Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.

Excessive leukocyte infiltration causes severe tissue damage in a variety of inflammatory diseases. The initial step in leukocyte extravasation is mediated by selectins and oligosaccharides on their glycoconjugate ligands. Human milk is a rich source of lactose-derived oligosaccharides that are partly absorbed in the intestine and excreted with the urine. As these components contain binding determinants for the selectins we investigated whether human milk oligosaccharides are able to affect leukocyte rolling and adhesion to endothelial cells under dynamic conditions. Therefore, monocytes, lymphocytes, or neutrophils isolated from human peripheral blood were passed over TNF-alpha-activated HUVEC under shear stress. The influence of different oligosaccharide fractions was determined by video-microscopy and compared with the effects of various individual oligosaccharides. Within a physiological range (12.5 - 125 microg/ml) the acidic fraction significantly inhibited leukocyte rolling and adhesion (up to 24.0% and 52.8%, respectively) in a concentration-dependent manner. These effects were even more pronounced than those achieved by soluble sialyl-Lewis x, a physiological binding determinant for selectins. Several active components within the oligosaccharide fraction of human milk were identified, e.g. 3'-sialyl-lactose and 3'-sialyl-3-fucosyl-lactose. These results indicate that specific oligosaccharides in human milk may serve as anti-inflammatory components and might therefore contribute to the lower incidence of inflammatory diseases in human milk-fed infants.

Background: The majority of the human genome comprises noncoding sequences, which are in part transcribed as long noncoding RNAs (lncRNAs). lncRNAs exhibit multiple functions, including the epigenetic control of gene expression. In this study, the effect of the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) on atherosclerosis was examined. Methods: The effect of MALAT1 on atherosclerosis was determined in apolipoprotein E–deficient (Apoe − /− ) MALAT1-deficient (Malat1 −/− ) mice that were fed with a high-fat diet and by studying the regulation of MALAT1 in human plaques. Results: Apoe −/− Malat1 −/− mice that were fed a high-fat diet showed increased plaque size and infiltration of inflammatory CD45 + cells compared with Apoe −/− Malat1 +/+ control mice. Bone marrow transplantation of Apoe −/− Malat1 −/− bone marrow cells in Apoe −/− Malat1 +/+ mice enhanced atherosclerotic lesion formation, which suggests that hematopoietic cells mediate the proatherosclerotic phenotype. Indeed, bone marrow cells isolated from Malat1 −/− mice showed increased adhesion to endothelial cells and elevated levels of proinflammatory mediators. Moreover, myeloid cells of Malat1 −/− mice displayed enhanced adhesion to atherosclerotic arteries in vivo. The anti-inflammatory effects of MALAT1 were attributed in part to reduction of the microRNA miR-503. MALAT1 expression was further significantly decreased in human plaques compared with normal arteries and was lower in symptomatic versus asymptomatic patients. Lower levels of MALAT1 in human plaques were associated with a worse prognosis. Conclusions: Reduced levels of MALAT1 augment atherosclerotic lesion formation in mice and are associated with human atherosclerotic disease. The proatherosclerotic effects observed in Malat1 −/− mice were mainly caused by enhanced accumulation of hematopoietic cells.

Single-nucleus RNA-sequencing reveals cellular heterogeneity of cardiac cells in aging. To comprehensively decipher the expected cellular responses to intrinsic cardiac aging, we applied microdroplet-based single-nucleus RNA-sequencing (9) of cross-sections of snap-frozen heart samples from 3 syngeneic young male mice (12 weeks old) and 3 aged male mice (18 months old). In total, we analyzed 14,827 nuclei from young hearts and 12,981 nuclei from old hearts (Supplemental Table 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.131092DS1). Using t-distributed stochastic neighbor embedding (tSNE) (10), global dimension reduction was constructed from all samples to visualize clusters that were defined by cell-specific gene markers (Figure 1A and Supplemental Table 2). Alignment of samples indicated high reproducibility across samples (Supplemental Figure 1). Most of the cells were in G 1 phase, and no influence of aging on cell cycle activity was observed (Supplemental Figure 2). Unsupervised clustering revealed 15 distinct gene expression patterns (Figure 1A and Supplemental Figure 3). Using cell type-specific gene markers (Supplemental Table 2) and published mouse single-cell gene expression data (11, 12), 7 major cell types could be annotated, including fibroblasts (A, B), cardiomyocytes (A, B, C), endothelial cells (A, B, C), immune cells (A, B, C), pericytes, epicardial cells, and adipocytes (Figure 1A and Supplemental Figure 3). In particular, for fibroblasts, the unsupervised clustering revealed 2 main clusters, fibroblast A (79.42%) and fibroblast B (20.58%). Separation of these 2 clusters was not significant (Supplemental Figure 3B), and gene markers were very similar (Supplemental Table 2); moreover, these 2 clusters were almost equally populated by young and old cells. Analysis of the cell numbers in clusters of other cell types than fibroblasts showed in part trends for changes during aging (Supplemental Figure 4) but did not reveal statistically significant differences. In general, 128 differentially expressed nonredundant genes (DEGs) were found between young and aged hearts (Figure 1B and Supplemental Table 3). Considering the DEGs in all cell clusters, 107 genes showed significantly increased expression (adjusted P < 0.1), and 21 genes showed significantly decreased expression (adjusted P < 0.1) in aged versus young hearts (Supplemental Table 3). Interestingly, aging predominantly affected gene expression patterns in fibroblasts (Figure 1B). Several highly differentially expressed genes could be confirmed by quantitative reverse transcription PCR of isolated cardiac fibroblasts (Supplemental Figure 5). Gene Ontology (GO) analysis of DEGs revealed a cell type-specific enrichment of genes associated with various pathways, such as angiogenesis, chemotaxis/migration, inflammation/immune response, and cell/matrix association (Figure 1C). Only a few coexpression networks and significantly regulated genes were shared between the main cell types. Among them, the e...

