itga8  (R&D Systems)

Journal: bioRxiv

Article Title: Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Baseline LMOD1 knockout phenotyping. ( A ) Nucleotide substitutions (red) in exon 2 of Lmod1 generate the orthologous R365* premature stop codon previously reported in a human patient. ( B ) Gastroparesis (yellow arrow) and megabladder (below) in Lmod1 R365* mouse phenocopies the same visceral myopathy described in a human. ( C ) Design of floxed Lmod1 mouse. Quantitative Lmod1 mRNA ( D ) and LMOD1 protein ( E ) expression in mice with indicated genotypes (n=3 mice). ( F ) Percent recombination of floxed Lmod1 locus with Itga8-CreER T2 driver ( Lmod1 SMKO ) using conventional PCR ( top ) and quantitative PCR ( bottom ) of genomic DNA derived from mouse aorta. ( G ) Quantitative RT-PCR of Lmod1 mRNA in aorta with primers depicted in panel C (n=3 mice). The reduced signal with primers F2+R2 signify the absence of an internal promoter yielding a mature Lmod1 mRNA, thus validating this conditional knockout as a true null allele. 4 ( H ) Intestinal myopathy (arrow) in conditional knockout of Lmod1 using Myh11-CreER T2 (labeled as Lmod1 KO-Myh11 ), but not in mice where the Itga8-CreER T2 driver was used (labeled Lmod1 KO-Itga8 ). ( I ) Body weights of homozygous floxed Lmod1 / Itga8Cre-ER T2 mice treated with oil (black) or tamoxifen (Tmx; red) beginning at eight weeks of age. The number of mice in each arm of the experiment is indicated in parentheses. ( J ) Systolic blood pressure, assessed by tail cuff method in oil (black) versus Tmx (red) homozygous floxed Lmod1 / Itga8Cre-ER T2 mice (n=6 mice). ( K ) Serum lipopolysaccharide (LPS) levels in conditional Lmod1 knockout mice using the indicated Cre drivers.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Knock-Out, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Labeling

Journal: bioRxiv

Article Title: Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) Confocal immunofluorescence microscopy (CIFM) of LMOD1 and ACTA2 in aorta and intestine of the indicated mice. Scale bars are 20 μm. Western blotting for LMOD1 in aorta ( B ) and intestine ( C ) of indicated mouse genotype. ( D ) Survival curves for Oil versus Tamoxifen (Tmx) treated floxed Lmod1 / Myh11-CreER T2 (left) and floxed Lmod1 / Itga8-CreER T2 (right) mice. The black arrow indicates the time of Oil or Tmx administration. Western blotting ( E ) and quantitation ( F ) of indicated proteins after six months of Oil or Tmx administration; n=5 independent mice per genotype ( Lmod1 WT is Lmod1 fl/fl ).

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Immunofluorescence, Microscopy, Western Blot, Quantitation Assay

Journal: bioRxiv

Article Title: Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) Experimental study starting at seven weeks of age. Beginning here, all Lmod1 WT mice carried the Itga8-CreER T2 allele and were treated with the same atherogenic regimen as Lmod1 SMKO mice. ( B ) Total serum cholesterol in Lmod1 WT (black dots) and Lmod1 SMKO (red dots) mice at indicated ages. ( C ) Total serum triglycerides and HDL and LDL. ( D ) Cumulative survival curves for Lmod1 WT and Lmod1 SMKO mice across multiple cohorts. Oil-Red-O (ORO) staining ( E ) and quantitation of percent ORO staining ( F ) across the aorta of Lmod1 WT (n=5) and Lmod1 SMKO (n=9) mice. ( G ) Gross cardiac images showing tofu-like appearance of coronary arteries (yellow arrows) in Lmod1 SMKO mice. Masson trichrome staining ( H ) and CIFM imaging of LMOD1 and ACTA2 protein ( I ) in sections of coronary artery from Lmod1 WT (top panels) and Lmod1 SMKO mice (bottom) under the PCSK9/HFD regimen for 52 days. Note the prominent ACTA2 positive fibrous cap in Lmod1 SMKO section. Scales bars, 50 μm.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Staining, Quantitation Assay, Imaging

