Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Fatty acid metabolism is significantly disrupted in cerebrospinal fluid (CSF) of SAH patients, with fatty acid‐binding protein (A‐FABP) enrichment. (A) Schematic workflow of targeted fatty acid metabolomics analysis based on human CSF samples (subarachnoid haemorrhage [SAH] group, n = 40; control group, n = 30). (B and C) The orthogonal partial least squares discrimination analysis (OPLS‐DA) reveals clear separation between the SAH (blue) and control (red) groups. (D) Heatmap of the differentially expressed fatty acid metabolites between the two groups. Rows represent individual fatty acids; colour intensity reflects relative abundance (red: high; blue: low). (E) The volcano plot presents significantly altered fatty acids, including C20:4n6 (arachidonic acid [AA]), C18:2n6c (linoleic acid [LA]) and C16:0 (palmitic acid [PA]). (F) Kyoto Encyclopaedia of Genes and Genomes ( https://www.kegg.jp/kegg/pathway.html ) (KEGG) pathway enrichment analysis of dysregulated fatty acid metabolism. (G) The regulation of lipolysis in adipocytes in KEGG pathway database (map04923), with A‐FABP highlighted in red. Source : Schematic workflow created with BioRender.com.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Control

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Elevated levels of fatty acid‐binding protein (A‐FABP) in cerebrospinal fluid (CSF) predict subarachnoid haemorrhage (SAH) severity and poor outcomes. (A) Schematic of cohort grouping (SAH: n = 40; control: n = 30). (B–D) Violin plots present levels of significantly altered fatty acids in CSF: (B) arachidonic acid (AA, C20:4n6), (C) linoleic acid (LA, C18:2n6c) and (D) palmitic acid (PA, C16:0). (E) Schematic of cohort grouping by SAH severity: (Hunt and Hess [H–H] group 1–2 level, n = 25; H–H group 3–5 level, n = 15). (F–H) Levels of (F) AA, (G) LA and (H) PA between severity low‐grade (H–H 1–2) and high‐grade (H–H 3–5) subgroups. (I–K) Correlation between significantly altered fatty acids and A‐FABP levels in CSF of SAH patients [(I) AA, (J) LA and (K) PA, n = 40]. (L) CSF levels of A‐FABP between severity low‐grade (H–H 1–2) and high‐grade (H–H 3–5) subgroups ( n = 48). (M and N) The relationship between CSF levels of A‐FABP and functional outcomes (modified Rankin Scale [mRS]) in SAH patients ( n = 48). Data are presented as means ± SD. * p < .05, ** p < .01, *** p < .001. Source : Schematic diagrams created with BioRender.com.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Control, Functional Assay, Modification

