Journal: bioRxiv

Article Title: Characterizing the SASP-Dependent Paracrine Spreading of Senescence Between Human Brain Cell Types

doi: 10.64898/2026.02.10.705129

Figure Lengend Snippet: A) Volcano plot of Nomic Bio Core Immune panel (268 cytokines) in DMSO and BrdU CM from astrocytes showing factors that are upregulated in BrdU CM treated (red) (log2fc > 1, p-value < 0.05) and downregulated in BrdU CM treated (blue) (log2fc < -1, p-value < 0.05). Black data points indicate factors that do not reach the threshold (−1 < log2fc < 1 | p-value > 0.05). B) Volcano plot of Nomic Bio Core Immune panel cytokines in DMSO and BrdU CM from endothelial cells showing factors that are upregulated in BrdU CM treated (red) (log2fc > 1, p-value < 0.05) and downregulated in BrdU CM treated (blue) (log2fc < -1, p-value < 0.05). Black data points indicate factors that do not reach the threshold (−1 < log2fc < 1 | p-value > 0.05). C) Volcano plot of Nomic Bio Core Immune panel cytokines in DMSO and BrdU CM from microglia showing factors that are upregulated in BrdU CM treated (red) (log2fc > 1, p-value < 0.05) and downregulated in BrdU CM treated (blue) (log2fc < -1, p-value < 0.05). Black data points indicate factors that do not reach the threshold (−1 < log2fc < 1 | p-value > 0.05). D) Volcano plot of Nomic Bio Core Immune panel cytokines in DMSO and BrdU CM from oligodendrocytes showing factors that are upregulated in BrdU CM treated (red) (log2fc > 1, p-value < 0.05) and downregulated in BrdU CM treated (blue) (log2fc < -1, p-value < 0.05). Black data points indicate factors that do not reach the threshold (−1 < log2fc < 1 | p-value > 0.05). E) Volcano plot of Nomic Bio Core Immune panel cytokines in DMSO and BrdU CM from neurons showing factors that are upregulated in BrdU CM treated (red) (log2fc > 1, p-value < 0.05) and downregulated in BrdU CM treated (blue) (log2fc < -1, p-value < 0.05). Black data points indicate factors that do not reach the threshold (−1 < log2fc < 1 | p-value > 0.05). F) Venn diagram showing the number of upregulated factors specifically in astrocyte BrdU CM and microglia BrdU CM (69) as well as the commonly upregulated factors . G) Z-score heatmap showing selected Core Immune panel (Nomic Bio) cytokine expression in all cell types (endothelial cells (pink), microglia (yellow), oligodendrocytes (green), astrocytes (purple), and neurons (blue)) based on nELISA analysis of CM from DMSO (grey) and BrdU (red) treated cell lines (n=3 replicates). H) Normalized expression of CCL2 based on human cytokine array in DMSO CM (grey) and BrdU CM (red) from astrocytes, endothelial cells, and microglia (n=4 replicates). Normalized expression of MIF based on human cytokine array in DMSO CM (grey) and BrdU CM (red) from astrocytes, endothelial cells, and microglia (n=4 replicates). I) Normalized nELISA signal of CCL2 in DMSO CM (grey) and BrdU CM (red) from astrocytes, endothelial cells, microglia, oligodendrocytes, and neurons (n=3 replicates). Normalized nELISA signal of MIF in DMSO CM (grey) and BrdU CM (red) from astrocytes, endothelial cells, microglia, oligodendrocytes, and neurons (n=3 replicates). Data was analyzed by two-way ANOVA with Tukey’s multiple comparisons test (H) and two-way ANOVA with Šídák’s multiple comparisons test (I). All graphs show mean with error bars depicting standard deviation (ns, p>0.05, *** p<0.001).

Article Snippet: Treatment with the CCL2 synthesis inhibitor Bindarit (MedChemExpress, Catalog No. HY-B0498) ( ) was used to specifically target CCL2 production in senescent cells.

