4, IL-5, IL-9, IL-13 deletion does not affect the neuropathologic response to ischemic stroke in the subacute and chronic phases. Our findings indicate that Th2 cytokines are not an essential neuroimmunological cue able to drive the brain's ischemic outcome.

Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Regulatory T (Treg) cells offer new therapeutic options for controlling undesired systemic and local immune responses. The aim of the current study was to determine the impact of therapeutic Treg administration on systemic and cardiac inflammation and remodeling in coxsackievirus B3 (CVB3) -induced myocarditis. Therefore, syngeneic Treg cells were applied intravenously in CVB3-infected mice 3 d after infection. Compared with CVB3 + PBS mice, CVB3 + Treg mice exhibited lower left ventricular (LV) chemokine expression, accompanied by reduced cardiac presence of proinflammatory Ly6ChighCCR2highCx3Cr1low monocytes and higher retention of proinflammatory Ly6CmidCCR2highCx3Cr1low monocytes in the spleen. In addition, splenic myelopoiesis was reduced in CVB3 + Treg compared with CVB3 + PBS mice. Coculture of Treg cells with splenocytes isolated from mice 3 d post-CVB3 infection further demonstrated the ability of Treg cells to modulate monocyte differentiation in favor of the anti-inflammatory Ly6ClowCCR2lowCx3Cr1high subset. Treg-mediated immunomodulation was paralleled by lower collagen 1 protein expression and decreased levels of soluble and insoluble collagen in LV of CVB3 + Treg compared with CVB3 + PBS mice. In agreement with these findings, LV systolic and diastolic function was improved in CVB3 + Treg mice compared with CVB3 + PBS mice. In summary, adoptive Treg transfer in the inflammatory phase of viral-induced myocarditis protects the heart against inflammatory damage and fibrosis via modulation of monocyte subsets.-Pappritz, K., Savvatis, K., Miteva, K., Kerim, B., Dong, F., Fechner, H., Müller, I., Brandt, C., Lopez, B., González, A., Ravassa, S., Klingel, K., Diez, J., Reinke, P., Volk, H.-D., Van Linthout, S., Tschöpe, C. Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

High-density lipoproteins reduce endothelial-to-mesenchymal transition.

OBJECTIVE: Endothelial-to-mesenchymal transition is an inflammation-induced process by which endothelial cells can transdifferentiate into fibroblasts. Based on the endothelial-protective and antifibrotic effects of high-density lipoproteins (HDL), we aimed to investigate whether HDL can reduce endothelial-to-mesenchymal transition. APPROACH AND RESULTS: Therefore, human aortic endothelial cells were stimulated with the profibrotic factor transforming growth factor (TGF)-ß1 in the presence or absence of HDL. Their impact on the transition of endothelial cells to mesenchymal-like cells was analyzed. Phase contrast microscopy demonstrated that HDL abrogated the TGF-ß1-induced spindle-shape morphology in human aortic endothelial cells. Furthermore, HDL decreased the TGF-ß1-mediated induction of α-smooth muscle actin expression and concomitant loss in endothelial cadherin expression, as shown by immunofluorescence staining and flow cytometry. In addition, HDL decreased the TGF-ß1-induced collagen deposition in human aortic endothelial cells involving the scavenger receptor class B, type 1 and downstream phosphatidyl inositol-3-kinase following the findings that the HDL-mediated reduction was abrogated by scavenger receptor class B, type 1 siRNA knockdown and phosphatidyl inositol-3-kinase inhibition, respectively. The HDL-mediated reduction in endothelial-to-mesenchymal transition was associated with an induction of the inhibitory Smad, Smad 7. CONCLUSIONS: We provide the first in vitro evidence that the endothelial-protective and antifibrotic effects of HDL include the reduction in endothelial-to-mesenchymal transition.

Cardiac migration of endogenous mesenchymal stromal cells in patients with inflammatory cardiomyopathy.