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Cerebral fatty acid‐binding protein (A‐FABP) is increased in mice and predominantly colocalizes with microglia. (A and B) Representative photographs from the bottom of mice brains and H&E staining from sham and subarachnoid haemorrhage (SAH) groups. (C) Quantification of FABPs family mRNA level in the mice cortex 24 h after SAH ( n = 3). (D) Representative images of immunohistochemical staining of A‐FABP (scale bar = 100 µm, n = 6). (E) Quantification of A‐FABP mRNA level in the mice cortex after SAH ( n = 6). (F and G) Western blot images and quantitative analysis of A‐FABP expressions in the mice cortex at 3, 12, 24, 48 and 72 h post‐SAH ( n = 6). (H–L) Representative microphotographs and quantitative analyses of immunofluorescence co‐staining for A‐FABP (red) with microglia (IBA1, green), astrocyte cells (GFAP, green), neuron cells (NeuN, green) and endothelial cells (CD31, green). Scale bar = 100 µm, n = 6. Data are presented as means ± SD. * p < .05, ** p < .01, *** p < .001.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Staining, Immunohistochemical staining, Western Blot, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Genetic ablation of fatty acid‐binding protein (A‐FABP) ameliorates brain injury in mice with subarachnoid haemorrhage (SAH). (A and B) Rotarod test: (A) latency to fall on the accelerating rotarod, (B) speed at fall on the accelerating rotarod ( n = 8). (C and D) Open field test: (C) distance travelled and (D) fast movement proportion ( n = 8). (E) Percentage of brain water content ( n = 6). (F and G) Representative photographs and quantitative analysis of mice brain stained with Evans Blue ( n = 6). (H and I) Representative images and quantitative analysis of TUNEL staining with co‐staining with NeuN (red) and TUNEL‐positive neurons (green, scale bar = 100 µm, n = 6). (J and L) Representative western blot images and quantitative analyses of (K) cleaved Caspase‐3/Caspase‐3 and (L) Bcl‐2/Bax ( n = 6). Data are presented as means ± SD. * p < .05, ** p < .01, *** p < .001, **** p < .0001.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Staining, TUNEL Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Fatty acid‐binding protein (A‐FABP) exacerbates microglia‐mediated neuroinflammation in mice with subarachnoid haemorrhage (SAH). (A) The mRNA levels of pro‐inflammatory cytokines TNF‐α, IL‐1β and IL‐6 are measured in different groups ( n = 6). (B–E) Representative western blot images and quantitative analyses of (C) TNF‐α, (D) IL‐1β and (E) IL‐6 in different groups ( n = 6). (F) The mRNA levels of pro‐inflammatory cytokines TNF‐α, IL‐1β and IL‐6 in WT and knockout (KO) mice after SAH ( n = 6). (G–J) Representative western blot images and quantitative analyses of pro‐inflammatory cytokines (H) TNF‐α, (I) IL‐1β and (J) IL‐6 in WT and KO mice after SAH ( n = 6). (K) Representative microphotographs of immunofluorescence co‐staining for pro‐inflammatory cytokines (red) with microglia (IBA1, green) in WT and KO mice after SAH (scale bar = 100 µm, n = 6). Data are presented as means ± SD. * p < .05, ** p < .01, *** p < .001.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Western Blot, Knock-Out, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Inhibition of fatty acid‐binding protein (A‐FABP) in microglia attenuates neuronal apoptosis and blood–brain barrier disruption. (A and B) Representative western blot image and quantitative analysis of A‐FABP expression at 0, 12 and 24 h post‐PA (200 µM) treatment ( n = 6). (C and D) Representative western blot image and quantitative analysis of A‐FABP expression at 0, 12 and 24 h post‐AA (100 µM) treatment ( n = 6). (E) Schematic diagram of microglial culture supernatant transfer model (PA, 200 µM; BMS, 20 µM). (F and G) Representative flow cytometry images of primary neuron cells apoptosis in PA‐induced microglial culture supernatant transfer model ( n = 6). (H) Schematic representation of bEnd.3 endothelial cell co‐culture system with BV2 microglial conditioned medium (CM) treated with PA (200 µM), with or without BMS (20 µM). (I and J) Representative microphotographs of immunofluorescence and quantitative analysis in bEnd.3 cells co‐cultured with the CM of BV2 cells (scale bar = 100 µm, n = 6). Data are presented as means ± SD. * p < .05, ** p < .01, **** p < .0001.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Inhibition, Binding Assay, Disruption, Western Blot, Expressing, Flow Cytometry, Co-Culture Assay, Immunofluorescence, Cell Culture

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Fatty acid‐binding protein (A‐FABP) suppression promotes fatty acid β‐oxidation reprogramming in microglia. (A) Schematic illustration of experimental design. Control group: PBS treatment. FFA group: 200 µM palmitic acid (PA). BMS group: Co‐treatment with PA (200 µM) and BMS (20 µM). (B–D) Oxygen consumption rate (OCR) was determined in BV2 cells treated with PA, with or without BMS ( n = 6). Quantitative analyses of (C) basal respiration and (D) maximal respiration. (E–H) Glycolytic proton efflux rate (GlycoPER) was determined in BV2 cells treated with PA, with or without BMS ( n = 6). Quantitative analyses of (F) basal glycolysis, (G) basal proton efflux rate and (H) compensatory glycolysis. (I–K) Representative western blot images and quantitative analyses of (J) CPT1A and (K) ACADL in different groups ( n = 6). (L) ATP content assay in different groups ( n = 6). Data are presented as means ± SD. * p < .05, **** p < .0001. glycoPER, glycolytic proton efflux rate. Source : Schematic diagram created with BioRender.com.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Control, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Adipocyte fatty acid‐binding protein as a cerebrospinal fluid–accessible biomarker and druggable target in subarachnoid haemorrhage: Linking fatty acid dysregulation to microglial neuroinflammation

doi: 10.1002/ctm2.70607

Figure Lengend Snippet: Fatty acid‐binding protein (A‐FABP) mediates neuroinflammation via the JAK2/STAT3 signalling pathway. (A) Workflow diagram. Proteomic analysis of brain tissue around the subarachnoid space from the subarachnoid haemorrhage (SAH)‐treated Veh group and the SAH‐treated BMS group ( n = 4). (B) KEGG enrichment highlights the JAK‐STAT signalling pathway. (C) Volcano plot demonstrates the results of 85 differentially expressed proteins. STAT3 is labelled in green. (D–H) Representative western blot images and quantitative analyses of (E) p‐STAT3/STAT3, (F) p‐JAK1/JAK1, (G) p‐JAK2/JAK2 and (H) p‐JAK3/JAK3 in WT and knockout (KO) mice after SAH. (I–L) Representative western blot images and quantitative analyses of (J) TNF‐α, (K) IL‐1β and (L) IL‐6 in different groups. (M and N) Representative images and quantitative analysis of TUNEL staining with co‐staining with NeuN (red) and TUNEL‐positive neurons (green, scale bar = 100 µm, n = 6). Data are presented as means ± SD. * p < .05, ** p < .01, *** p < .001. C‐A1, coumermycin A1; ns, no significance. Source : Schematic diagram created with BioRender.com.