Techniques: Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Characterizing the SASP-Dependent Paracrine Spreading of Senescence Between Human Brain Cell Types

doi: 10.64898/2026.02.10.705129

Figure Lengend Snippet: A) Schematic depicting BulkSignalR pipeline which uses known ligand-receptor interactions and affected downstream pathways to analyze their activation based on our bulk RNAseq data from DMSO and BrdU treated human cell lines (created with BioRender). B) Venn diagram showing the number of receptors inferred from BulkSignalR to be activated across each of the five human cell types. Three receptors were identified in common between astrocytes (purple), endothelial cells (pink), and microglia (yellow) which were the cell types shown to be capable of receiving senescence signals and becoming SA β-gal positive: CXCR7, KREMEN2, and GIPR. Only CXCR7 was expressed in the cell types capable of entering secondary senescence (astrocytes, endothelial cells, microglia) ( , S3B). C) TPM expression values of CXCR7 , its ligand CXCL12 , and DPP4 which cleaves and inactivates CXCL12 in DMSO (grey) and BrdU (red) treated cell lines (n=3 replicates). D) Schematic of the four selected SASP inhibitors mechanisms of action: Bindarit is a CCL2 synthesis inhibitor which prevents p65 activation of the CCL2 gene at the promoter region, ISO-1 is a MIF antagonist, ACT-1004-1239 is a CXCR7 antagonist, and Sitagliptin inhibits DPP4 preventing its action of cleaving and inactivating CXCL12 (created with BioRender). Data was analyzed by two-way ANOVA with Tukey’s multiple comparisons test (C). All graphs show mean with error bars depicting standard deviation (ns, p>0.05, ** p<0.01, *** p<0.001).

Article Snippet: Treatment with the CCL2 synthesis inhibitor Bindarit (MedChemExpress, Catalog No. HY-B0498) ( ) was used to specifically target CCL2 production in senescent cells.

Techniques: Activation Assay, RNA sequencing, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Characterizing the SASP-Dependent Paracrine Spreading of Senescence Between Human Brain Cell Types

doi: 10.64898/2026.02.10.705129

Figure Lengend Snippet: A) TPM expression values of CCL2 receptors CCR1, CCR4, CCRL2, and ACKR1 in DMSO (grey) and BrdU (red) treated cell lines (n=3 replicates). B) TPM expression values of receptors KREMEN2 and GIPR in DMSO (grey) and BrdU (red) treated cell lines (n=3 replicates). Data was analyzed by two-way ANOVA with Tukey’s multiple comparisons test (A-B). All graphs show mean with error bars depicting standard deviation (ns, p>0.05, * p<0.05, ** p<0.01, *** p<0.001).

Article Snippet: Treatment with the CCL2 synthesis inhibitor Bindarit (MedChemExpress, Catalog No. HY-B0498) ( ) was used to specifically target CCL2 production in senescent cells.

Techniques: Expressing, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Elevated CCL2 levels in the decidua and serum of OAPS patients and OAPS mice compared with HCs and control mice. A) Representative immunohistochemistry images for CCL2 in decidua from HCs (n = 6) and OAPS patients (n = 8). Three random fields of each sample were used for semiquantitative analyses. Scale bars: 50 µm. B) Scatter plot showing the average OD of CCL2 in decidua. A point represents a random field. C) The expression levels of CCL2 in medium supernatant of primary decidual cells from HCs (n = 6) and OAPS patients (n = 6). D) The expression levels of CCL2 in medium supernatant of primary decidual cells from HCs (n = 4) after stimulation of PBS, HC‐IgG/β2GPI complex, and aPL‐IgG/β2GPI complex. E) Flow chart depicting the methods of establishing OAPS mouse model by intravenous injection of aPL‐IgG. F) Representative figures for the uterus from D15.5 pregnant mice after injection of 0.9% NaCI, HC‐IgG, and aPL‐IgG. G) The embryo absorption rates of D15.5 pregnant mice after injection of 0.9% NaCI, HC‐IgG, and aPL‐IgG (n = 6). H) Representative HE images for decidua and placenta from D15.5 pregnant mice after injection of 0.9% NaCI, HC‐IgG, and aPL‐IgG. The decidua and placenta were detected for sites where embryos were live; for sites where embryos were absorbed, the whole uterus was detected. Scale bars: 1 mm. I) The levels of serum CCL2 in mice of different groups (n = 6). J) The expression of the Ccl2 gene was detected by RT‐qPCR in the decidua from mice in different groups (n = 6). K) Representative immunohistochemistry images for CCL2 in the decidua from mice of different groups. Three random fields of each sample were used for semiquantitative analyses. Scale bars: 5 µm. L) Bar plot showing the average OD of CCL2 in decidua. A point represents a sample. M) The proportion of macrophages in the decidua of mice from different groups, as detected by FCM (n = 6). N) Representative immunofluorescence images for CCR2 + macrophage in decidua from mice of different groups. Scale bars = 100 µm. O) The Tnf and Ifng gene expression in decidua from mice in different groups, detected by RT‐qPCR (n = 6). Data in (B) and (C) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (D), (G), (J), (L), (M), and (O) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. For (I), boxplot features: minimum whisker, 25th percentile – 1.5 × IQR or the lowest value within; minimum box, 25th percentile; center, median; maximum box, 75th percentile; maximum whisker, 75th percentile + 1.5 × IQR or the greatest value within. * p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; OD, optical density; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; FCM, flow cytometry; IQR, interquartile range; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: Decidual cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin‐streptomycin (Gibco, USA) at 37 °C and 5% CO 2 for 24 h. Primary decidual cells were stimulated with human β 2 ‐glycoprotein I (2 μg mL −1 , MCE, USA), aPL‐IgG (200 μg mL −1 ), HC‐IgG (200 μg mL −1 ), or CCL2 inhibitor Bindarit (30 μM, MCE, USA) in separate experiments.