Abstract-Circulating blood-derived vasculogenic cells improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. By using the inducible suicide gene thymidine kinase to deplete transplanted cells, we determined the contribution of physical incorporation in 3 animal models. After acute myocardial infarction, depletion of cells 14 days after infusion resulted in a reduction of capillary density and a substantial deterioration of heart function. Likewise, neovascularization of Matrigel plugs and ischemic limbs was significantly suppressed when infused cells were depleted 7 days after infusion. Key Words: progenitor cells Ⅲ neovascularization Ⅲ cell therapy C ell therapy is a promising option for treating ischemic diseases. Adult stem and progenitor cells from various sources have experimentally been shown to augment the functional recovery after ischemia. Clinical trials confirmed that autologous cell therapy using bone marrow-derived or circulating blood-derived progenitor cells is safe and provides beneficial effects 1-3 (see also recent metaanalysis 4 ). Endothelial progenitor cells (EPCs) were initially isolated from bone marrow or peripheral blood and were characterized by the expression of the hematopoietic stem cell marker CD34 or CD133 and endothelial markers such as vascular endothelial growth factor receptor 2 (KDR). 5,6 Isolated CD34 ϩ cells differentiate to endothelial cells in vitro and incorporate into newly formed vessels during angiogenesis in vivo. 5 These pioneering studies suggested that bone marrow-derived cells can participate in vascular repair. Indeed, a single hematopoietic stem cell was shown to give rise to both blood cells and vascular endothelium. [7][8][9] The contribution of circulating cells to the endothelium was further supported by sexmismatched heart transplantation in humans. 10,11 Other investigators used culture assays to enrich for EPC-yielding cells, which coexpress endothelial and myeloid marker ("early EPCs," proangiogenic cells), or to show a more mature endothelial cell phenotype on further culture and expansion ("late or outgrowing EPCs"). 12,13 Although the origin and the phenotype of the cultured EPCs is not entirely clear and may vary depending on the defined specific culture conditions, 14 the therapeutic potential and improved neovascularization mediated by the cultured cells has been documented extensively in ischemic models. 12,15,16 Progenitor cell-mediated improved neovascularization of ischemic tissue might be attributable to various therapeutic actions including a physical incorporation of the infused cells in the endothelium leading to the formation of new capillaries. 16 -18 Transplanted or infused hema...

Monocyte-endothelium interaction is a fundamental process in many acute and chronic inflammatory diseases. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are fish oil-derived alternative (ω-3) precursor fatty acids implicated in the suppression of inflammatory events. We investigated their influence on rolling and adhesion of monocytes to human umbilical vein endothelial cells (HUVEC) under laminar flow conditions in vitro. Exposure of HUVEC to tumor necrosis factor (TNF-α) strongly increased 1) surface expression of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1), and E-selectin, 2) platelet-activating factor (PAF) synthesis as assessed by thrombin challenge, and 3) rate of rolling and adhesion of monocytes. Preincubation of HUVEC with EPA or DHA markedly suppressed PAF synthesis, monocyte rolling, and adherence, whereas expression of endothelial adhesion molecules was unchanged. Also, PAF receptor antagonists markedly suppressed the adhesion rate of monocytes, and EPA or DHA revealed no additional inhibitory capacity. In contrast, arachidonic acid partially reversed the effect of the antagonist. We conclude that ω-3 fatty acids suppress rolling and adherence of monocytes on activated endothelial cells in vitro by affecting endothelial PAF generation.

Rationale: Cell therapy is a promising option for the treatment of acute or chronic myocardial ischemia. The intracoronary infusion of cells imposes the potential risk of cell clotting, which may be prevented by the addition of anticoagulants. However, a comprehensive analysis of the effects of anticoagulants on the function of the cells is missing.Objective: Here, we investigated the effects of heparin and the thrombin inhibitor bivalirudin on bone marrow-derived mononuclear cell (BMC) functional activity and homing capacity. Methods and Results:

Objective: To assess the effect of long-term pharmacological inhibition of miR-21 in a model of metabolic syndrome and obesity. Methods: Aged db/db mice were treated with locked nucleic acid-modified anti-miRs directed against miR-21 (LNA-21), control LNAs or PBS for 18 weeks. Cardiac function was assessed by echocardiography and the effect on body weight and white adipose tissue (WAT) was evaluated. Results: MiR-21 expression was efficiently inhibited in the heart and WAT with no apparent liver toxicity or deterioration of kidney function. MiR-21 inhibition had no effect on cardiac hypertrophy as well as systolic and diastolic cardiac functions. However, levels of cardiac collagen 1 were modestly reduced in LNA-21 treated mice. MiR-21 inhibition reduced body weight, as well as adipocyte size and serum triglycerides were significantly decreased. The miR-21 targets TGFb-receptor 2 (TGFBR2) and phosphatase and tensin homolog (PTEN) were derepressed in WAT of LNA-21 treated mice and Sprouty1 and 2 were increased after miR-21 inhibition. Conclusions: Long-term treatment with LNA-21 is safe and efficiently suppresses miR-21 expression. Cardiac function was not affected. LNA-21 treatment led to a significant weight loss and reduces adipocyte size as well as derepression of the targets TGFRB2, PTEN, and Sprouty1 and 2.