Article Snippet: Mice were gavaged orally with a selective A‐FABP inhibitor (BMS309403, #HY‐101903, MCE, USA; 15 mg/kg/day; dissolved in 4% Tween 80 and phosphate‐buffered saline) or vehicle (without BMS309403) one and 12 h after the sham or SAH surgery and then once per day for the next six consecutive days (concentration as previously described).

Techniques: Binding Assay, Western Blot, Knock-Out, TUNEL Assay, Staining

Journal: EMBO Molecular Medicine

Article Title: Saturated fatty acids induce lipotoxicity in lymphatic endothelial cells contributing to secondary lymphedema development

doi: 10.1038/s44321-025-00286-4

Figure Lengend Snippet: ( A ) Circulating levels of fatty acid-binding protein 4 (FABP4) measured by ELISA in plasma samples from patients with lymphedema ( n = 51) and non-lymphedema controls ( n = 18). ( B ) Quantification of cell death (trypan blue assay) in HDLECs treated with SA (50 µM) or palmitic acid (PA, 250 µM) with or without the FABP4 inhibitor BMS-309403 (BMS, 5 µM) pre-treatment ( n = 4 independent experiments). ( C ) Representative western blot of ER stress markers (sXBP1 and CHOP) in HDLECs treated with SA at increasing concentrations (10–50 µM) with or without BMS pre-treatment. ( D ) Representative immunofluorescence images of HDLECs treated with SA and BMS showing FABP4 (red) and the endoplasmic reticulum marker calreticulin (green). DAPI stains the nucleus (blue). Scale bars: 50 µm. ( E ) Colocalization quantification between FABP4 and calreticulin using Pearson’s correlation coefficient, showing no significant differences between treatment groups ( n = 3 independent experiments). ( F ) Quantification of cell death in HDLECs transfected with siFABP4 or scrambled siRNA (Sc-siRNA), with or without SA treatment, showing reduced cell death following FABP4 knockdown ( n = 6 independent experiments). ( G ) Western blot confirming FABP4 knockdown in HDLECs transfected with FABP4-targeting siRNA (siFABP4), with no effect on FABP5 expression ( n = 2). Data are presented as mean ± SEM. Statistical analysis: two-tailed unpaired t test for ( A ); one-way ANOVA with Tukey’s post hoc test for ( B , E , F ). Significance: ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values for the statistical comparisons are shown in Appendix Table . FABP4 fatty acid binding protein 1, sXBP-1 spliced X-box binding protein 1, CHOP C/EBP homologous protein, DAPI 4′,6-diamidino-2-phenylindole. .

Article Snippet: To determine the effects of pharmacological inhibition of FABP4, either the FABP4 inhibitor BMS-309403 (15 mg·kg − 1·day − 1; HY-101903A, MedChemExpress, NJ, USA) or vehicle (phosphate-buffered saline, PBS) was administered chronically by daily oral gavage for 4 weeks starting on the day of the lymphatic surgery.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Western Blot, Immunofluorescence, Marker, Transfection, Knockdown, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Saturated fatty acids induce lipotoxicity in lymphatic endothelial cells contributing to secondary lymphedema development

doi: 10.1038/s44321-025-00286-4

Figure Lengend Snippet: ( A , B ) Oil Red O staining of tail tissue from sham and lymphedema (LE) mice maintained on a high saturated fat diet (HSFD) or treated with the FABP4 inhibitor BMS-309403 (HSFD + BMS) or switched to a control diet (HSFD → CD) after lymphatic injury. ( A ) Representative images showing lipid accumulation (red) in lymphedematous tails. Scale bars: 100 μm. ( B ) Quantification of Oil Red O positive area reveals increased lipid deposition in lymphedema tissue under HSFD, which is significantly reduced by BMS treatment or dietary transition ( n = 4 mice). ( C – E ) Immunofluorescence staining for FABP4 (red) in tail tissue from CD, HSFD-, HSFD + BMS-, or HSFD → CD-fed lymphedema mice. DAPI (blue) marks nuclei. ( C ) Representative images showing increased FABP4 positive adipocytes in HSFD-fed mice. Scale bars: 50 μm. ( D ) Quantification of FABP4 positive adipocyte number. ( E ) Quantification of FABP4 positive adipocyte area ( n = 4 mice). ( F ) Circulating FABP4 levels measured in mice with lymphedema, showing increased plasma FABP4 in HSFD-fed mice and partial reduction with BMS treatment ( n = 7 mice). Data are presented as mean ± SEM. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. Significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values for the statistical comparisons are shown in Appendix Table . HSFD high saturated fat diet, CD control diet, FABP4 fatty acid-binding protein 4, BMS BMS-309403, DAPI 4′,6-diamidino-2-phenylindole. .