Techniques: Control, Immunohistochemistry, Expressing, Injection, Quantitative RT-PCR, Immunofluorescence, Gene Expression, Whisker Assay, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: CCL2 promotes CCR2 + macrophage infiltration in OAPS decidua. A) Schematic diagram of an in vitro chemotaxis system where primary decidual cells chemoattract THP‐1 or THP‐1‐derived M0 macrophages. Briefly, the culture supernatant of primary decidual cells was added to the lower chamber of a 3.0 µm Transwell insert, and THP‐1 or THP‐1‐derived M0 macrophages, resuspended in FBS‐free medium, were added to the upper chamber. B) Bar plot showing the number of THP‐1 cells in the lower chamber after chemotaxis induced by decidual cells from OAPS patients and HCs (n = 6). C) Representative images and statistical results showing the number of THP‐1 M0 macrophages that migrated through the Transwell membrane (n = 5). Each sample was analyzed in three fields for quantification. D) Bar plot showing the number of THP‐1 cells in the lower chamber after chemotaxis induced by decidual cells from OAPS patients with or without Bindarit treatment (n = 4). E) Representative images and statistical results showing the number of THP‐1 M0 macrophages that migrated through the Transwell membrane (n = 5). Each sample was analyzed in three fields for quantification. F) Bar plot showing the number of THP‐1 cells in the lower chamber after chemotaxis induced by decidual cells from OAPS patients, with or without RS504393 treatment (n = 6). G) Representative images and statistical results showing the number of THP‐1 M0 macrophages that migrated through the Transwell membrane (n = 5). Each sample was analyzed in three fields for quantification. H) Flow chart depicting the methods of treating the OAPS mice model by intravenous injection of PF‐4136309. I) Representative images of uteri from D15.5 pregnant mice after injection of solvent or PF‐4136309. J) Embryo resorption rates in D15.5 pregnant mice following injection of solvent or PF‐4136309 (n = 6). K) Representative HE images for decidua and placenta from D15.5 pregnant mice after injection of solvent and PF‐4136309. L) The levels of serum CCL2 in mice of different groups (n = 6). M) The expression of the Ccl2 gene was detected by RT‐qPCR in the decidua from mice (n = 5 and 6, respectively). N) Representative immunohistochemistry images for CCL2 in decidua from mice of different groups (n = 6). Three random fields of each sample were used for semiquantitative analyses. Scale bars: 50 µm. O) Bar plot showing the average OD of CCL2 in decidua. A point represents a sample. P) The proportion of macrophage in the decidua of mice that received different treatments was detected by FCM (n = 6). Q) Representative immunofluorescence images for CCR2 + macrophage in the decidua from mice that received different treatments. Scale bars: 50 µm. Data in (B), (C), (D), (E), (F), (G), (J), (K), (M), and (P) are presented as mean ± SD and analyzed using Student's t ‐test. For (K), boxplot features: minimum whisker, 25th percentile – 1.5 × IQR or the lowest value within; minimum box, 25th percentile; center, median; maximum box, 75th percentile; maximum whisker, 75th percentile + 1.5 × IQR or the greatest value within. Data in (O) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; OD, optical density; FCM, flow cytometry; SD, standard deviation; IQR, interquartile range; ANOVA, analysis of variance.