Journal: bioRxiv

Article Title: Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Coronary artery SMC lineage tracing in Lmod1 SMKO mice. Lmod1 WT ( A-C ) and Lmod1 SMKO ( D-J ) coronary arteries from mice carrying the membrane tomato/membrane GFP (mTmG) reporter. Red fluorescence ( A,B,D,E,G,H ) indicates surrounding cardiomyocytes and endothelial and immune cells as well as fibroblasts whose mTmG reporter did not undergo recombination (i.e., Itga8-CreER T2 was inactive in these cells). Conversely, green fluorescence represents medial and plaque cells of SMC origin (i.e., Itga8-CreER T2 was active in these cells and recombined out the tomato reporter allowing for membrane GFP expression). Note GFP+ cells beginning to populate early atheroma ( D,E ; see also, 20F ) and comprising a large portion of advanced plaques ( G,H ). White asterisks indicate necrotic core. Immunogold electron microscopy lineage tracing (IEMLT) of a Lmod1 WT control coronary artery ( C ); an Lmod1 SMKO coronary from 16-day lesion with two intimal core-centric GFP+ cells (labeled 1 and 2) ( F ); and an advanced, 63-day lesion showing several GFP+ cells at the fibrous cap (I). ( J ) Higher magnification of red boxed region in panel I showing three fibrous cap cells (labeled 1-3), two of which (1 and 2) are GFP+. ( K ) Percentage of GFP+ cells in plaques of seven independent Lmod1 SMKO coronaries (red bar) and the percentage of Core (green bar) and Cap (blue bar) derived foam cells that were GFP+. Scale bars are 50 μm. Adv, adventitia; CM, cardiomyocyte; EC, endothelial cell; Int, intima; Med, media; RBC, red blood cell. See Supplemental Figures 19 and 20 for additional IEMLT images.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Membrane, Fluorescence, Expressing, Electron Microscopy, Control, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: ( A ) UCSC genome browser screenshot of human LMOD1 gene structure and position of annotated SNV. The approximate position of sgRNAs used to generate an 11.35 kb intronic deletion in mice is shown. Note that this deletion removes the rs34091558 SNV and an SRF-binding consensus CArG box (indicated by ChIP-seq peak in human coronary artery SMCs and an arrow pointing to a red [conserved CArG] line on the “CArGs track”). This region also coincides with active chromatin marks. Western blot ( B ) and CIFM ( C ) of aortic LMOD1 in mice homozygous for the intronic deletion. Western blot ( D ) and quantitation ( E ) of aortic LMOD1 and ITGA8 in the indicated genotypes. Oil-Red-O staining ( F,H ) and CIFM of LMOD1 ( G,I ) in coronary arteries from Lmod1 WT ( F,G ) and Lmod1 SMKO ( H,I ) mice. Scale bars, 50 μm.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Binding Assay, ChIP-sequencing, Western Blot, Quantitation Assay, Staining

Journal: bioRxiv

Article Title: Loss of the Coronary Artery Disease Risk Gene Leiomodin1 in Vascular Smooth Muscle Cells Triggers Rapid Onset Coronary Atherosclerosis

doi: 10.64898/2026.02.15.705944

Figure Lengend Snippet: Effect of Lmod1 iND on CAD phenotype in Lmod1 SMKO mice. (A) Western blot of LMOD1 protein t1/2 in Lmod1 WT (top) and Lmod1 iND (bottom) MASMCs following various times of Cycloheximide (CHX) exposure. (B) Quantitation of LMOD1 bands in panel A (n=3 independent experiments). (C) Electropherogram demonstrating sequence fidelity of the five DNA sequence-encoding amino acid substitutions (indicated in red at bottom with arrows). (D) IFM of LMOD1 and ORO staining of coronary arteries from each mouse model after 10 weeks of PCSK9/HFD. (E) Western blotting and quantitation of aortic LMOD1 from each genotype. (F) Total cholesterol measures in Lmod1 iND mice crossed with Tagln-Cre (black dots) or Itga8-CreER T2 (red dots) to induce the Lmod1 iND mutant.

Article Snippet: Membranes were blocked and incubated overnight at 4 °C with primary antibodies against LMOD1 (Proteintech #15117-1-AP), SRF (Cell Signaling Technology #5147S), ACTA2 (Sigma-Aldrich #A2547-0.2ML), ACTB (Affinity #T0022), GAPDH (Millipore #MAB374), ITGA8 (Santa Cruz #SC-365798), TAGLN (Abcam #ab14106), and Thrombospondin-1 (THBS1) (Abcam #ab85762) ( Table S3 ).

Techniques: Western Blot, Quantitation Assay, Sequencing, Staining, Mutagenesis

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Itga8 + fibroblasts represent a critical subpopulation driving neurogenic bladder fibrosis. A) UMAP plot of subclustered fibroblasts. B) Dot plot of markers for each subset. C) Frequency of each cluster at different time points. D) Representative immunofluorescence images and quantitative analysis of Col1a2 (green), Itga8 (red), and DAPI (blue) expression in different groups, n = 3 per group. E) Representative Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of the marker genes expressed in each subset of fibroblasts. F) Box plots of the collagen signature score for each fibroblast subset. G) UMAP plot of Acta2 . H) UMAP plot of Cthrc1 . I,J) Scatter plot demonstrating a significant positive correlation between Itga8 and (I) Acta2 or J) Cthrc1 . K) Dot plots demonstrated the expression dynamics of Acta2 , Cthrc1 , Itga8 , and Myl9 along pseudo‐time. L) The hdWGCNA dendrogram of seven identified modules. M) A bubble plot represented the scores of the seven modules in each subset. N) Networks of the representative genes from module 7. O) Enrichment analysis of each module. Fib. = Fibroblasts. Data represent mean ± SD. One‐way ANOVA was used for D. *** p < 0.001.

