Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: Distinct chemotactic SMCs express high levels of MΦ chemoattractants, ameliorating atheroprogression (A–C) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780 . (A) Violin plots (calculated on all cells expressing detectable baseline levels of the respective gene) of highly expressed cytokines and chemokines in chemotactic SMCs. Dots represent single cells, only cells exhibiting detectable expression of the particular gene are included (B) interactome depicting cell-cell interactions between MΦ and SMC subsets, prominent SMC → MΦ interactions are depicted in red. Intensity of red color depicts the respective portion of the CCL2-CCR2 axis for the concrete interaction (the darker the red color, the more the CCL2-CCR2 axis accounts for the respective inter-cluster interplay among all detected chemokine-receptor interactions). (C) Heatmap further unraveling SMC → MΦ chemokine:chemokine-receptor interactions. Blue box depicts interactions of chemotactic SMC subset, red box depicts CCL2-mediated interactions between SMC and MΦ subsets. (D) Ccl2 and Mif expression in Ng2 + SMCs FACS-sorted from western-diet fed atherosclerotic MC RFP-rep mice compared to chow-diet fed non-atherosclerotic control mice. n = 3–4 mice per group. (E) Representative images of BCA sections from Ccl2 SMC +/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet stained for ACTA2 (green), LGALS3 (red), and Hoechst (blue). Scale bars, 100 μm. (F and G) Morphometric analysis of plaque size (F) and vascular remodeling (G) from BCA sections at three consecutive locations from Ccl2 SMC +/+ (n = 11) and Ccl2 SMCΔ/Δ (n = 10) littermates. (H and I) Quantification of ACTA2 + smooth muscle cell content as ACTA2 + area in percentage of total plaque area and percentage of 30 μm plaque surface area in valves (H) and in the BCA at three consecutive locations (I). (H and I) n = 10–11 mice per group. (J and K) Analysis of intimal LGALS3 + area as percentage of plaque size in BCA sections at three consecutive locations (J) and in plaques from aortic valves (K) (n = 10–11 each). (L) Schematic illustration of media and intima processing from aortae of Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet (left). Heatmap displaying expression of differentially regulated genes in bulk RNA-seq of Ccl2 SMC+/+ mice (n = 3) and Ccl2 SMCΔ/Δ mice (n = 4). Rows represent individual replicates, differentially expressed genes are illustrated in columns (right). (M) Volcano plots of intima/media RNA-seq showing differentially expressed genes in Ccl2 SMC+/+ mice (n = 3) and Ccl2 SMCΔ/Δ mice (n = 4), x-axis depicts Log2FC, y-axis depicts -Log10(adj. p-value). Data are shown as mean and SEM. (H and K), Student’s t test was used. (F, G, I, and J) Repeated measures two-way ANOVA or mixed-effects model was used. ∗ p < 0.05; NS, not significant. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression.

Article Snippet: SMCs were cultivated with SMC medium (PELOBiotech® PB-MH-200-2190) for at least 1 week before they were split.