Article Snippet: Decidual cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin‐streptomycin (Gibco, USA) at 37 °C and 5% CO 2 for 24 h. Primary decidual cells were stimulated with human β 2 ‐glycoprotein I (2 μg mL −1 , MCE, USA), aPL‐IgG (200 μg mL −1 ), HC‐IgG (200 μg mL −1 ), or CCL2 inhibitor Bindarit (30 μM, MCE, USA) in separate experiments.

Techniques: In Vitro, Chemotaxis Assay, Derivative Assay, Membrane, Injection, Solvent, Expressing, Quantitative RT-PCR, Immunohistochemistry, Immunofluorescence, Whisker Assay, Reverse Transcription, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: The aPL‐IgG/β2GPI complex promotes macrophages to express more CCL2 via the NF‐κB pathway in vitro. (A) UMAP presenting the expression of CCL2 gene in the integrated single‐cell landscape. (B) FCM analysis of CCL2 secretion in major decidual immune cells after 18 h LPS stimulation (n = 6). (C) FCM analysis of CCL2 secretion in CCR2 + and CCR2 − macrophages after 18 h LPS stimulation (n = 6). (D) Violin plot showing CCL2 gene expression levels in decidual macrophages from OAPS patients compared with HCs. (E) Representative immunofluorescence images of CD14 (red), CCL2 (green), and DAPI (blue) co‐staining in the decidua from OAPS patients and HCs. Scale bar: 100 µm. (F) Bar plot showing the proportion of CCL2 + macrophages, as determined by semi‐quantitative immunofluorescence staining (n = 4 for HCs, n = 5 for OAPS). (G) FCM analysis of CCL2 secretion in macrophages from the decidua of HCs after stimulation with HC‐IgG/β2GPI and aPL‐IgG/β2GPI complexes (n = 6). (H) Expression levels of CCL2 , TNF, IL1B , and IL6 genes in THP‐1 M0 macrophages, as detected by RT‐qPCR following stimulation with PBS, HC‐IgG/β2GPI, and aPL‐IgG/β2GPI complexes (n = 3). (I) Levels of CCL2 and TNF‐α in the culture supernatant of THP‐1 M0 macrophages, as detected by ELISA following stimulation with PBS, HC‐IgG/β2GPI, and aPL‐IgG/β2GPI complexes (n = 3). (J) Volcano plot of DEGs in THP‐1 M0 macrophages under the stimulation of HC‐IgG/β2GPI and aPL‐IgG/β2GPI complex. (K) GO terms of upregulated DEGs in (J). (L) KEGG terms of upregulated DEGs in (J). (M) Representative immunoblot showing the expression of TLR4, NF‐κB p65, IκBα, and IKKα in THP‐1 M0 macrophages stimulated with PBS, HC‐IgG/β2GPI, and aPL‐IgG/β2GPI complexes. (N) Bar plot showing the levels of TLR4 in THP‐1 M0 macrophages after different stimulations (n = 3). (O) Bar plot showing the phosphorylation levels of NF‐κB P65, IκBα, and IKKα in THP‐1 M0 macrophages with different stimulations (n = 3). (P) Representative immunofluorescence images for NF‐κB P65 (green) and DAPI (blue) in THP‐1 M0 macrophages after different stimulations. Scale bar: 50 µm. Data in (C) and (G) are presented as mean ± SD and analyzed using a paired t ‐test. Data in (D) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (B), (H), (I), (N), and (O) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; DEGs, differentially expressed genes; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; FCM, flow cytometry; ANOVA, analysis of variance; SD, standard deviation.