Introduction. Mesenchymal stromal cells (MSC) have immunomodulatory features. The aim of this study was to investigate the migration and homing potential of endogenous circulating MSC in virus negative inflammatory cardiomyopathy (CMi). Methods. In 29 patients with (n = 23) or without (n = 6) CMi undergoing endomyocardial biopsies (EMB), transcardiac gradients (TCGs) of circulating MSC were measured by flow cytometry from blood simultaneously sampled from aorta and coronary sinus. The presence of MSC in EMB, cardiac inflammation, and SDF-1α mRNA expression were detected via immunohistochemistry and real-time PCR. Results. MSC defined as CD45(-)CD34(-)CD11b(-)CD73(+)CD90(+) cells accounted for 0.010 [0.0025-0.048]%/peripheral mononuclear cell (PMNC) and as CD45(-)CD34(-)CD11b(-)CD73(+)CD105(+) cells for 0.019 [0.0026-0.067]%/PMNC, both with similar counts in patients with or without cardiac inflammation. There was a 29.9% (P < 0.01) transcardiac reduction of circulating MSC in patients with CMi, correlating with the extent of cardiac inflammation (P < 0.05, multivariate analysis). A strong correlation was found between the TCG of circulating MSC and numbers of MSC (CD45(-)CD34(-)CD90(+)CD105(+)) in EMB (r = -0.73, P < 0.005). SDF-1α was the strongest predictor for increased MSC in EMB (P < 0.005, multivariate analysis). Conclusions. Endogenous MSC continuously migrate to the heart in patients with CMi triggered by cardiac inflammation.

Sex- and age-related differences in the inflammatory properties of cardiac fibroblasts: impact on the cardiosplenic axis and cardiac fibrosis.

Background: Age and sex are prominent risk factors for heart failure and determinants of structural and functional changes of the heart. Cardiac fibroblasts (cFB) are beyond their task as extracellular matrix-producing cells further recognized as inflammation-supporting cells. The present study aimed to evaluate the impact of sex and age on the inflammatory potential of cFB and its impact on the cardiosplenic axis and cardiac fibrosis. Materials: Left ventricles (LV) of 3- and 12-months old male and female C57BL/6J mice were harvested for immunohistochemistry, immunofluorescence and cFB outgrowth culture and the spleen for flow cytometry. LV-derived cFB and respective supernatants were characterized. Results: LV-derived cFB from 3-months old male mice exhibited a higher inflammatory capacity, as indicated by a higher gene expression of CC-chemokine ligand (CCL) 2, and CCL7 compared to cFB derived from 3-months old female mice. The resulting higher CCL2/chemokine C-X3-C motif ligand (Cx3CL1) and CCL7/Cx3CL1 protein ratio in cell culture supernatants of 3-months old male vs. female cFB was reflected by a higher migration of Ly6Chigh monocytes towards supernatant from 3-months old male vs. female cFB. In vivo a lower ratio of splenic pro-inflammatory Ly6Chigh to anti-inflammatory Ly6Clow monocytes was found in 3-months old male vs. female mice, suggesting a higher attraction of Ly6Chigh compared to Ly6Clow monocytes towards the heart in male vs. female mice. In agreement, the percentage of pro-inflammatory CD68+ CD206- macrophages was higher in the LV of male vs. female mice at this age, whereas the percentage of anti-inflammatory CD68+ CD206+ macrophages was higher in the LV of 3-months old female mice compared to age-matched male animals. In parallel, the percentage of splenic TGF-ß+ cells was higher in both 3- and 12-months old female vs. male mice, as further reflected by the higher pro-fibrotic potential of female vs. male splenocytes at both ages. In addition, female mice displayed a higher total LV collagen content compared to age-matched male mice, whereby collagen content of female cFB was higher compared to male cFB at the age of 12-months. Conclusion: Age- and sex-dependent differences in cardiac fibrosis and inflammation are related to age- and sex-dependent variations in the inflammatory properties of cardiac fibroblasts.

Identification of noncytotoxic and IL-10-producing CD8+AT2R+ T cell population in response to ischemic heart injury.