Techniques: RNA Sequencing, Expressing, Western Blot, Control, Staining

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: SMCs exert chemotactic cues on plaque MΦs (A–F) In vivo imaging of an atherosclerotic lesions within the carotid artery in atherosclerotic MC RFP-rep ; Lyz-MΦ GFP-rep mice after 14 weeks western diet by multi-photon microscopy. (A) Time-series with a focus on the shoulder region of the plaque, arrows depicting locally confined but dynamic protrusions formed by Lyz + MΦs (green) toward SMCs (red). Images from . Scale bars, 20 μm. (B) In vivo imaging of Lyz + MΦ-SMC contacts during Lyz + MΦ migration within the intima. Rendered illustration of Lyz + MΦs (green) migrating along SMCs (red), including exemplary migration tracks of 2 cells. Scale bars, 20 μm. (C) Analysis of the duration of the interactions between SMCs (red) and Lyz + MΦs (green). (D) Analysis of the displacement length during interaction and during free migration. (E) Velocity profile of cells 1 and 2 (labeled in the migration tracks above under B) over time: boxes indicate interactions; horizontal lines indicate mean velocity of the time period included. (F) Left: meandering index (track straightness) of Lyz + MΦs during interaction with SMCs and during free migration without interaction. Right: displacement rate of Lyz + MΦs during interaction with SMCs and during free migration without interaction with SMCs. (C–F) n = 46–52 cell tracks covering free migration (−) or subsequent SMC interaction (+) or vice versa from n = 3 mice, Mann-Whitney test used to compare groups, ∗∗∗ p < 0.001, ∗∗ p < 0.001. (G) In vivo imaging of static SMC-Cx3cr1 + MΦ contacts in a MC RPF-rep ; Cx3cr1-MΦ GFP-rep mouse, arrow depicting a SMC embedded in two Cx3cr1 + MΦs. Scale bars, 20 μm. (H) Ex vivo confocal imaging of cross-sections of atherosclerotic valves in MC RFP-rep ; Cx3cr1-MΦ GFP-rep mice after 12 weeks western diet, MΦs in green, SMCs in red, nuclei in blue, arrowheads depicting SMC-MΦ contacts. Scale bars, 40 μm. (I and J) Ex vivo confocal imaging of SMC lin ; Cx3cr1-MΦ GFP-rep mice after 22–24 weeks of western diet, (I) atherosclerotic valve cross-sections, MΦs in green, SMC and SMC-progeny in red, nuclei in blue, arrowheads depicting SMC-MΦ contacts, dashed line outlining SMC enveloping MΦ. Scale bars, 10 μm. (J) En face confocal z stacks of the atherosclerotic intima in SMC-tdT lin ; Cx3cr1-MΦ GFP-rep mouse, Cx3cr1 + MΦs in green, SMC lin cells in red, blue arrowheads pointing toward SMC-MΦ contacts observable in the cross- and longitudinal-sections of the z stack, most frequent within the plaque surface. Scale bars, 50 μm (left), 5 μm (right) (K), left: representative immunohistochemical images of human aortic plaques stained for CD68 and α-SMA, scale bars, 300 μm, right: immunofluorescence staining for CD68 (red) and α-SMA (green) and with Hoechst (blue), dashed lines represent macrophage and SMCs, scale bars, 20 μm on the left and 10 μm on the right immunofluorescence image. (L) Pearson correlation of the relative CD68 + area in fibrous cap environment (defined as the plaque area within the top 30% plaque surface) with, left: the relative necrotic core size, middle: the α-SMA content and right: the plaque vulnerability index (further elaborated in methods). Intermediate (n = 5) and advanced (n = 8) human plaques, graded accordingly by the pathology department, were included. Pearson r and two-tailed p value are included for every Pearson correlation. (M) Summary illustration of the natural MΦ distribution within atherosclerotic lesions. MΦs mainly localize at areas of the plaque surface adjacent to SMCs in murine and in human atherosclerotic lesions. Bar graphs show mean with SEM.

Article Snippet: SMCs were cultivated with SMC medium (PELOBiotech® PB-MH-200-2190) for at least 1 week before they were split.

Techniques: In Vivo Imaging, Western Blot, Microscopy, Migration, Labeling, MANN-WHITNEY, Ex Vivo, Imaging, Immunohistochemical staining, Staining, Immunofluorescence, Two Tailed Test