Article Snippet: Decidual cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin‐streptomycin (Gibco, USA) at 37 °C and 5% CO 2 for 24 h. Primary decidual cells were stimulated with human β 2 ‐glycoprotein I (2 μg mL −1 , MCE, USA), aPL‐IgG (200 μg mL −1 ), HC‐IgG (200 μg mL −1 ), or CCL2 inhibitor Bindarit (30 μM, MCE, USA) in separate experiments.

Techniques: In Vitro, Expressing, Gene Expression, Immunofluorescence, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Inhibition of TLR4 decreases CCL2 expression in macrophages in vitro and reduces decidual CCR2 + macrophage infiltration and abortion in vivo. (A) Expression levels of CCL2, TNF, IL1B, and IL6 genes in THP‐1 M0 macrophages stimulated with aPL‐IgG/β2GPI complex and inhibited by TAK‐242, as detected by RT‐qPCR (n = 3). (B) Levels of CCL2 and TNF‐α in THP‐1 M0 macrophage culture supernatant stimulated with aPL‐IgG/β2GPI complex and inhibited by TAK‐242, detected by ELISA (n = 3). (C) Representative immunoblot visualizing the expressions of NF‐κB P65, IκBα, and IKKα in THP‐1 M0 macrophages of different groups (n = 3). (D) Bar plot showing the phosphorylation levels of NF‐κB P65, IκBα, and IKKα in THP‐1 M0 macrophages from different groups (n = 3). (E) Representative immunofluorescence images for NF‐κB P65 (green) and DAPI (blue) in THP‐1 M0 macrophages from different groups. Scale bar: 50 µm. (F) Flow chart depicting the methods of treating the OAPS mice model by intraperitoneal injection of TAK‐242. (G) Representative images of uteri from D15.5 pregnant mice after injection of solvent or TAK‐242. (H) Embryo resorption rates in D15.5 pregnant mice following injection of solvent or TAK‐242 (n = 6). (I) Representative HE‐stained images of decidua and placenta from D15.5 pregnant mice in different groups. (J) The levels of serum CCL2 in mice of different groups (n = 6). (K) Expression levels of Ccl2 gene in the decidua of mice, as detected by RT‐qPCR (n = 6). (L) Representative immunohistochemistry images showing CCL2 expression in the decidua of mice from different groups. Three random fields per sample were used for semiquantitative analysis. Scale bars: 50 µm. (M) Bar plot showing the average OD of CCL2 in decidua. A point represents a sample. (N) Proportion of macrophages in the decidua of mice with different treatments, as detected by FCM (n = 6). (O) Representative immunofluorescence images showing CCR2 + macrophages in the decidua of mice from different groups. Scale bars: 50 µm. (P) Schematic diagram of the OAPS mouse model following treatment with PF‐4136309 and TAK‐242. Data in (H), (I), (K), and (N) are presented as mean ± SD and analyzed using Student's t ‐test. For (I), boxplot features: minimum whisker, 25th percentile – 1.5 × IQR or the lowest value within; minimum box, 25th percentile; center, median; maximum box, 75th percentile; maximum whisker, 75th percentile + 1.5 × IQR or the greatest value within. Data in (A), (B), (D), and (M) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.001, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; RT‐qPCR, real time‐quantitative reverse transcription polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; TNF, tumor mecrosis factor; FCM, flow cytometry; IQR, interquartile range; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: Decidual cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin‐streptomycin (Gibco, USA) at 37 °C and 5% CO 2 for 24 h. Primary decidual cells were stimulated with human β 2 ‐glycoprotein I (2 μg mL −1 , MCE, USA), aPL‐IgG (200 μg mL −1 ), HC‐IgG (200 μg mL −1 ), or CCL2 inhibitor Bindarit (30 μM, MCE, USA) in separate experiments.