Emerging evidence suggests a cardioprotective role of the angiotensin AT2R, albeit the underlying cellular mechanisms are not well understood. We aimed in this article to elucidate a potential role of cardiac angiotensin AT2R in regulating cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, double-immunofluorescence staining showed that AT2R was detected in a fraction of CD8(+) T cells infiltrating in the peri-infarct myocardium. We developed a method that allowed the isolation of myocardial infiltrating CD8(+)AT2R(+) T cells using modified MACS, and further characterization and purification with flow cytometry. Although the CD8(+)AT2R(-) T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes (CMs), the CD8(+)AT2R(+) T cells were noncytotoxic to these CMs. The CD8(+)AT2R(+) T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-γ expression when compared with CD8(+)AT2R(-) T cells. We further showed that IL-10 gene expression was enhanced in CD8(+) T cells on in vitro AT2R stimulation. Importantly, in vivo AT2R activation engendered an increment of CD8(+)AT2R(+) T cells and IL-10 production in the ischemic myocardium. In addition, intramyocardial transplantation of CD8(+)AT2R(+) T cells (versus CD8(+)AT2R(-)) led to reduced ischemic heart injury. Moreover, the CD8(+)AT2R(+) T cell population was also demonstrated in human peripheral blood. Thus, we have defined the cardioprotective CD8(+)AT2R(+) T cell population, which increases during ischemic heart injury and contributes to maintaining CM viability and providing IL-10, hence revealing an AT2R-mediated cellular mechanism in modulating adaptive immune response in the heart.

Staurosporine-induced differentiation of the RGC-5 cell line leads to apoptosis and cell death at the lowest differentiating concentration.

BACKGROUND: Supplementation of staurosporine is the method of choice for differentiating the solely existing retinal ganglion cell (RGC)-5 cell line. This differentiation was initially claimed to be in the absence of apoptosis, but some publications supposed the induction of apoptosis during staurosporine induced RGC-5 differentiation. In respect to these inconsistencies in the literature, we investigated in detail whether RGC-5 cell differentiation by staurosporine induces apoptosis or not. METHODS: Amounts of 50 nM, 200 nM, 300 nM, and 600 nM of staurosporine were supplemented on RGC-5 cells for 24 h. Cell morphology and cell death, via propidium iodide staining, were evaluated with phase contrast and fluorescence microscopy, respectively. Cell amount, cell proliferation, and cell viability were analyzed by crystal violet staining, CFSE flow cytometry, and MTS assay, respectively. Apoptosis was determined by analyzing caspase 3/7 activity, Annexin-V+/ 7AAD- cells and the quotient of Bax to Bcl-2 mRNA expression via caspase 3/7 activity assay, flow cytometry, and real-time PCR, respectively. RESULTS: RGC-5 cells started to change their morphology and their expression of neuronal markers at 50 nM of staurosporine. This was associated with apoptosis and cell death, as indicated by a 2.1-fold (p < 0.0005) increase in caspase 3/7 activity, a 1.2-fold (p < 0.05) induction of Annexin-V+/ 7AAD- cells, and a 12-fold (p < 0.0005) increase in propidium iodide positive cells, respectively. Furthermore, staurosporine led to a dose-dependent increase in apoptosis and reduction in cell viability, cell density, and cell proliferation. CONCLUSIONS: The lowest staurosporine concentration inducing RGC-5 cell differentiation is accompanied by apoptosis and cell death.

The E3 Ubiquitin Ligase Peli1 Deficiency Promotes Atherosclerosis Progression.