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Immunofluorescence, Expressing, Marker

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Itga8 promotes fibroblast contraction and migration via the FAK and RhoA/ROCK signaling pathways. A) Representative images of the contraction assay in fibroblasts transduced with OE‐NC or OE‐Itga8 lentivirus. B) Quantification of cell contraction in (A), n = 4 per group. C) Representative images of the scratch assay in fibroblasts transduced with OE‐NC or OE‐Itga8 lentivirus. D) Statistical analysis of the wound area over time in (C), n = 3 per group. E) Representative images of immunofluorescence staining in fibroblasts transduced with OE‐NC or OE‐Itga8 lentivirus. F) Quantification of α‐SMA/F‐actin in (E), n = 3 per group. G) qRT‐PCR analysis of Acta2 mRNA expression, n = 3 per group. H) qRT‐PCR analysis of Cthrc1 mRNA expression, n = 3 per group. I) Representative images of the contraction assay in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus. J) Quantification of cell contraction in (I), n = 6 per group. K) Representative images of the scratch assay in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus. L) Statistical analysis of the wound area over time in (K), n = 3 per group. M) Representative images of immunofluorescence staining in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus. N) Quantification of α‐SMA/F‐actin in (M), n = 3 per group. O) RNA sequencing of primary bladder fibroblasts transduced with sh‐Itga8 or sh‐NC. The heatmap shows the downregulation of fibroblast activation‐related genes in sh‐Itga8 transduced cells. P) Gene set enrichment analysis (GSEA) revealed downregulation of the pathway regulating the actin cytoskeleton in the sh‐Itga8 group. Q) Representative images of Western blots of Itga8 and downstream molecules. R) Representative immunofluorescence images of FAK. S) Quantification of FAK fluorescence intensity in (R). T) Western blot analysis of phosphorylated FAK (p‐FAK), total FAK, and RhoA in primary bladder fibroblasts transfected with either a kinase‐dead FAK mutant (K454R). U) Representative images and quantification of the contraction assay in fibroblasts transduced with FAK K454R mutant plasmid. V) Representative images and quantification of the scratch assay in fibroblasts transduced with FAK K454R mutant plasmid. W,X) Representative Western blot images of fibroblasts overexpressing Itga8 treated with the (W) FAK inhibitor PF‐573228 or the (X) Rho kinase inhibitor CCG‐1423. n.s. = not statistically significant; NPNT = nephronectin; PF = PF‐573228; CCG = CCG‐1423. Data represent mean ± SD. Unpaired two‐tailed t‐test was used for B, F, S, and U. One‐way ANOVA was used for G, H, J, and N. Two‐way ANOVA was used for D, L, and V. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Migration, Protein-Protein interactions, Contraction Assay, Transduction, Wound Healing Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, RNA Sequencing, Activation Assay, Western Blot, Fluorescence, Transfection, Mutagenesis, Plasmid Preparation, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Trem2⁺ macrophages interact with fibroblasts via the Fn1‐Itga8 ligand‐receptor pairing. A) UMAP plot of subclustered macrophages. B) Dot plot of markers for each subset. C) Frequency of each cluster at different time points. D) Representative immunofluorescence images and quantitative analysis of Cd68 (red), Trem2 (yellow), and DAPI (blue) expression in different groups, n = 3 per group. E) Representative GO and KEGG enrichment of the marker genes expressed in each subset of macrophages. F) Box plots of the ECM score for each subset. G) Box plots of the collagen score for each subset. H) Box plots of the pro‐fibrotic score for each subset. I) Box plots of the anti‐fibrotic score for each subset. J) Bubble plot showing the primary ligand‐receptor pairs involved in the interaction between Trem2⁺ macrophages and Itga8⁺ fibroblasts. K) Representative western blot images of Itga8, Itgb1, Itga5, and Fn1. L) Representative images of the immunoprecipitation assay in fibroblasts. M) Representative images of the immunoprecipitation assay in 293T cells overexpressing Itga8. N) Dot plots demonstrated the expression dynamics of Fn1 and Trem2 along pseudo‐time. Mφ = Macrophage. Data represent mean ± SD. One‐way ANOVA was used for D. *** p < 0.001.

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Immunofluorescence, Expressing, Marker, Western Blot, Immunoprecipitation

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Co‐culture experiments demonstrate that Fn1 derived from Trem2⁺ macrophages activates fibroblasts via Itga8. A) Schematic representation of the co‐culture experiment design. B) Flow cytometry confirms that IL‐17a induces the differentiation of BMDM into Trem2⁺ macrophages in vitro. C) Quantification of the percentage of Trem2 + macrophage in (B), n = 3 per group. D) Representative Western blot images of in vitro‐induced Trem2⁺ macrophages. E) ELISA analysis of Fn1 expression in the conditioned medium. F) Representative Western blot images of primary bladder fibroblasts treated with conditioned medium from IL‐17a‐induced BMDM. G,H) qRT‐PCR (G) and Western blot (H) analysis of Fn1 expression at the mRNA and protein levels following transfection with Fn1 siRNA. I) ELISA analysis of Fn1 expression in the culture supernatant following transfection with Fn1 siRNA. J) Representative Western blot images of fibroblasts treated with conditioned medium from IL‐17a‐induced BMDM transfected with either si‐NC or si‐Fn1. K) Representative images of the contraction assay in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with the conditioned medium. L) Quantification of cell contraction in (K), n = 4 per group. M) Representative Western blot images of fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with conditioned medium from IL‐17a‐induced BMDM. N) Representative images of the scratch assay in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with Fn1. O) Statistical analysis of the wound area over time in (N), n = 3 per group. P) Representative images of immunofluorescence staining in fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with Fn1. Q) Quantification of α‐SMA/F‐actin in (P), n = 3 per group. R,S) qRT‐PCR (R) and Western blot (S) analysis of Trem2 expression at the mRNA and protein levels following transfection with OE‐Trem2. T) Flow cytometry confirms that IL‐17a induces the differentiation of BMDM into Trem2⁺ macrophages in vitro. U) Quantification of the percentage of Trem2 + macrophage in (T), n = 3 per group. V) Representative Western blot images of fibroblasts transduced with sh‐NC or sh‐Itga8 lentivirus and treated with conditioned medium from Trem2‐overexpressing BMDM. Mφ = Macrophage. Data represent mean ± SD. Unpaired two‐tailed t‐test was used for C, E, G, I, R, and U. One‐way ANOVA was used for L, and Q. Two‐way ANOVA was used for O. * P < 0.05, ** P < 0.01, *** p < 0.001. Figure was created in BioRender. Wang, J. (2025) https://BioRender.com/kp68v7m .