Techniques: Inhibition, Expressing, In Vitro, In Vivo, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Immunofluorescence, Injection, Solvent, Staining, Immunohistochemistry, Whisker Assay, Reverse Transcription, Polymerase Chain Reaction, Flow Cytometry, Standard Deviation

Journal: Advanced Science

Article Title: Deciphering the Role and Mechanism of Decidual Monocyte‐Derived Macrophage Infiltration in Obstetric Antiphospholipid Syndrome at Single‐Cell Resolution

doi: 10.1002/advs.202503480

Figure Lengend Snippet: Vascular ECs express higher levels of ACKR1 to aggregate CCL2. (A) Representative immunofluorescence images for vWF (red), CCL2 (green), and DAPI (blue) co‐staining in decidua from OAPS patients and HCs. Scale bar: 100 µm. (B) UMAP plot of EC subclusters at single‐cell resolution. (C) Bar plot showing the proportions of EC subclusters in the decidua of OAPS patients and HCs. (D) Violin plot showing the marker genes of EC subclusters. (E) Bubble plot showing the intercellular communications mediated by ACKR1 and CCL2 between macrophages and vascular ECs in decidua from OAPS patients and HCs. (F) Violin plot of the expression of the ACKR1 gene in vascular ECs of OAPS patients compared with those of HCs decidua. (G) Representative immunofluorescence images for vWF (red), ACKR1 (green), and DAPI (blue) co‐staining in decidua from OAPS patients and HCs. Scale bar: 100 µm. (H) Volcano plot of DEGs for vascular ECs from OAPS patients’ decidua and those from HCs decidua. (I) GO terms of the upregulated DEGs in (H). (J) Heatmap showing the functional scores of vascular ECs in the OAPS and HC groups. (K) Representative immunohistochemistry images for IFN‐γ and TNF‐α in decidua from HCs (n = 6) and OAPS patients (n = 8). Three random fields of each sample were used for semiquantitative analyses. Scale bars: 50 µm. (L) Scatter plot showing the average OD of IFN‐γ and TNF‐α in the decidua. Each point represents a random field. (M) Expression levels of IFN‐γ and TNF‐α in the supernatant of primary decidual cells from HCs (n = 6) and OAPS patients (n = 6). (N) Expression levels of IFN‐γ and TNF‐α in the supernatant of primary decidual cells from HCs following stimulation with PBS, HC‐IgG/β2GPI complex, and aPL‐IgG/β2GPI complex (n = 4). (O) Line chart showing ACKR1 gene expression in EA.hy926 cells under different concentrations of TNF‐α (n = 3). (P) Line chart showing ACKR1 gene expression in EA.hy926 cells under different concentrations of IFN‐γ (n = 3). (Q, S) Representative immunoblot and semi‐quantified results showing ACKR1 protein expression in EA.hy926 cells under different concentrations of TNF‐α (n = 3). (R, T) Representative immunoblot and semi‐quantified results showing ACKR1 protein expression in EA.hy926 cells under different concentrations of IFN‐γ (n = 3). (U) Schematic diagram summarizing the findings of this study. In brief, the aPL‐IgG/β2GPI complex promotes decidual macrophages to express CCL2 via the TLR4‐NF‐κB pathway. CCL2 accumulates on the surface of vascular ECs through ACKR1, recruiting more monocytes into the decidua. Infiltrated CCR2 + macrophages exacerbate decidual inflammatory injury in OAPS patients. ACKR1 overexpression in vascular ECs is driven by increased IFN‐γ and TNF‐α levels in the decidual environment of OAPS patients. Data in (L) and (M) are presented as mean ± SD and analyzed using Student's t ‐test. Data in (N), (O), (P), (S), and (T) are presented as mean ± SD and analyzed using one‐way ANOVA with Dunnett's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. OAPS, obstetric antiphospholipid syndrome; HCs, healthy controls; aPL, antiphospholipid antibody; Ig, immunoglobin; β2GPI, β2 glycoprotein I; EC, endothelial cell; OD, optical density; TNF, tumor necrosis factor; IFN, interferon; ELISA, enzyme‐linked immunosorbent assay; DEGs, differential expressed genes; SD, standard deviation; ANOVA, analysis of variance.

Article Snippet: Decidual cells were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin‐streptomycin (Gibco, USA) at 37 °C and 5% CO 2 for 24 h. Primary decidual cells were stimulated with human β 2 ‐glycoprotein I (2 μg mL −1 , MCE, USA), aPL‐IgG (200 μg mL −1 ), HC‐IgG (200 μg mL −1 ), or CCL2 inhibitor Bindarit (30 μM, MCE, USA) in separate experiments.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Functional Assay, Immunohistochemistry, Gene Expression, Western Blot, Over Expression, Enzyme-linked Immunosorbent Assay, Standard Deviation