Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (A) or CCL2 (B) at concentrations of 5, 10, 50, or 100 ng/mL, and cell viability was determined by cell counting using a hemocytometer or MTT assay, respectively. (C) Flow cytometry results are presented as histograms of the average percentage of cells that expressed IGF-1R and CCR2 receptors (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 4/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to the control group: p < 0.05 (*) or p < 0.0001(***); significant value compared to control group and the other treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Cell Counting, MTT Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by fluorescence microscopy. ( A ) Photomicrographs show the expression of FN ascertained by immunofluorescence and fluorescence microscopy analysis. Magnification: 400× ( B ) Bars correspond to the quantitative analysis of FN expression in tEnd.1 cells in selected microscopic fields (n = 5/group). The results are expressed in pixels/μm 2 . ( C ) Flow cytometry results are presented as histograms of the average percentage of cells that expressed CD49e/VLA-5 and CD44 receptors for FN (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 5/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.0001 (***); significant values compared to control group and single treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: (A) tEnd.1 cells treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h on BSA or FN coating were stained with Alexa 488-phalloidin and analyzed by confocal microscopy with a 63× objective. ( B ) tEnd.1 cells were allowed to adhere on BSA- or FN-coated surfaces for 1 h after stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 24 h. ( C ) tEnd.1 cells were allowed to migrate through transwell chambers coated with BSA or FN after chemotactic stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 6 h. Photomicrographs demonstrate cells invading through the transwell membrane. Giemsa staining. Scale bar = 10 μm. ( D ) Bars represent the number of migrating cells in a transwell system. Data are represented as mean ± SEM (n = 5/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (***); significant values compared to control group and the IGF-1 treatment: p < 0.05 (#); and significant values compared to control group and single treatments: p < 0.01 (+).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Staining, Confocal Microscopy, Membrane, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: ( A ) tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by optical microscopy. Photomicrographs show intracellular lumina in tEnd.1 cells, indicated by arrows. Giemsa staining. Scale bar = 10 μm. ( B ) tEnd.1 cells were treated with IGF-1, CCL2, or IGF-1/CCL2 for 8 days on BSA or FN coating and analyzed by optical microscopy. Photomicrographs demonstrate capillary-like structures, indicated by asterisks. Giemsa staining. Scale bar = 10 μm. ( C ) Number of capillary-like structures. ( D ) Luminal area of capillary-like structures. Bars represent the mean ± SEM (n = 6/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant compared with control, p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Microscopy, Staining, Control

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 3. The accumulation of cells 24 h after CCL3 injection is hampered in NOD (5–7-week-old) mice (A). Granulocytes (B) and monocytes (C) are not attracted in NOD mice. In response to CCL2, NOD mice show a deficient accumulation of cells into the air pouch after 24 h (D). The accumulation of monocytes to CCL2 is almost absent in NOD (E). Data are cumulative of two individual experiments and represented as mean SEM (n=6/group in A and C; n=4/group in B, C, E). *p<0.05 relative to NOD as determined with Mann-Whitney (A, D) or Student's t-test (B, C, E).

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Injection, MANN-WHITNEY

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 6. The air pouch of C57BL/6 and BALB/c contains a high number of MU (A) and DC (B) after CCL2 injection, while those of NOD mice do not. MU and DC are defined as shown in Fig. 4. Data are cumulative of two experiments and are represented as mean SEM; each bar represents at least four animals (5–7 weeks old), *p<0.05 as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Injection

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 7. The air pouch of control mice at 24 h after CCL2 injection shows an increased production of IL-1b; however, this is not seen in NOD (A). NOD mice produce higher amounts of IL-10 in comparison to C57BL/6 and BALB/c mice (B). A representative flow cytometric analysis of intracellular staining for IL-10 (gated for CD11b+ cells) shows that monocytes (CD11bhiF4/80medCD11clow) are producing IL-10, while MU (CD11b+F4/80hiCD11clow) do not (C). Figures shown are representative of five NOD mice 24 h after injection with CCL2. The production of TNF-a in the air pouch of NOD mice is similar to control mice (D). Data are represented as mean SEM (n=5/group; 5–7 weeks old). *p<0.05 relative to NOD as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Control, Injection, Comparison, Staining

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 9. Peritoneal exudate MU of NOD mice show a decreased migration towards CCL2 compared to C57BL/6 control mice using a Boyden chamber chemotaxis assay. Data are represent mean SD (n=3), *p<0.05 relative to NOD as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Migration, Control, Chemotaxis Assay