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Co-Culture Assay, Derivative Assay, Flow Cytometry, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Transfection, Contraction Assay, Transduction, Wound Healing Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Knockdown of Itga8 improves urodynamic parameters and bladder fibrosis in rats. A) Schematic diagram of the animal experimental design. B) Representative urodynamic features during the acute and chronic phases of BPNI in the control, NB, NB + sh‐NC, and NB + sh‐Itga8 groups. The black arrow indicates a single voiding event. C) Bar graphs representing the maximum intravesical pressure, change in intravesical pressure, and voiding efficiency (calculated as voided volume/maximum bladder capacity) for each group, n = 6 per group. D) Representative images of Masson's trichrome staining for each group. E) Quantification of the ratio of smooth muscle to collagen in (E), n = 3 per group. F) Measurement of hydroxyproline content during the acute and chronic phase of BPNI, n = 3 per group. G,H) Representative western blot images (G) and quantitative analysis (H) of Itga8, FAK, RhoA, ROCK1, ROCK2, and Myl9. Data represent mean ± SD. One‐way ANOVA was used for C, E, F, and H. Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001. Compared with the sh‐NC group, # p < 0.05, ## p < 0.01, and ### p < 0.001. Figure was created in BioRender. Wang, J. (2025) https://BioRender.com/3xlbk8m .

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Knockdown, Control, Staining, Western Blot

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Col1a2 ‐CreERT; Itga8 fl/fl mice demonstrates that Itga8 deletion delays fibrosis progression and protects bladder function. A) Schematic diagram of the animal experimental design. B) Representative immunofluorescence images of Col1a2 (green), Itga8 (red), and DAPI (blue) expression in different groups. C) Quantitative analysis of percentage of Itga8 + fibroblasts, n = 3 per group. D) Representative flow cytometry plots of Itga8 + fibroblasts in each group. E) Quantification of the percentage of Itga8 + fibroblasts in (D), n = 3 per group. F) Representative urodynamic profiles during the acute and chronic phases of BPNI in Col1a2 ‐CreERT⁺; Itga8 fl/fl mice and their littermate controls. The black arrow indicates a single voiding event. G,H) Bar graphs representing (G) delta intravesical pressure and (H) voiding efficiency (calculated as voided volume/maximum bladder capacity) for each group, n = 6 per group. I) Representative images of Masson's trichrome staining for each group. J) Quantification of the ratio of smooth muscle to collagen in (I), n = 3 per group. K) Measurement of hydroxyproline content during the acute and chronic phase of BPNI, n = 3 per group. L,M) Representative western blot images (L) and quantitative analysis (M) of isolated fibroblasts from the acute and chronic phases of BPNI in Col1a2 ‐CreERT⁺; Itga8 fl/fl mice and their littermate controls. Data represent mean ± SD. An unpaired two‐tailed t ‐test was used for C, G, H, K, and M. One‐way ANOVA was used for E and J. * p < 0.05, ** p < 0.01, *** p < 0.001. Figure was created in BioRender. Wang, J. (2025) https://BioRender.com/avquqdv .

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Immunofluorescence, Expressing, Flow Cytometry, Staining, Western Blot, Isolation, Two Tailed Test

Journal: Advanced Science

Article Title: Targeting Itga8 Mitigates Neurogenic Bladder Fibrosis Driven by Trem2⁺ Macrophage‐Derived Fn1 via FAK/RhoA/ROCK Signaling

doi: 10.1002/advs.202510631

Figure Lengend Snippet: Schematic diagram for the process of Itga8 + fibroblast activation in the context of neurogenic bladder fibrosis. In the normal bladder, fibroblasts and macrophages were in a state of quiescence. In contrast, neurogenic bladder was characterized by a substantial augmentation in the population of Itga8⁺ fibroblasts during the acute phase post‐injury. Moreover, fibroblast activation was orchestrated by Trem2⁺ macrophages, which secreted Fn1 to engage Itga8 on fibroblasts, thereby reinforcing the pro‐fibrotic communication between fibroblasts and macrophages. This, in turn, activated the FAK/RhoA/ROCK pathway and increased the expression of pro‐fibrotic genes through cytoskeletal remodeling, leading to fibroblast activation and ultimately to bladder fibrosis. Inhibition of Itga8 was expected to block the pathways mentioned above, delay fibrogenesis, and preserve bladder function integrity. Created in BioRender. Wang, J. (2025) https://BioRender.com/m2gslzz .