BACKGROUND: Atherosclerosis is a chronic inflammatory vascular disease and the main cause of death and morbidity. Emerging evidence suggests that ubiquitination plays an important role in the pathogenesis of atherosclerosis including control of vascular inflammation, vascular smooth muscle cell (VSMC) function and atherosclerotic plaque stability. Peli1 a type of E3 ubiquitin ligase has emerged as a critical regulator of innate and adaptive immunity, however, its role in atherosclerosis remains to be elucidated. METHODS: Apoe-/- mice and Peli1-deficient Apoe-/- Peli1-/- mice were subject to high cholesterol diet. Post sacrifice, serum was collected, and atherosclerotic plaque size and parameters of atherosclerotic plaque stability were evaluated. Immunoprofiling and foam cell quantification were performed. RESULTS: Peli1 deficiency does not affect atherosclerosis lesion burden and cholesterol levels, but promotes VSMCs foam cells formation, necrotic core expansion, collagen, and fibrous cap reduction. Apoe-/- Peli1-/- mice exhibit a storm of inflammatory cytokines, expansion of Th1, Th1, Th17, and Tfh cells, a decrease in regulatory T and B cells and induction of pro-atherogenic serum level of IgG2a and IgE. CONCLUSIONS: In the present study, we uncover a crucial role for Peli1 in atherosclerosis as an important regulator of inflammation and VSMCs phenotypic modulation and subsequently atherosclerotic plaque destabilization.

Single-Cell RNA-Seq Reveals a Crosstalk between Hyaluronan Receptor LYVE-1-Expressing Macrophages and Vascular Smooth Muscle Cells.

Background: Atherosclerosis is a chronic inflammatory disease where macrophages participate in the progression of the disease. However, the role of resident-like macrophages (res-like) in the atherosclerotic aorta is not completely understood. Methods: A single-cell RNA sequencing analysis of CD45+ leukocytes in the atherosclerotic aorta of apolipoprotein E-deficient (Apoe-/-) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the side-to-specific predisposition to atherosclerosis, was performed. A population of res-like macrophages expressing hyaluronan receptor LYVE-1 was investigated via flow cytometry, co-culture experiments, and immunofluorescence in human atherosclerotic plaques from carotid artery disease patients (CAD). Results: We identified 12 principal leukocyte clusters with distinct atherosclerosis disease-relevant gene expression signatures. LYVE-1+ res-like macrophages, expressing a high level of CC motif chemokine ligand 24 (CCL24, eotaxin-2), expanded under hypercholesteremia in Apoe-/- mice and promoted VSMC phenotypic modulation to osteoblast/chondrocyte-like cells, ex vivo, in a CCL24-dependent manner. Moreover, the abundance of LYVE-1+CCL24+ macrophages and elevated systemic levels of CCL24 were associated with vascular calcification and CAD events. Conclusions: LYVE-1 res-like macrophages, via the secretion of CCL24, promote the transdifferentiation of VSMC to osteogenic-like cells with a possible role in vascular calcification and likely a detrimental role in atherosclerotic plaque destabilization.

Epitope of antiphospholipid antibodies retrieved from peptide microarray based on R39-R43 of ß2-glycoprotein I.

Background: Antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies and thromboembolic or pregnancy complications. Although cryptic epitope R39-R43 belonging to beta-2-glycoprotein 1 (ß2GP1) has been identified as the main antigenic determinant for aPLs, we have recently demonstrated that the epitope is a motif determined by the polarity, rather than by the sequence or charge of amino acids. Objective: In the present study, we wanted to identify the association of residues needed to obtain the highest aPL affinity. Methods: Based on the epitope R39-R43 and our identified motif, we generated a printed peptide microarray of 676 different peptides. These peptides have been then screened for their ability to interact with the plasmas from 11 well-characterized APS patients and confirmed by surface plasma resonance assay. Results and Conclusions: We identified a peptide that selectively bound immunoglobulin G (IgG) derived from APS patients with 100 times more affinity than ß2GP1, Domain I, or epitope R39-R43. This peptide is able to inhibit the activity of IgG derived from APS patients in vitro. We have also generated a monoclonal IgG antibody against this peptide. Using both peptide and monoclonal antibody, we have been able to develop a fully standardized indirect colorimetric immunoassay with highly sensitivity. The identification of the optimized peptide offers a new standardized and accurate tool for diagnostics of APS. Furthermore, having increased affinity for aPL, this peptide could represent a useful tool as prevention strategy for APS and an alternative to the use of anticoagulants.