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 8. Neutralization of IL-10 does not restore the cell recruitment towards CCL2 to the level of C57BL/6 mice (A). Addition of recombinant IL-10 to the air pouch of C57BL/6 mice decreases the recruitment towards the control level (A). Blocking IL-10 raises the level of IL-1b in the air pouch of NOD to a similar level of C57BL/6 mice (B). Addition of recombinant IL-10 in the air pouch of C57BL/6 does not lower the amount of IL-1b (B). Mean SD (n=6) are shown. Data in (B) are presented relative to the amount of IL-1b that is observed in control-injected animals. Statistical significance was deter- mined with Kruskall-Wallis test and differences between groups using the Mann-Whitney test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Neutralization, Recombinant, Control, Blocking Assay, Injection, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: BMP9 inhibits CCL2 expression and release by endothelial cells. Confluent HPAECs were serum-restricted for 16 h followed by treatment with BMP9 in 0.1% FBS. (A) HPAECs were treated with 1 ng/ml BMP9 for 2, 4, 8 or 12 h (3 experiments). Data show the fold change relative to 0.1% FBS at each time point. (B) HPAECs were treated with BMP9 (0-10 ng/ml) for 8 h (5 experiments). (C) HPAECs were treated with BMP9 (0-10 ng/ml) for 24 h. CCL2 immunoreactivity of conditioned media was normalized to cell number for each well ( n =4 wells per treatment) and is representative of 3 experiments. (D) HPAECs were treated with BMP10 (0-10 ng/ml) for 8 h (3 experiments). (E) HPAECs were treated with BMP9 (1 ng/ml) or BMP10 (1 ng/ml) for 24 h. CCL2 immunoreactivity of conditioned media was normalized to cell number for each well. Data ( n =4 wells per treatment) are representative of 3 experiments. (F,G) HPAECs were treated with BMP9 (1 ng/ml) or BMP10 (1 ng/ml) for 8 h and expression of CCL2 (F) and ID1 and ID2 (G) measured (6 experiments). (H,I) HAECs were treated with BMP9 (0-10 ng/ml) (H) or BMP10 (0-10 ng/ml) (I) for 8 h (3 experiments). All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to control. Significance was calculated using either one-way repeated measures ANOVA with post-hoc Tukey's HSD test (B,E-G) or Friedman multiple comparison test with post-hoc Dunn's analysis (C,D,H,I). * P <0.05, ** P <0.01, *** P <0.001, compared with control (0.1% FBS without added BMP9 or BMP10).

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Expressing, Control, Comparison

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: Reduction of ALK1 attenuates the induction of CCL2 by BMP9, and both ACTR-II and BMPR-II mediate the repression by BMP9 and BMP10. (A-C) HPAECs were transfected with siRNA for ALK1 (siA1), BMPR2 (siB2) or a non-targeting control pool (siCP) using DharmaFECT1 (DH1). (A) HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 8 h. Expression of CCL2 was normalized to ACTB . Data show the fold change relative to DH1/0.1% FBS (4 experiments). (B) HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 24 h. Conditioned media were collected, assayed for CCL2 by ELISA and normalized to cell number for each well. Data ( n =4 wells per treatment) are from a representative of 4 experiments. (C) Specific reduction of ALK1 and BMPR-II by their respective siRNAs was confirmed by western blotting, the numbers below the blots representing band density ratios relative to α-tubulin normalised to the DH1 control. Arrows indicate the positions of the molecular mass markers (kDa). (D-F) HPAECs were transfected with a non-targeting control siRNA pool (siCP) or siRNAs for ACVR2A (siA2A), BMPR2 (siB2) or both in combination (siA2AB2) using DharmaFECT1 (DH1). HPAECs were treated with 1 ng/ml BMP9 or BMP10 in 0.1% FBS for 8 h. Expression of ACTR-IIA ( ACVR2A ; D), BMPR-II ( BMPR2 ; E) and CCL2 (F) were normalized to ACTB . Data show the fold change relative to DH1/0.1% FBS (6 experiments). The key for D-F is provided in F. (G-I) Confluent serum-restricted HPAECs were pretreated with 250 nM LDN-193189 or 2µM SD208 for 1 h followed by 1 ng/ml BMP9 in 0.1% FBS for 8 h for mRNA extraction. Expression of CCL2 (G), CXCL8 (H) and ID1 (I) were determined by qPCR and normalized to ACTB . qPCR data are presented as the fold change relative to the DMSO control (1:2500 in 0.1% FBS) (3 experiments). All data are expressed as mean±s.e.m. Significance was calculated using either a paired Students t -test (A,B,G-I), comparing with 0.1% FBS control, or one-way repeated measures ANOVA with post-hoc Sidak test (D-F), comparing with siCP of same treatment. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Transfection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Extraction