Article Snippet: The Col1a2 ‐CreERT; Itga8 fl/fl mice were generated by Cyagen Bioscience (Suzhou, China).

Techniques: Activation Assay, Expressing, Inhibition, Blocking Assay

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Expression Profile of ITGA8 at Different Developmental Stage. A) RNA‐sequencing analysis reveals age‐dependent different genes in human brain microvessels ( n = 3 samples). B) qPCR analysis confirms the age‐dependent modulation of pericyte related genes expression in human brain microvessels ( n = 4 samples, all p < 0.0001). C) Representative immunostaining of ITGA8 in human brain sections. Scale bars: 100 µm (upper), 50 µm (lower). ITGA8 expression was quantified ( n = 4 samples, p = 0.0028, 0.0015). D) Western blotting analysis of ITGA8 protein levels at various developmental stages, normalized to sham controls. “E” denotes embryonic days, “P” denotes postnatal days, and “M” denotes age in months ( n = 4 mice, p = 0.9999, 0.0022, 0.0014, 0.0098, and 0.9998). E) Western blotting indicates the predominant expression of ITGA8 in human brain pericytes (PC) compared to astrocytes (AS) and endothelial cells (EC) ( n = 3 cell samples, all p < 0.0001). F) Immunofluorescence showed the distinct temporal expression profile of ITGA8 ‐expressing cells during BBB development, with the right panels providing magnified views (scale bar: 15 µm) of the areas marked in the left panels (scale bar: 40 µm). G) The temporal expression profile of Pdgfrb ‐expressing cells during BBB development, using immunofluorescence staining, and the right panels offer magnified views (scale bar: 15 µm) of the selected areas in the left panels (scale bar: 40 µm). Arrowheads point to scattered PDGFRβ+ cells within the brain parenchyma. H) Quantification of the density of tdTomato+ cells per 100 µm^2 of CD31 area in various brain regions, presenting a comparative analysis of tdTomato+ cell density between ITGA8 + cells and PDGFRβ+ cells across different ages. The number of tdTomato+ cells is normalized to the CD31 area, reflecting regional vascular density variations ( n = 5 mice, p = 0.0022, 0.0467, 0.0428, 0.0460, and 0.5382). I) Quantification of the coverage of tdTomato+ cells on CD31+ vessels, comparing the coverage between ITGA8 + cells and PDGFRβ+ cells across various ages normalized to vascular area ( n = 5 mice, p = 0.0028, 0.0035, 0.0468, 0.3657, and 0.6354). Data represent mean ± SEM. Significance notations: ns = not significant ( p > 0.05), * p < 0.05, ** p < 0.01, **** p < 0.0001; Unpaired, 2‐tailed Student t‐ test was used to compare groups in (H,I). Comparisons between multiple groups were made using one‐way ANOVA test followed by Tukey's HSD post hoc test in (B–D).

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Expressing, RNA Sequencing, Immunostaining, Western Blot, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Deletion of Pericyte Itga8 Impairs Neural Function. A) Schematic illustration of the experimental paradigm for conditional knockout of pericyte ITGA8 using tamoxifen intraperitoneal injection, followed by functional analysis. B) Western blot analysis showing ITGA8 protein levels in brain microvessels of ITGA8 iPCKO and ITGA8 fl/fl mice at 12 days post‐tamoxifen administration. M indicates marker ( n = 4 mice, p = 0.0001). C) Kaplan–Meier survival curve comparing survival rates between ITGA8 iPCKO and ITGA8 fl/fl mice (> 95%, 60 days). Survival percentages are indicated ( n = 20 mice, p < 0.0001). Log‐rank test. D–F) Gait analysis using the Catwalk XT10.0 system. Quantification of regularity index D), maximum contact area E), and swing speed F) in ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days. Right forelimb (RF), right hindlimb (RH), left forelimb (LF), and left hindlimb (LH) are assessed ( n = 20 mice, p = < 0.0168, 0.0001, 0.0012, 0.0002, 0.0148, 0.0008, 0.0001, 0.0001, and 0.0001). G) Rotarod test results showing motor coordination and learning capabilities, with latency to fall for ITGA8 iPCKO and ITGA8 fl/fl mice at 12‐ and 40‐days ( n = 20 mice, p = 0.0023). H,I) Pole Climbing Test (PCT) results measuring turnaround time (iTurn) H) and total climbing duration (TD) I) at 12‐ and 40‐days ( n = 18 mice, all p < 0.0001). J) Timeline of Morris Water Maze (MWM) test was conducted on ITGA8 iPCKO mice and ITGA8 fl/fl mice. K) Initial swimming distances was measured in the MWM test before training trials at 40 days. Data from ITGA8 fl/fl ( n = 13 mice) and ITGA8 iPCKO ( n = 27 mice, p = 0.5132). L) Comparison of escape latency during the acquisition phase of the MWM test between ITGA8 iPCKO and control mice at different time points. Data from ITGA8 fl/fl ( n = 13 mice) and ITGA8 iPCKO ( n = 27 mice, p = 0.6962, 0.0212, 0.0873, 0.0042, 0.013). M) Representative swimming traces of ITGA8 iPCKO and ITGA8 fl/fl mice during the probe trial at 45 days. N,O) Quantification of platform zone crossings N) and latency to first reach the platform zone O) during the probe trial at 45 days in the MWM test. Data from ITGA8 fl/fl ( n = 13 mice) and ITGA8 iPCKO ( n = 27 mice, p = 0.0388, 0.0332). P) Golgi staining of pyramidal neurons in the motor cortex and hippocampus CA1 at 40 days. Scale bar: 50 µm. Higher magnification of dendritic spines shown in right panels. Scale bar: 15 µm. Q) Quantitative analysis of dendritic spine density in neurons of the motor cortex and hippocampus CA1 at 40 days. Data points represent mice. ITGA8 iPCKO ( n = 8 mice), ITGA8 fl/fl ( n = 5 mice, all p < 0.0001). R) Fluoro‐Jade B (FJB) staining of brain sections at 40 days. FJB positive neurons are indicated by white arrowheads. (Scale bar: 30 µm, n = 6 mice). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All intergroup comparisons were analyzed using unpaired 2‐tailed Student t‐ test.

