Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy.

doi: 10.1073/pnas.2112561118

Figure Lengend Snippet: Fig. 5. Vasoactive gene expression from retinal microglia and fractalkine-induced vasoconstriction are altered after 4 wk of STZ-induced diabetes. (A) The responsiveness of retinal vessels to hyperoxic challenge was explored in vivo using OCTA (Insets show OCTA images from baseline and after expo- sure to O2). (Scale bar, 200 μm.) While hyperoxic challenge (filled bars) lead to constriction in the control group (n = 10 normoxia, n = 6 100% O2), no constriction was observed in the STZ cohort (n = 12 normoxia, n = 7 100% O2). (B) Microglial vasoregulation was investigated during diabetes, with 4-wk STZ-treated and control retinae exposed to fractalkine ex vivo (representative control and STZ images in Inset) (Scale bar, 50 μm.) While vessels from con- trol retinae showed fractalkine-induced vasoconstriction (filled bar), STZ retinae exhibited no change (n = 5 animals). (C) Differential microglial gene expression data from 4 wk control and STZ-treated animals were compared to vasomodulatory gene lists (vasoconstriction, GO:0097746; angiogenesis, GO:0001525; vasodilation, GO:0097746), with the RAS positive regulator angiotensinogen (Agt), and negative regulator (Ahr) significantly altered (FDR- adjusted, citrate control n = 5, STZ n = 4). (D) OCTA was used to quantify retinal superficial capillary diameter in 4-wk control and STZ-treated animals (unfilled and filled bars, respectively) exposed to candesartan or vehicle. In STZ-treated animals, capillary diameter returned to baseline in the candesartan-treated group (n = 7 control, n = 8, 5 STZ vehicle and candesartan, respectively). (E) Retinal blood flow was quantified using arterio-venous transit time and showed increased transit time (slower blood flow) in STZ-treated animals independent of candesartan treatment (n = 8 control, n = 11 and 8 STZ vehicle and candesartan, respectively). (F) Quantification of the arteriovenous ratio showed candesartan treatment increased the diameter of larger vessels in STZ-treated retinae relative to control and vehicle-treated tissues (n = 8 control, n = 11 and 8 STZ vehicle and candesartan, respectively). Data expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant rat fractalkine (200 ng/mL; R&D Systems, #537-FT-025/CF) or vehicle (PBS) was introduced after 10 min of baseline recording and imaged for an additional 10 min. At the end of this incubation, vessel diameter was measured at sites with and without microglial contact (2 to 4 individual capillary sites per retina, n = 4 to 6 retinae) and measurements expressed as a percentage of baseline diameter of the same vessel region (taken as the average vessel diameter over the initial 10-min baseline).

Techniques: Gene Expression, In Vivo, Control, Ex Vivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy.

doi: 10.1073/pnas.2112561118

Figure Lengend Snippet: Fig. 6. Schematic representation of microglial regulation of ret- inal capillary constriction. Data from this study show microglia are structurally and functionally capable of involvement in the neurovascular unit. Microglia contact neuronal synapses and reti- nal capillaries (including pericytes) and activation of fractalkine- Cx3cr1 signaling results in capillary constriction, which is via an AT1R-dependent mechanism. Ultimately, capillary regulation may occur via direct microglial mechanism or may involve contri- butions from pericytes and M€uller cells. EC, endothelial cell; PC, pericyte.

Article Snippet: Recombinant rat fractalkine (200 ng/mL; R&D Systems, #537-FT-025/CF) or vehicle (PBS) was introduced after 10 min of baseline recording and imaged for an additional 10 min. At the end of this incubation, vessel diameter was measured at sites with and without microglial contact (2 to 4 individual capillary sites per retina, n = 4 to 6 retinae) and measurements expressed as a percentage of baseline diameter of the same vessel region (taken as the average vessel diameter over the initial 10-min baseline).