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: CCL2 repression by BMP9 in HPAECs is dependent on Smad4, but not Smad2 or Smad3. (A) Confluent serum-restricted HPAECs were treated with 0.01-10 ng/ml BMP9 in 0.1% FBS for 1 h. Protein lysates were immunoblotted for phospho-Smad1/5, Smad1, phospho-Smad2 or Smad2. Blots are representative of 4 experiments. (B) Quantification of the blots in A, calculated as the ratio of the density of the phospho-Smad band to the Smad band for each sample and normalised to the 0.1% FBS control. (C-E) HPAECs were transfected with SMAD4 siRNA (siS4) or a non-targeting control pool (siCP) using DharmaFECT1 (DH1). (C) Reduced Smad4 protein was confirmed by western blotting. (D) Confluent serum-restricted HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 8 h (3 experiments). (E) Confluent serum-restricted HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 24 h. CCL2 release was measured by ELISA and normalised to cell number. Data are from a representative of 3 experiments ( n =4 wells per treatment). (F,G) HPAECs were transfected with SMAD2 siRNA (siS2) or siCP using DH1 and treated with BMP9 for 8 h as described above. (F) CCL2 (top panel) and CXCL8 (bottom panel) expression (3 experiments). (G) Smad2 protein knockdown was confirmed by western blotting. (H,I) HPAECs were transfected with SMAD3 siRNA (siS3) or siCP using DH1 and treated with BMP9 for 8 h. (H) Smad3 protein knockdown was confirmed by western blotting. (I) CCL2 expression (3 experiments). For western blots, the migration positions of the relevant protein molecular mass markers (kDa) are indicated by arrows. The numbers below each blot panel represent band density ratios relative to α-tubulin normalised to the DH1 control. All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to DHI/0.1% FBS. Significance was calculated using one-way repeated measures ANOVA with post-hoc Tukey’s HSD test. * P <0.05.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Control, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Knockdown, Migration

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: CCL2 repression by BMP9 in HPAECs is not dependent on Smad1, Smad5 or Smad9. HPAECs were transfected with siRNAs for SMAD1 (siS1), SMAD5 (siS5) or SMAD9 (siS9) alone or in combination using DharmaFECT1 (DH1). In parallel, cells were transfected with a non-targeting control pool (siCP). (A,B) Knockdown of Smad1 and Smad5 were confirmed by western blotting of cells transfected with siRNAs targeting individual (A) and combinations (B) of Smads. The migration positions of the relevant protein molecular mass markers (kDa) are indicated by arrows. The numbers below each blot panel represent band density ratios relative to α-tubulin normalised to the DH1 control. (C-E) Transfected HPAECs were serum-restricted, followed by treatment with 1 ng/ml BMP9 in 0.1% FBS for 8 h. CCL2 expression (C) and ID2 and CXCL8 expression (D) were determined in cells in which individual Smads were knocked down (5 experiments). CCL2 and ID2 expression (E) were determined in HPAECs in which combinations of Smads were knocked down (4 experiments). (F) Transfected HPAECs were serum-restricted, followed by treatment with 0.3 ng/ml BMP9 or BMP10 in 0.1% FBS for 4h (5 experiments). All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to DH1/0.1% FBS. Significance was calculated using one-way repeated measures ANOVA with post-hoc Tukey's HSD (C-E) or Sidak (F) test. * P <0.05, ** P <0.01, *** P <0.001. For CCL2 , data were compared with siCP/0.1% FBS. For ID2 and CXCL8 , data were compared with siCP/BMP9.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Transfection, Control, Knockdown, Western Blot, Migration, Expressing