Journal: Immunity

Article Title: Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation

doi: 10.1016/j.immuni.2023.08.002

Figure Lengend Snippet: SMCs within the fibrous cap preserve a strategic positioning of plaque MΦs and secure homeostatic MΦ functions (A and B) Reanalyzed single-cell RNA-seq data from mouse aortic roots from atherosclerotic SMC lin mice from Wirka et al., GEO: GSE131780 . (A) UMAP based dimensionality reduction of analyzed cells (left), heatmap illustrating cytokine and chemokine expression of different SMC subsets (right). (B) Marker genes of SMC clusters illustrated in a heatmap, composed by ClustVis. (C) Representative confocal image depicting the spatial distribution of the key cSMC marker PDGFRβ within an atherosclerotic valve in SMC lin ; Cx3cr1-MΦ GFP-rep mice after 22–24 weeks of western diet, SMC lin cells in red, MΦs in green, and PDGFRβ in white. Scale bars: 30 μm (left) and 15 μm (right images). (D) Illustration of the experimental setup of the migration assay: macrophages undergo a migratory decision either moving toward the artificially composed SMC-rich fibrous cap below or residing at the artificially composed, necrotic cell rich, necrotic core. SMCs (representing the fibrous cap) are located in the lower chamber, whereas peritoneal macrophages have been attached on the transwell of the upper chamber. Necrotic Jurkat cells (representing the necrotic core) have been added to the upper chamber. (E) Number of peritoneal MΦs from Lyz-MΦ GFP-rep mice that transmigrated toward the lower chamber per field of view (FOV). Isotype or anti-CCL2 blocking antibody was simultaneously added to the lower chamber. MΦ numbers per FOV counted at 4 subsequent time points (n = 4 independent experiments). (F) Distribution of macrophages as percentage of LGALS3 + area in 30 μm plaque surface area in percentage of total plaque LGALS3 + area at three subsequent locations (n = 10 each). (G) Left: quantification of LGALS3 + surface macrophage content as relative LGALS3 + area in percentage of total plaque surface area (defined as the upper 30 μm of the plaque) from BCA sections at three consecutive locations (n = 10 each). Right: representative immunofluorescent images of BCA sections for ACTA2 (green), LGALS3 (red), and Hoechst (blue) with highlighted 30 μm plaque surface area from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western-diet feeding. Scale bars, 100 μm. (H) Volcano plot depicting differentially regulated genes analyzed by RNA-seq of FACS-sort enriched peritoneal MΦs, coincubated either with live or dead Jurkat cell supernatant for 12 h. (I) Quantification of peritoneal macrophages 12 h after addition of live or dead Jurkat cell supernatant (n = 6). (J–L) Efferocytosis assay, analyzing the efferocytotic capacity of the MΦ population, isolated from Lyz-MΦ GFP-rep mice. Apoptotic Jurkat cells were added for 1 h after 6 h incubation either with or without CCL2. (J) Quantification of MΦs with engulfed apoptotic cells upon presence or absence of CCL2 (n = 5 independent experiments). (K) Quantification of the total number of engulfed apoptotic cells upon CCL2 presence of absence. (L) Representative epifluorescence images of the efferocytosis assay with peritoneal macrophages (green) and apoptotic Jurkat cells (red), 1 h after Jurkat cell addition. Scale bars, 50 μm. (M–O) Necrotic core analysis as total necrotic area in μm 2 (M) and in percentage of plaque area (N), assessed with Masson Trichrom’s staining of valve sections, from Ccl2 SMC+/+ (n = 9) and Ccl2 SMCΔ/Δ (n = 10) littermates after 14 weeks of western diet. (O) Left: representative images of necrotic core content analyzed by Masson Trichrom’s staining of valve sections from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet. ∗ indicates necrotic areas. Scale bars, 100 μm. Right: representative images of immunofluorescence stainings of valve sections from Ccl2 SMC+/+ and Ccl2 SMCΔ/Δ littermates after 14 weeks of western diet for ACTA2 (green), LGALS3 (red), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (yellow), and DAPI (blue). Scale bars, 100 μm. (P and Q) Quantification of cell apoptosis as total amount of TUNEL + LGALS3 + Hoechst + MΦs in plaque (P) and as total amount of TUNEL + Hoechst + apoptotic cells (Q) in Ccl2 SMC+/+ (n = 9) and Ccl2 SMCΔ/Δ (n = 10) individual littermates in total after 14 weeks of western diet, only including plaques at the proximal and intermediate BCA, without distal BCA areas with its early lesions. (R) Quantification of valve atherosclerotic plaques for (left) total and relative TUNEL + cells. Data are shown as mean and SEM. (I, J, K, M, N, and R) Student’s t test was used for normally distributed data and Wilcoxon matched-pairs signed rank test for not normally distributed data. (E, F, G, P, and Q) Repeated measure two-way ANOVA or mixed-effects model was used. ∗ p < 0.05; ∗∗ p < 0.01; NS, not significant. Bar graphs show mean with SEM.

Article Snippet: SMCs were cultivated with SMC medium (PELOBiotech® PB-MH-200-2190) for at least 1 week before they were split.

Techniques: RNA Sequencing, Expressing, Marker, Western Blot, Migration, Blocking Assay, Isolation, Incubation, Staining, Immunofluorescence, End Labeling, TUNEL Assay

Journal: iScience

Article Title: Distinct brain and lung endothelial miRNA/mRNA profiles after exposure to Plasmodium falciparum- infected red blood cells

doi: 10.1016/j.isci.2024.111265

Figure Lengend Snippet:

Article Snippet: HMVEC-L (lung ECs) (Provitro- #1210144) cultivated in microvascular EC growth medium (Provitro- #2010102) according to Provitro guidelines.

Techniques: Cell Culture, Software