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Knock-Out, Injection, Functional Assay, Western Blot, Marker, Comparison, Control, Staining

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Role of Pericyte Itga8 in Maintaining BBB Integrity. A) Hematoxylin and eosin–stained coronal sections. The framed areas show the location used for all histological measurements. Scale bar: 1000 µm. B) The brain vessels in ITGA8 iPCKO mice and ITGA8 fl/fl mice show the comparable permeability to dextran‐2000 kDa at 12 days. Higher magnification views in right panels. Scale bar: 25 µm. Quantification of vessel permeability to dextran‐2000 kDa ( n = 6 mice, p = 0.5768). C) Brain sections of ITGA8 iPCKO and ITGA8 fl/fl mice at 12 days showing dextran‐70 kDa leakage from blood vessels. Higher magnification views in right panels. Scale bars: 25 µm (left), 10 µm (right). Arrowheads point to dextran‐70 kDa tracer leakage outside blood vessels. Vessel permeability was quantified in brain sections ( n = 6 mice, p = 0.0021). D) Brain sections of ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days showing dextran‐70 kDa leakage from blood vessels. Higher magnification views in right panels. Scale bar: 25 µm (left). Arrowheads point to dextran‐70 kDa tracer leakage outside blood vessels. Vessel permeability was quantified in brain sections ( n = 6 mice, p < 0.0001). E) Brain sections of ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days showing leakage of dextran‐2000 kDa from blood vessels. Higher magnification in right panels; Arrowheads point to dextran‐2000KD tracer leakage outside blood vessels. Scale bar: 25 µm. Quantification of vessel permeability in brain sections ( n = 6 mice, p < 0.0001). F) Transmission electron microscopy of brain capillaries in ITGA8 iPCKO and control mice at 40 days. EC: endothelial cell; PC: pericyte; L: lumen. Arrows indicate the basement membrane; the red asterisk (*) highlights perivascular edema. G,H) Double immunostaining of CD31 and Occludin in brain sections at 12 days G) and 40 days H). Scale bar: 20 µm. Right panels: Quantification of Occludin mean fluorescence intensity (MFI) on CD31+ vessels ( n = 6 mice, p = < 0.5932, 0.0001). I,J) Double immunostaining of CD31 and Claudin5 in brain sections at 12 days I) and 40 days J). Scale bar: 20 µm. Right panels: Quantification of Claudin5 MFI on CD31+ vessels ( n = 6 mice, p = < 0.0062, 0.0001). Data represent mean ± SEM. Significance notations: ns ( p > 0.05); ** p < 0.01; *** p < 0.001; **** p < 0.0001. All intergroup comparisons were analyzed using unpaired 2‐tailed Student t‐ test.

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Staining, Permeability, Transmission Assay, Electron Microscopy, Control, Membrane, Double Immunostaining, Fluorescence