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: BMP9 does not affect CCL2 induction by TNF-α in HPAECs and HAECs. (A) Confluent serum-restricted HPAECs were treated with BMP9 (5 ng/ml) alone or with TNF-α (5 ng/ml) in 0.1% FBS for 6 h. Co-treatments were added without BMP9 pre-incubation or after 1 h or 16 h pre-incubation with 5 ng/ml BMP9 (3 experiments). (B) Confluent serum-restricted HPAECs were treated with BMP9 (5 ng/ml) and TNF-α (5 ng/ml) in 0.1% FBS for 6 h. Conditioned media were assayed for CCL2 by ELISA. Data are from a representative of 3 experiments ( n =4 wells per treatment). (C) Confluent serum-restricted HPAECs were treated with BMP9 (0-5 ng/ml) and TNF-α (0-2 ng/ml) in 0.1% FBS for 6 h (4 experiments). (D,E) Confluent serum-restricted HAECs were treated with BMP9 (5 ng/ml) alone or with TNF-α (5 ng/ml) in 0.1% FBS for 6 h and assayed for CCL2 expression (D) (4 experiments) and CCL2 release (E). ELISA data are from a representative of 3 experiments ( n =4 wells per treatment). (F) Confluent serum-restricted HAECs were treated with BMP9 (0-5 ng/ml) and TNF-α (0-5 ng/ml) in 0.1% FBS for 6 h. All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to 0.1% FBS (no additions). Significance was calculated using Friedman multiple comparisons test with post-hoc Dunn’s analysis. * P <0.05, ** P <0.01, *** P <0.001, compared with control (0.1% FBS).

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Control

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: Mural cells sustain a vascular MΦ niche (A) In vivo multi-photon imaging of Ca 2+ signal and morphological changes of MΦs in Cx3cr1-MΦ Ca-rep mice in an environment of laser-induced microinjuries. MΦs are depicted in red; Ca 2+ signal and vascular flow are depicted in green. MΦs have been rendered additionally below, with a pseudocolored depiction of the Ca 2+ signal. Images are derived from . (B) In vivo and ex vivo confocal and airy-scan imaging of MC-MΦ contacts across organs in MC RFP-rep ; Cx3cr1-MΦ GFP-rep mice, arrows depicting cell-cell contacts: top left: intravital imaging of the microvasculature in the mesentery, the dashed line is depicting MCs (scale bars, 5 μm); top middle: ex vivo imaging of the heart microvasculature (scale bars, 50 μm); top right: en face ex vivo imaging of the aortic atherosclerotic intima after 3 months of western-diet feeding (macrovasculature), the dashed line is subdividing the plaque core from the shoulder region (scale bars, 10 μm); bottom left: ex vivo imaging of the kidney microvasculature (scale bars, 50 μm), including higher magnification below (dashed line depicting MCs) (scale bars, 7 μm); bottom middle: ex vivo imaging of the lung microvasculature (scale bars, 20 μm), including higher magnification below (Cx3cr1 hi CD68 lo interstitial MΦs (iMΦs) in green, CD68 hi Cx3cr1 lo alveolar MΦs (aMΦs) in white, MCs in red) (dashed line depicting MCs) (scale bars, 5 μm); bottom right: ex vivo imaging of the stomach microvasculature (scale bars, 50 μm), including higher magnification below (dashed line depicting MCs) (scale bars, 10 μm). Interstitial MΦs are shown in green, and MCs are shown in red for all organs, with further subdifferentiation of MΦs in the lung (as depicted above). (C) Analysis of the time until MΦs form their first dendrites (left) and time which MΦs require to reach injury (right), as the time in minutes after laser injury, in MΦ GFP-rep mice treated locally (subcutaneously) and systemically with isotype or CCL2-neutralizing antibody (n = 17–37 individual cells analyzed from 3 to –4 mice/group). (D) Reanalyzed single-cell RNA-seq data from human coronary arteries from Wirka et al., GEO: GSE131780 . Uniform Manifold Approximation and Projection (UMAP) based dimensionality reduction of analyzed cells. (E) Highly expressed cytokines and chemokines in human SMCs from coronary arteries analyzed from cells shown in (D) CCL2 is highlighted as the most prominently expressed chemokine. (F) Percentage of peritoneal macrophage survival upon CCL2 stimulation at different time points under starvation stress conditions (n = 3 experiments). (G) Quantification of CD68 + perivascular macrophage content in Ccl2 MC+/+ and Ccl2 MCΔ/Δ mice in percentage of total perivascular area (15 μm radius around the vessel) in the kidney (n = 5–6 mice/group). (H) Quantification of cell proliferation as EdU + cells relative to CD68 + area (as number of proliferating cells/μm 2 ). (I) Quantification of blood monocyte counts by automated blood counter (n = 5–6). (J) Representative images from immunofluorescence staining of kidney sections in Ccl2 MCΔ/Δ and Ccl2 MC+/+ mice for ACTA2 (red), CD68 (green). Scale bars, 50 μm (left: Ccl2 MC+/+ ; right: Ccl2 MCΔ/Δ ). (C, G, H, and I) Student’s t test was used. (F) Repeated measures two-way ANOVA was -808990139890500used. ∗ p < 0.05. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: In Vivo, Imaging, Derivative Assay, Ex Vivo, Western Blot, Single Cell, RNA Sequencing, Immunofluorescence, 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: MC-derived CCL2 sustains a homeostatic MΦ phenotype across the vascular tree (A and B) UMAP based dimensionality reduction of single-cell RNA-seq of FACS-sort enriched CD45 + CD11b hi CD64 hi F4/80 hi cells in kidney (A) and lung (B) of Ccl2 MCΔ/Δ and Ccl2 MC+/+ mice (n = 4/group). (C–G) Volcano and violin plots depicting selected significantly differentially regulated genes in (C) kidney resident MΦ cluster 2, (D) kidney resident MΦ cluster 0, (E) lung monocyte cluster 1, (F) lung alveolar MΦ cluster 2, (G) lung Folr2 hi Mrc1 hi interstitial MΦ cluster 3. (H) Frequency of lung Zeb2 hi interstitial MΦ cluster 4 cells among all analyzed cells. (I) Significantly differentially regulated genes, associated with a functionally differentiated, efferocytotic MΦ phenotype in Ccl2 MC+/+ and Ccl2 MCΔ/Δ chimera mice. Low-input RNA-seq of FACS-sorted Cx3cr1 + MΦs from Ccl2 MC+/+ or Ccl2 MCΔ/Δ chimera mice with MC RFP-ep ; Cx3cr1-MΦ GFP-rep bone marrow after 20 weeks western diet (experimental setup further depicted in Figure S3 ) (n = 3–4 chimera mice). Student’s t test was used. Expression levels of depicted genes normalized to sample with highest expression (set as 1) across all samples. Bar graphs show mean with SEM. Violin plots with matching boxplot and mean expression. ∗ p < 0.05.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Derivative Assay, Single Cell, RNA Sequencing, Western Blot, Expressing