Follicular regulatory helper T cells control the response of regulatory B cells to a high-cholesterol diet.

AIMS: B cell functions in the process of atherogenesis have been investigated but several aspects remain to be clarified. METHODS AND RESULTS: In this study, we show that follicular regulatory helper T cells (TFR) control regulatory B cell (BREG) populations in Apoe-/- mice models on a high-cholesterol diet (HCD). Feeding mice with HCD resulted in up-regulation of TFR and BREG cell populations, causing the suppression of proatherogenic follicular helper T cell (TFH) response. TFH cell modulation is correlated with the growth of atherosclerotic plaque size in thoracoabdominal aortas and aortic root plaques, suggesting that TFR cells are atheroprotective. During adoptive transfer experiments, TFR cells transferred into HCD mice decreased TFH cell populations, atherosclerotic plaque size, while BREG cell population and lymphangiogenesis are significantly increased. CONCLUSION: Our results demonstrate that, through different strategies, both TFR and TFH cells modulate anti- and pro-atherosclerotic immune processes in an Apoe-/- mice model since TFR cells are able to regulate both TFH and BREG cell populations as well as lymphangiogenesis and lipoprotein metabolism.

Impact of Syndecan-2-Selected Mesenchymal Stromal Cells on the Early Onset of Diabetic Cardiomyopathy in Diabetic db/db Mice.

Background: Mesenchymal stromal cells (MSCs) are an attractive cell type for cell therapy given their immunomodulatory, anti-fibrotic, and endothelial-protective features. The heparin sulfate proteoglycan, syndecan-2/CD362, has been identified as a functional marker for MSC isolation, allowing one to obtain a homogeneous cell product that meets regulatory requirements for clinical use. We previously assessed the impact of wild-type (WT), CD362-, and CD362+ MSCs on local changes in protein distribution in left ventricular (LV) tissue and on LV function in an experimental model of early-onset diabetic cardiomyopathy. The present study aimed to further explore their impact on mechanisms underlying diastolic dysfunction in this model. Materials: For this purpose, 1 × 106 WT, CD362-, or CD362+ MSCs were intravenously (i.v.) injected into 20-week-old diabetic BKS.Cg-m+/+Leprdb/BomTac, i.e., db/db mice. Control animals (db+/db) were injected with the equivalent volume of phosphate-buffered saline (PBS) alone. After 4 weeks, mice were sacrificed for further analysis. Results: Treatment with all three MSC populations had no impact on blood glucose levels in db/db mice. WT, CD362-, and CD362+ MSC application restored LV nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels in db/db mice, which correlated with a reduction in cardiomyocyte stiffness. Furthermore, all stromal cells were able to increase arteriole density in db/db mice. The effect of CD362+ MSCs on NO and cGMP levels, cardiomyocyte stiffness, and arteriole density was less pronounced than in mice treated with WT or CD362- MSCs. Analysis of collagen I and III protein expression revealed that fibrosis had not yet developed at this stage of experimental diabetic cardiomyopathy. All MSCs reduced the number of cardiac CD3+ and CD68+ cells in db/db mice, whereas only splenocytes from CD362-- and CD362+-db/db mice exhibited a lower pro-fibrotic potential compared to splenocytes from db/db mice. Conclusion: CD362+ MSC application decreased cardiomyocyte stiffness, increased myocardial NO and cGMP levels, and increased arteriole density, although to a lesser extent than WT and CD362- MSCs in an experimental model of early-onset diabetic cardiomyopathy without cardiac fibrosis. These findings suggest that the degree in improvement of cardiomyocyte stiffness following CD362+ MSC application was insufficient to improve diastolic function.

Atherosclerotic plaque vulnerability is increased in mouse model of lupus.