Article Snippet: To determine the effects of pharmacological inhibition of FABP4, either the FABP4 inhibitor BMS-309403 (15 mg·kg − 1·day − 1; HY-101903A, MedChemExpress, NJ, USA) or vehicle (phosphate-buffered saline, PBS) was administered chronically by daily oral gavage for 4 weeks starting on the day of the lymphatic surgery.

Techniques: Staining, Control, Immunofluorescence, Clinical Proteomics, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Saturated fatty acids induce lipotoxicity in lymphatic endothelial cells contributing to secondary lymphedema development

doi: 10.1038/s44321-025-00286-4

Figure Lengend Snippet: ( A ) Tail volume measurements over time in mice with lymphedema (LE), showing that prolonged swelling induced by HSFD is significantly reduced with treatment using the FABP4 inhibitor BMS-309403 (HSFD + BMS) compared to HSFD + PBS controls ( n = 3–7 mice). ( B ) Scatter plots showing the changes in tail volume 28 days post-lymphatic surgery ( n = 3–7 mice). ( C ) Representative immunofluorescence staining of LYVE-1-positive cells (green) and markers of ER stress (sXBP-1, CHOP) and apoptosis (TUNEL) (red) in tail tissues from lymphedema mice across diets, 28 days post-surgery. DAPI stains the nucleus (blue). Scale bars: 25 μm. Scatter plots showing ( D , G ) sXBP-1 + LYVE-1+ colocalization and perilymphatic sXBP-1 intensities, ( E , H ) CHOP + LYVE-1+ colocalization and perilymphatic CHOP intensities, and ( F , I ) TUNEL + LYVE-1+ colocalization and perilymphatic TUNEL intensities comparing the groups ( n = 3–7 mice). Data are presented as mean ± SEM. Statistical analysis: two-way ANOVA with Šídák’s post hoc test for ( A ); one-way ANOVA with Tukey’s post hoc test for ( B , D – I ). Significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values for the statistical comparisons are shown in Appendix Table . LYVE-1 lymphatic vessel endothelial hyaluronan receptor-1, sXBP-1 spliced X-box binding protein 1, CHOP C/EBP homologous protein, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, DAPI 4′,6-diamidino-2-phenylindole. .

Article Snippet: To determine the effects of pharmacological inhibition of FABP4, either the FABP4 inhibitor BMS-309403 (15 mg·kg − 1·day − 1; HY-101903A, MedChemExpress, NJ, USA) or vehicle (phosphate-buffered saline, PBS) was administered chronically by daily oral gavage for 4 weeks starting on the day of the lymphatic surgery.

Techniques: Immunofluorescence, Staining, TUNEL Assay, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Saturated fatty acids induce lipotoxicity in lymphatic endothelial cells contributing to secondary lymphedema development

doi: 10.1038/s44321-025-00286-4

Figure Lengend Snippet: Representative immunohistochemistry images showing ( A ) F4/80 positive macrophages and ( B ) CD4 positive T cells in the perilymphatic region of tail tissue from mice with lymphedema fed a high saturated fat diet (HSFD), treated with the FABP4 inhibitor BMS-309403 (HSFD + BMS), or switched to a control diet after lymphatic injury (HSFD → CD). Scale bars: 50 μm. Quantification of immune cell infiltration for ( C ) F4/80 positive macrophages and ( D ) CD4 positive T cells ( n = 5 mice). HSFD-fed mice exhibited significantly higher perilymphatic immune cell infiltration, which was attenuated by FABP4 inhibition or dietary transition. Data are presented as mean ± SEM. Statistical analysis: one-way ANOVA with Tukey’s post hoc test. Significance: * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values for the statistical comparisons are shown in Appendix Table . HSFD high saturated fat diet, CD control diet, FABP4 fatty acid-binding protein 4. .

Article Snippet: To determine the effects of pharmacological inhibition of FABP4, either the FABP4 inhibitor BMS-309403 (15 mg·kg − 1·day − 1; HY-101903A, MedChemExpress, NJ, USA) or vehicle (phosphate-buffered saline, PBS) was administered chronically by daily oral gavage for 4 weeks starting on the day of the lymphatic surgery.

Techniques: Immunohistochemistry, Control, Inhibition, Binding Assay