Techniques: Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: CXCR4 + CD163 + monocytes/CXCR4 + CD68 + macrophages are specific cells in AAGN. A UMAP of monocyte subsets from peripheral blood transcriptome sequencing and the proportion of cells in each sample (AAV = 15,CTRL = 3). B Classical and nonclassical monocyte marker genes. C Expression of common chemokine receptors in classical and non-classical monocytes, with CXCR4 being highly expressed in classical monocytes and significantly reduced after treatment. After 3 days AAGN serum incubation with THP-1, the expression of CXCR4 and CD163 mRNA ( D ) and protein levels ( E ) increased significantly, while CD86 and CD68 did not change ( n = 3). F Under the light microscope, THP-1 cells incubated with AAGN serum were still in a suspended round state, and did not differentiate into a adherent spindle-shaped state of macrophages (20 ×, scale bar = 100 μm, n = 3). G Spatial transcriptome sequencing of renal tissue in AAGN revealed a marked increase in CXCR4 mRNA level, which was more pronounced at the site of crescent formation. H Double immunofluorescence staining of CD68 and CXCR4 showed that CXCR4 + CD68. + macrophages increased in MPO + AAGN and PR3 + AAGN crescents compared with IgAVN, LN and IgAN, and the MPO + AAGN was more obvious than PR3 + AAGN (600 ×, scale bar = 20 μm). I Soluble CXCR4 level was found to be increased in AAGN plasma (AAGN = 23, CTRL = 14) but not urine (AAGN = 8, CTRL = 13) by ELISA. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Sequencing, Marker, Expressing, Incubation, Light Microscopy, Double Immunofluorescence Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: CXCR4 activates the ELMO1/DOCK180/RAC1 efferocytosis pathway to promote M2-Mφ polarization. Histograms of KEGG gene enrichment of up-regulated differential genes in peripheral blood single-cell transcriptome sequencing A and renal tissue spatial transcriptome sequencing B showed that CXCR4 was enriched in “Leukocyte transepithelial migration” and “Endocytosis” pathways. C Both mRNA and protein levels of MERTK were increased after 3 days of incubation of THP-1 with AAGN serum. D Both mRNA and protein levels of MERTK were increased after 3 days of incubation with AAGN serum of Mo-Mφ. E Cellular immunofluorescence showed an increase in the number of CD68 + macrophages phagocytosed apoptotic Jurkat cells (DiD. + ) after incubation with AAGN serum (400 ×, scale bar = 10 μm). F After incubation with AAGN serum of Mo-Mφ, the expression of CD163 was increased, but that of CD86 was not changed. G Protein levels of CXCR4, ELMO1, DOCK180, RAC1 and TGF-β1 were significantly increased in Mo-Mφ after 3 days of incubation with AAGN serum by western blotting. Co-IP experiments confirmed the interaction between CXCR4 and ELMO1 ( H and I ) and CXCR4 being stronger after Mφs were stimulated with AAGN serum in the ELMO1 antibody-bound CXCR4 group ( H ). J As verified by CHX protein half-life test, with the extension time of CXCR4 knockdown, the protein half-life level of ELMO1 gradually decreased. N = 3 in each in vitro experiment. * P < 0.05 and ** P < 0.01

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Sequencing, Migration, Incubation, Immunofluorescence, Expressing, Western Blot, Co-Immunoprecipitation Assay, Knockdown, In Vitro

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Inhibition of CXCL12/CXCR4 alleviates AAGN progression. A Suitable concentrations for LIT927 (CXCL12 neutral ligand antagonist) and AMD3100 (CXCR4 inhibitor) were determined as 30 μM and 100 μM, respectively, using CCK-8 assays ( n = 3). B When mixed individually with AAGN serum to stimulate macrophages (Mφs), both drugs reduced MERTK, CD163, TGF-β1 and ELMO1/DOCK180/RAC1 axis components at transcriptional and protein levels ( n = 3). C Schematic of the EAV rat model establishment. Drug administration, either concurrently with modeling or from the third week post-modeling, did not significantly affect rat body weight D but alleviated hematuria E and proteinuria F , with AMD3100 showing greater efficacy. Starting treatment at the third week was as effective as concurrent administration. G LIT927 administration at the onset of modeling reduced cellular and fibrous crescent formation. When started from the third week, it alleviated fibrocellular and fibrous crescents without affecting cellular crescents. AMD3100 reduced various types of crescent formation when given at modeling onset but primarily alleviated fibrocellular and fibrous crescents when started three weeks later (80 ×, scale bar = 10 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Inhibition, CCK-8 Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Pathogenic Functions of Tumor Necrosis Factor Receptor- Associated Factor 6 Signaling Following Traumatic Brain Injury

doi: 10.3389/fnmol.2021.629910

Figure Lengend Snippet: Schematic of the possible mechanism of neuroinflammation following TBI. TRAF6 expression is upregulated after TBI, which activates downstream MAPKs or NF-κB intracellular signaling pathways, induces the expression of chemokines CCL2 and CXCL1, and acts on the corresponding receptors CCR2 and CXCR2, contributes to neuroinflammation, and then leads to pathological changes such as neurologic function injury and nerve cell apoptosis.

Article Snippet: A rat CXCL1 ELISA kit was purchased from Hangzhou MultiSciences (Lianke) Biotech (EK396/2-96, Hangzhou, Zhejiang, China), a rat CCL2 ELISA kit from R&D Systems (MJE00, Minneapolis, MN, United States), a rat CCR2 ELISA kit from CUSABIO TECHNOLOGY (CSB-EL004841RA, Wuhan, Hubei, China), and a rat CXCR2 ELISA kit from Cloud-Clone Corp (SEC006Ra, Katy, TX, United States).