Anti-apolipoprotein A-1 (anti-apoA-1 IgG) and anti-double stranded DNA (anti-dsDNA IgG) autoantibodies have been described as mediators of atherogenesis in mice and humans. In the present study, we aim to investigate the association between atherosclerotic parameters, autoantibodies and plaque vulnerability in the context of systemic lupus erythematosus (SLE). We therefore bred a lupus prone-mouse model (Nba2.Yaa mice) with Apoe-/- mice resulting in Apoe-/-Nba2.Yaa mice spontaneously producing anti-apoA-1 IgG antibodies. Although Apoe-/-Nba2.Yaa and Apoe-/- mice subject to a high cholesterol diet displayed similar atherosclerosis lesions size in aortic roots and abdominal aorta, the levels of macrophage and neutrophil infiltration, collagen, MMP-8 and MMP-9 and pro-MMP-9 expression in Apoe-/-Nba2.Yaa mice indicated features of atherosclerotic plaque vulnerability. Even though Apoe-/-Nba2.Yaa mice and Apoe-/- mice had similar lipid levels, Apoe-/-Nba2.Yaa mice showed higher anti-apoA-1 and anti-dsDNA IgG levels. Apoe-/-Nba2.Yaa mice displayed a reduction of the size of the kidney, splenomegaly and lymph nodes (LN) hypertrophy. In addition, anti-apoA-1 and anti-dsDNA IgG increased also in relation with mRNA levels of GATA3, IL-4, Bcl-6 and CD20 in the spleen and aortic arch of Apoe-/-Nba2.Yaa mice. Our data show that although atherosclerosis-lupus-prone Apoe-/-Nba2.Yaa mice did not exhibit exacerbated atherosclerotic lesion size, they did show features of atherosclerotic plaque destabilization in correlation with the increase of pro-atherogenic autoantibodies.

Cardiotrophin-1 Deficiency Abrogates Atherosclerosis Progression.

Cardiotrophin-1 (CT-1) is associated with cardiovascular (CV) diseases. We investigated the effect of CT-1 deficiency in the development and progression of atherosclerosis in double knockout Apoe-/-ct-1-/- mice. Apoe-/- C57Bl/6 or Apoe-/-ct-1-/- C57Bl/6 mice were fed a normal chow diet (NCD) or a high-cholesterol diet (HCD). After sacrifice, serum triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), free fatty acids and systemic paracrine factors were measured. Intraplaque lipid and collagen content were quantified in the aortic sections. Immune cell populations in spleen, lymph nodes and aorta were analysis by flow cytometry. Apoe-/-ct-1-/- mice in accelerated atherosclerosis exhibited a reduction of total cholesterol, LDL-C, atherosclerotic plaques size in the aortic root and in the abdominal aorta and improved plaque stability in comparison to Apoe-/- mice. CT-1 deficiency in Apoe-/- mice on (HCD) promoted atheroprotective immune cell responses, as demonstrated by a rise in plasma anti-inflammatory immune cell populations (regulatory T cells, Tregs; regulatory B cells, Bregs and B1a cells) and atheroprotective IgM antibodies. CT-1 deficiency in advanced atherosclerosis mediated regulation of paracrine factors, such as interleukin (IL)-3, IL-6, IL-9, IL-15, IL-27, CXCL5, MCP-3, MIP-1α and MIP-1ß. In a model of advanced atherosclerosis, CT-1 deficiency induced anti-inflammatory and atheroprotective effects which resulted in abrogation of atheroprogression.

Innate and adaptive immunity in atherosclerosis.

Atherosclerosis is a chronic inflammatory disorder of the large and medium-size arteries characterized by the subendothelial accumulation of cholesterol, immune cells, and extracellular matrix. At the early onset of atherogenesis, endothelial dysfunction takes place. Atherogenesis is further triggered by the accumulation of cholesterol-carrying low-density lipoproteins, which acquire properties of damage-associated molecular patterns and thereby trigger an inflammatory response. Following activation of the innate immune response, mainly governed by monocytes and macrophages, the adaptive immune response is started which further promotes atherosclerotic plaque formation. In this review, an overview is given describing the role of damage-associated molecular patterns, NLRP3 inflammasome activation, and innate and adaptive immune cells in the atherogenesis process.