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: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Single Cell, 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 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: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

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

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: Short-term CCL2 inhibition in advanced atherosclerosis triggers detrimental changes in plaque phenotype (A) Acute pharmacological CCL2 inhibition in ApoE −/− mice after 6 months of western type diet. The anti-CCL2 or isotype control antibody was injected intravenous (i.v.) 2 weeks before sacrifice every 48 h (n = 7–8 / group). (B) Quantification of fibrous cap coverage as continuity (percentage of fibrous cap covered plaque surface length relative to complete plaque surface length) at three subsequent BCA locations. (C) Quantification of ACTA2 + area within plaque surface as % of plaque surface area (defined as the top 30 μm stripe of the lesion) at three subsequent BCA locations. (D) Quantification of absolute ACTA2 + area in μm 2 at three subsequent BCA locations. (E) Quantification of macrophage area as LGALS3 area in μm 2 at three subsequent BCA locations. (F) Quantification of total plaque size as absolute plaque area in μm 2 at three subsequent BCA locations. (G) Quantification of cell apoptosis as total amount of TUNEL + cells in plaque at three subsequent BCA locations. (H) Representative images of BCA sections from ApoE −/− mice after 6 months of western diet stained for ACTA2 (green), LGALS3 (far red), TUNEL (red), and Hoechst (blue). Scale bars, 50 μm. (I) Quantification of blood leukocytes, neutrophils, lymphocytes, monocytes, and plasma cholesterol (n = 7–8). (I) Student’s t test was used. (B–G) Repeated measures two-way ANOVA or mixed-effects model, with subsequent Šídák’s multiple comparisons test in (B)–(D), was used. ∗ p < 0.05; ∗∗ p < 0.01 NS, not significant. Bar graphs show mean with SEM.

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Inhibition, Western Blot, Control, Injection, TUNEL Assay, Staining, Clinical Proteomics

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:

Article Snippet: MIF inhibitor 1600 μM (ISO-1 HY-16692, MedChemExpress), CCL2 specific monoclonal antibody 20 μg/ml (MCP-1 16-7096-85, eBioscience) and IgG isotype control 20 μg/ml (16-4888-85, Invitrogen ®) reagents were supplemented to the different conditions as described.

Techniques: Control, Blocking Assay, Selection, Recombinant, Western Blot, In Situ, Staining, Single Cell, DNA HS Assay, Picogreen Assay, cDNA Synthesis, Clinical Proteomics, SYBR Green Assay, Lysis, Enzyme-linked Immunosorbent Assay, Isolation, Software