Techniques: Expressing, Protein-Protein interactions

Journal: Frontiers in Molecular Neuroscience

Article Title: Pathogenic Functions of Tumor Necrosis Factor Receptor- Associated Factor 6 Signaling Following Traumatic Brain Injury

doi: 10.3389/fnmol.2021.629910

Figure Lengend Snippet: Number of animals used for each experiment.

Article Snippet: A rat CXCL1 ELISA kit was purchased from Hangzhou MultiSciences (Lianke) Biotech (EK396/2-96, Hangzhou, Zhejiang, China), a rat CCL2 ELISA kit from R&D Systems (MJE00, Minneapolis, MN, United States), a rat CCR2 ELISA kit from CUSABIO TECHNOLOGY (CSB-EL004841RA, Wuhan, Hubei, China), and a rat CXCR2 ELISA kit from Cloud-Clone Corp (SEC006Ra, Katy, TX, United States).

Techniques: Fluorescence, TUNEL Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Pathogenic Functions of Tumor Necrosis Factor Receptor- Associated Factor 6 Signaling Following Traumatic Brain Injury

doi: 10.3389/fnmol.2021.629910

Figure Lengend Snippet: Primer sequences.

Article Snippet: A rat CXCL1 ELISA kit was purchased from Hangzhou MultiSciences (Lianke) Biotech (EK396/2-96, Hangzhou, Zhejiang, China), a rat CCL2 ELISA kit from R&D Systems (MJE00, Minneapolis, MN, United States), a rat CCR2 ELISA kit from CUSABIO TECHNOLOGY (CSB-EL004841RA, Wuhan, Hubei, China), and a rat CXCR2 ELISA kit from Cloud-Clone Corp (SEC006Ra, Katy, TX, United States).

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Pathogenic Functions of Tumor Necrosis Factor Receptor- Associated Factor 6 Signaling Following Traumatic Brain Injury

doi: 10.3389/fnmol.2021.629910

Figure Lengend Snippet: TRAF6 knockdown suppressed CCL2, CCR2, CXCL1, and CXCR2 expression at both mRNA and protein levels in injured cortex after TBI. (A–D) AAV9-TRAF6-RNAi downregulated (A,B) CCL2 and CCR2 mRNA, (C,D) CXCL1 and CXCR2 mRNA, (E,F) CCL2 and CCR2 protein, and (G,H) CXCL1 and CXCR2 protein expression in injured cortex on day 3 post-TBI. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. AAV9-TRAF6-RNAi group. # P < 0.05, ## P < 0.01, and ### P < 0.001 vs. sham group.

Article Snippet: A rat CXCL1 ELISA kit was purchased from Hangzhou MultiSciences (Lianke) Biotech (EK396/2-96, Hangzhou, Zhejiang, China), a rat CCL2 ELISA kit from R&D Systems (MJE00, Minneapolis, MN, United States), a rat CCR2 ELISA kit from CUSABIO TECHNOLOGY (CSB-EL004841RA, Wuhan, Hubei, China), and a rat CXCR2 ELISA kit from Cloud-Clone Corp (SEC006Ra, Katy, TX, United States).

Techniques: Knockdown, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Pathogenic Functions of Tumor Necrosis Factor Receptor- Associated Factor 6 Signaling Following Traumatic Brain Injury

doi: 10.3389/fnmol.2021.629910

Figure Lengend Snippet: Inhibitors of p-NF-κB and MAPKs reduced upregulation of CCL2, CCR2, CXCL1, and CXCR2 following TBI. A 25 mg/10 ml dose of the p-NF-κB inhibitor BAY117082, p-JNK inhibitor PD98059, p-ERK inhibitor SP600125, or p-p38 inhibitor SB203580 reduced CCL2 (A–D) , CCR2 (E–H) , CXCL1 (I–L) , and CXCR2 (M–P) protein expression in the injured cortex as measured by ELISA, while lower doses (2.5 mg/10 ml) had no significant effect. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. high dose group. ### P < 0.001 vs. sham group.

Article Snippet: A rat CXCL1 ELISA kit was purchased from Hangzhou MultiSciences (Lianke) Biotech (EK396/2-96, Hangzhou, Zhejiang, China), a rat CCL2 ELISA kit from R&D Systems (MJE00, Minneapolis, MN, United States), a rat CCR2 ELISA kit from CUSABIO TECHNOLOGY (CSB-EL004841RA, Wuhan, Hubei, China), and a rat CXCR2 ELISA kit from Cloud-Clone Corp (SEC006Ra, Katy, TX, United States).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay