Fig 1: Visceral High APs Secrete Adipogenic Inhibitors and Acquired a Smooth Muscle Fate upon Adipogenic Differentiation(A) Hierarchical clustering of differentially abundant proteins in CM from 3-days-DIFF VH and VL APs, respectively.(B) Relative mRNA expression of Igfbp1–7 in VIS low and VIS high APs at day 2 post-differentiation. Levels are expressed as fold change relative to VIS low APs.(C) Igfbp2 levels in CM of SUB, VIS low, and VIS high cultures at day 6 post-differentiation.(D–F) Oil red O quantification (D) and relative mRNA expression of Pparγ (E) and C/ebpα (F) in SUB and VIS low AP cultures DIFF in the presence of PBS control, 25 ng/mL mouse recombinant Igfbp2 alone or combined with 30 μg/mL Igfbp2 neutralizing antibody during the first 3 days of differentiation.(G–I) Oil red O quantification (G) and relative mRNA expression of Pparγ (H) and C/ebpα (I) in SUB and VIS low AP cultures DIFF in the presence of 20% of PBS or VIS high CM from cells treated or not with 30 μg/mL Igfbp2 neutralizing antibody during differentiation.(J) Relative mRNA expression of genes encoding extracellular matrix (ECM) components and fibroblast markers in 6 days UND VIS low and VIS high APs expressed as fold change from VIS low.(K) Representative Western blots of periostin, α-Sma, and α-Tubulin in UND and 6-days-DIFF SUB, VH, and VL APs, respectively.(L and M) The corresponding densitometry of (L) periostin and (M) α-Sma expression normalized by α-Tubulin and expressed as fold change from UND VH.(N) Representative images of immunocytochemistry in 6-days-DIFF SUB, VH, and VL APs stained for caldesmon, 4′,6-diamidino-2-phenylindole (DAPI), and BODIPY.(O and P) Relative mRNA expression of (O) α-Sma and (P) Sm22a in UND and 6 days DIFF VH and VL APs expressed as fold change from UND VH.Values are mean ± SEM (*p < 0.05; **p < 0.005 versus VH under the same condition; #p < 0.05; ##p < 0.005 versus UND within the same progenitors subset; $p <0.05; $ $p < 0.005 versus SUB under the same condition (n = 3–4 independent experiments, each including 10–15 mice). Scale bar size is 50 μm.
Fig 2: Summary of the main findings.P2Y1R is upregulated in reactive astrocytes in several neurological disorders such as epilepsy, stroke, Alzheimer’s disease, and traumatic brain injury. The increase in P2Y1R signaling in astrocytes leads to neuronal hyperexcitability by increased expression of IGFBP2, which acts as an excitatory signal on neurons. Our data indicate that the functional phenotype of reactive astrocytes with upregulated P2Y1R-IGFBP2 signaling is shared in several neurological diseases including epilepsy and stroke and may contribute to neuronal hyperexcitability.
Fig 3: IGFBP2 and P2Y1R are co-upregulated in reactive astrocytes in mouse models of seizure and stroke.A Representative images of GFAP, P2Y1R, and IGFBP2 immunohistochemistry in the hippocampal CA1 region. The images were taken from the side contralateral to the kainic acid injection. Note that P2Y1R and IGFBP2 were upregulated in reactive astrocytes. These astrocytes were similarly distributed in the hippocampal CA1 region. The bar graph shows a summary of P2Y1R- and IGFBP2-positive GFAP-positive astrocytes. n = 6–7 slices from 3 mice for each group (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. B Immunohistochemistry for IGFBP2 and P2Y1R in astrocytes was performed in brains 3 days after MCAO. Expression of IGFBP2 and P2Y1R appeared in GFAP-positive astrocytes in the striatum ipsilateral to MCAO. There was no obvious expression of either P2Y1R or IGFBP2 in the side contralateral to MCAO. The bar graph shows a summary of P2Y1R- and IGFBP2-positive GFAP-positive astrocytes. n = 6 slices from 4 mice (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. Source data are provided as a Source Data file.
Fig 4: Effects of pharmacological IGFBP2 signaling blockade on neuronal Ca2+ signals.Stimulus-evoked neuronal Ca2+ signals in AstroP2Y1OE or control mice, measured using jGCaMP8s. The Schaffer collaterals were stimulated 300 times with 40 Hz at 0.06 mA. A Linsitinib (10 μM), an IGF-1R antagonist, was treated for 1 h at room temperature. Linsitinib significantly reduced neuronal Ca2+ signal in AstroP2Y1OE slices. ACSF, n = 51 ROIs from 4 slices, 3 mice; Linsitinib, n = 52 ROIs from 4 slices, 3 mice (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. B Linstinib did not affect neuronal Ca2+ signals in control slices. ACSF, n = 53 ROIs from 6 slices, 4 mice; Linstinib, n = 71 ROIs from 6 slices, 4 mice (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. C IGF-2R antibody (5 μg/mL) was treated for 2 h at room temperature. IgG was used as a control. IgG control, n = 50 ROIs from 5 slices, 3 mice; IGF-2R antibody, n = 50 ROIs from 5 slices, 3 mice (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. D NBI 31772 (10 μM), which blocks IGFBP binding to IGF-1, was treated for 1 h at room temperature. ACSF, n = 35 ROIs from 5 slices, 4 mice; NBI 31772, n = 52 ROIs from 6 slices, 4 mice (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. Source data are provided as a Source Data file.
Fig 5: IGFBP2 in astrocytes leads to enhanced neuronal activities in AstroP2Y1OE mice.A, B Immunohistochemical analysis of IGFBP2 expression in the hippocampus. GFAP was used as an astrocytic marker. IGFBP2 signals that were colocalized with GFAP were quantified. Control, n = 293 ROIs from 15FOVs, 3 mice; AstroP2Y1OE, n = 331 ROIs from 12 FOVs, 3 mice (two-sided two-sample t-test). Data are presented as mean ± s.d. C Representative traces of EFS-evoked neuronal Ca2+ signals (magenta) and astrocytic Ca2+ signals (green). Compared with control IgG (n = 20 ROIs from 3 slices, 3 mice), IGFBP2 antibodies reduced neuronal Ca2+ signals without affecting astrocytic Ca2+ signals in AstroP2Y1OE mice (n = 40 ROIs from 6 slices, 3 mice) (two-sided two-sample t-test). Data are presented as mean ± s.d. D IGFBP2 at 10 ng/mL (1 h at room temperature) enhanced EFS-evoked neuronal Ca2+ signals in the soma. ACSF (control), n = 42 cells from 5 slices, 3 mice; IGFBP2, n = 61 cells from 6 slices, 3 mice (two-sided two-sample t-test). Data are presented as mean ± s.d. E Schematic diagram for the AAV-mediated knockdown of Igfbp2 in astrocytes. The schematic diagram in Fig. 6E was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. F Traces of neuronal Ca2+ signals in the soma of AstroP2Y1OE mice with or without Igfbp2 knockdown in astrocytes. Control, n = 55 cells from 9 slices, 4 mice; Igfbp2 gRNA, n = 33 cells from 5 slices, 3 mice (two-sided Mann–Whitney U test). Data are presented as mean ± s.d. Source data are provided as a Source Data file.
Supplier Page from BioLegend for Recombinant Mouse IGFBP-2 (carrier-free)