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Impact of ITGA8 Deficiency on Pericyte Morphology and Interactions with Endothelial Cells. A) Brain sections of ITGA8 iPCKO; H11‐GFP‐tdTomato mice and control mice at 40 days, immunostained for vessels. Scale bar: 40 µm. Higher magnification images in the right panels indicated by yellow boxes, Scale bar: 20 µm. B–D) Quantification of B) pericyte projection length, C) coverage, and D) number in brain sections at 40 days ( n = 6 mice, p = < 0.0001, 0.0001, 0.8753). E) Representative images of CD13+ pericyte coverage on CD31+ vessels in brain sections of ITGA8 iPCKO mice and control mice at 40 days. Scale bar: 40 µm. Higher magnification in right panels with yellow boxes, Scale bar: 20 µm. F) Transmission electron microscopy of brain microvessels in ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days. EC: endothelial cell; PC: pericyte; L: lumen. The arrowhead indicates contracted pericyte bodies and diminished pericyte coverage on microvasculature, with pericytes appearing reduced interactions with neighboring endothelial cells in ITGA8 iPCKO mice. G) Double immunostaining for CD31 and N‐cadherin in brain sections at 40 days. Scale bar: 25 µm. Quantitative analysis of N‐cadherin relative MFI on CD31+ vessels in brain sections at 40 days ( n = 6 mice, p = 0.0018). H) GTP‐loaded RhoA levels measurement using a RhoA activation assay kit, which employs the pull‐down method to quantify active RhoA levels in HBPCs following hITGA8 and its knockdown ( n = 3 independent experiments). I) Quantification of RhoA activation status in HBPCs under ITGA8 overexpression versus knockdown conditions was performed using the G‐LISA RhoA activation assay ( n = 3 independent experiments, p = <0.0001, 0.0470). J) Images of ITGA8 manipulated HBPCs show that ITGA8 knockdown in HBPCs results in altered cell shapes and reduced projections compared to control cells, while ITGA8 overexpression enhanced cellular extensions. Additionally, the application of the ROCK inhibitor Y27632 to ITGA8 ‐overexpressing HBPCs reveals a reduction in the ITGA8 ‐induced morphological changes ( n = 3 independent experiments, all p < 0.0001). K) Western blot analysis of p‐MLC/MLC levels in the pericytes of ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days ( n = 4 mice, p = 0.0003). L) Western Blot Analysis of p‐MLC in HBPCs depicting the levels of p‐MLC/MLC in HBPCs following ITGA8 knockdown or overexpression or treated with RhoA inhibitor Y27632 ( n = 3 independent experiments, all p < 0.0001). M) Confocal images of HBPC‐HBEC mixed cultures in fibrin gel on day 3, showing cellular interactions. HBPCs were labeled with a red CellTracker dye and HBECs with a green one ( n = 3 independent experiments, p = 0.0012). N) Confocal image showing interactions between GFP‐expressing HBPCs (green) and unlabeled HBECs following a 16 h co‐culture in Matrigel. Arrowheads point to altered cellular projections in pericytes. Arrowheads denote pericyte processes and intercellular networks, while arrows highlight endothelial‐aligned pericyte somata ( n = 3 independent experiments, p = 0.0009). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), ** p < 0.01, **** p < 0.0001; Unpaired, 2‐tailed Student t‐ test was used to compare groups in (B–D), (G), (K), and (N). Comparisons between multiple groups were made using one‐way ANOVA test followed by Tukey's HSD post hoc test in (I–J) and (L).

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Control, Transmission Assay, Electron Microscopy, Double Immunostaining, Activation Assay, Knockdown, Over Expression, Western Blot, Labeling, Expressing, Co-Culture Assay

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Pericyte Cytoskeletal Tension and Interaction with ECM Activating TGFβ Signaling. A) Immunofluorescence analysis of F‐actin (phalloidin) and α‐SMA in pericytes demonstrated ITGA8 expression‐dependent cytoskeletal remodeling, with quantitative assessment of phalloidin‐stained cellular projections ( n = 4 independent experiments, p = < 0.0421, 0.0001). B) Representative images of the vascular network formed by HBECs (lenti‐mCherry‐labeled) and HBPCs (lenti‐EGFP‐labeled) in microfluidic chips are shown. HBPCs with ITGA8 knockdown or overexpression exhibited morphological changes and altered interaction with endothelial cells ( n = 4 independent experiments, p = < 0.0286, 0.0001). C) Representative fluorescent images showing the leaked FITC‐microbeads outside of vascular at 60 min post‐perfusion. The asterisks indicate the area outside of the vascular ( n = 4 independent experiments, p = < 0.0001, 0.9325). D) Western blotting analysis showing the total TGFβ1 levels after ITGA8 knockdown or overexpression in HBPCs. The total TGFβ1 levels were quantified in different group ( n = 3 independent experiments, p = 0.9686). E) Elisa analysis showing the active TGFβ in the HBPCs conditional medium following ITGA8 knockdown or overexpression ( n = 3 independent experiments, p = 0.0123, 0.0066). F,G) Western blot analysis of p‐SMAD2/3 / SMAD2/3 levels in HBPCs following ITGA8 knockdown or overexpression ( n = 3 independent experiments, all p < 0.0001). H) Elisa analysis of active TGFβ1 levels in the condition medium of pericyte‐endothelial cell transwell co‐culture system ( n = 3 independent experiments, p = 0.9971, 0.0102). I) Elisa analysis showing the active TGFβ levels in the HBPCs conditional medium following ITGA8 overexpression or treatment with CytoD ( n = 3 independent experiments, p = < 0.0083, 0.0276, 0.0001). J) Co‐immunoprecipitation (Co‐IP) showing the binding between ITGA8 and TGFβ1 . K) Elisa analysis showing the mature TGFβ in the HBPCs conditional medium after pretreatment with RGD peptide ( n = 3 independent experiments, p = < 0.0194, 0.3252, 0.0001). L) Representative images of the vascular network formed by HBECs (lenti‐mCherry‐labeled) and HBPCs (lenti‐EGFP‐labeled) in microfluidic chips are shown. HBPCs were transfected with lenti‐ ITGA8 or the co‐culture system were treated with RGD peptide ( n = 4 independent experiments, p = < 0.0446, 0.0001). M) Representative fluorescent images showing the leaked FITC‐microbeads outside of vascular at 60 min post‐perfusion in Blank, RGD, ITGA8 OE , and ITGA8 OE + RGD group. The asterisks indicate the area outside of the vascular. The vascular permeability was quantified ( n = 4 independent experiments, p = < 0.0001, 0.0006). N) Representative images of the vascular network formed by HBECs (lenti‐mCherry‐labeled) and HBPCs (lenti‐EGFP‐labeled) in microfluidic chips of Blank, SB431542, ITGA8 OE , and ITGA8 OE + SB431542 group. HBPCs were transfected with lenti‐ ITGA8 or the co‐culture system were treated with SB431542 ( n = 4 independent experiments, p = < 0.0373, 0.0001). O) Representative fluorescent images showing the leaked FITC‐microbeads outside of vascular at 60 min post‐perfusion in Blank, SB431542, ITGA8 OE , and ITGA8 OE + SB431542 group. The asterisks indicate the area outside of the vascular. The vascular permeability was quantified ( n = 4 independent experiments, p = < 0.0001, 0.0012). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), * p < 0.05, ** p < 0.01, **** p < 0.0001; Multi‐group comparisons were analyzed by one‐way ANOVA with Tukey's HSD post‐hoc testing.

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Immunofluorescence, Expressing, Staining, Labeling, Knockdown, Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Transfection, Permeability

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Temporal Dynamics of ITGA8 Expression and Localization Post‐Stroke. A) tSNE plots of total 71040 cells derived from MCAO model and sham‐operated controls, revealing 16 major clusters from brain tissues collected at days 1, 3, and 7 post‐MCAO, as well as from the sham group with three mice included in each group. Each point in the plot represents an individual cell, indicated by a droplet barcode and assigned a color based on its cluster. 1804 mural cells were identified. B) tSNE‐based unsupervised clustering revealing nine unique mural cell subtypes, each differentiated by distinct gene expression profiles. C) Violin plots show the expression of top cell‐type specific marker genes in each cluster of mural cells. D) Dynamic changes in mural cell subtype proportions post‐ischemia. Graph showing the temporal variation of specific mural cell subtypes (including cluster 0) at different time points (Sham, Day 1, 3, and 7 following MCAO surgery). E) Western blotting analysis of ITGA8 expression post‐stroke ( n = 6 mice, p = < 0.0001, 0.0001, 0.0001, 0.0012, 0.8101). F) Immunofluorescence staining demonstrates the distinct temporal pattern of ITGA8+ cell in post stroke. Scale bar: 40 µm. Right panels are magnified views of boxes indicated in left panels. Scale bar: 15 µm. Arrowhead indicates arteriole, arrow indicates venule. G) Immunofluorescence staining demonstrates the temporal pattern of PDGFRβ+ cell after stroke. Scale bar: 40 µm. Right panels are magnified views of boxes indicated in left panels. Scale bar: 15 µm. H) Quantification of tdTomato+ cells coverage on vessels of ITGA8‐ Cre; H11‐tdTomato‐GFP mice and Pdgfrb ‐Cre; H11‐tdTomato‐GFP mice at the same time timepoint, including Sham, D1, D3, D5 and D7 post‐stroke ( n = 6 mice, p = 0.9841, 0.1232, 0.0056, 0.1523, 0.6488). I) Quantification of tdTomato+ cells per 100 µm 2 CD31+ area in different brain regions of ITGA8 ‐Cre; H11‐tdTomato‐GFP mice. Comparison of tdTomato+ cell density between two genotypes at the same timepoint ( n = 6 mice, p = 0.0431, 0.0737, 0.0003, 0.0378, 0.0484). J) Quantification of Scattered PDGFRβ+ cells at different time point. The number of tdTomato+ cells was compared across different time points ( n = 6 mice, p = < 0.0342, 0.0001, 0.0001, 0.0002, 0.0001). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), ** p < 0.01, *** p < 0.001, **** p < 0.0001; Unpaired, 2‐tailed Student t‐ test was used to compare groups in (H,I). Comparisons between multiple groups were made using one‐way ANOVA test followed by Tukey's HSD post hoc test in (E) and (J).

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Expressing, Derivative Assay, Gene Expression, Marker, Western Blot, Immunofluorescence, Staining, Comparison

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: ITGA8 Regulates Pericyte Remodeling and Neovascularization Post‐Stroke. A) Experimental setup for studying ITGA8 deletion in pericytes post‐MCAO surgery. B) Kaplan‐Meier survival analysis of ITGA8 iPCKO mice and control post‐MCAO (> 95%, 20 days). Values are expressed as percentage of surviving mice ( n = 15 mice, p = 0.0010). Log‐rank test. C) Statistical analysis of the neurological deficit score in ITGA8 iPCKO and ITGA8 fl/fl mice after MCAO surgery at day 7 ( n = 8 mice, p = 0.0233). D) MAP2 immunostaining showing the infarct size of ITGA8 iPCKO and ITGA8 fl/fl mice, the infarct area was indicated by the yellow broken line. Scale bar: 1000 µm. The infarct size was quantified ( n = 6 mice, p = 0.0030). E) Hematoxylin and eosin–staining showing the abnormal structure of microvascular in ITGA8 iPCKO mice at day 7. The areas framed by white broken line indicate infarct area. The peri‐infarct area, demarcated by the black frame, was analyzed for vascular phenotypes. The arrowheads indicated the microvascular in the peri‐infarct area. * indicates the perivascular edema. F) Brain sections of ITGA8 iPCKO; H11‐GFP‐tdTomato mice and control mice at 7 days, immunostained for vessels. Scale bar: 20 µm. The image, with yellow boxes indicating areas of higher magnification in the right panel. Scale bar: 20 µm. Representative image shows abnormalities in pericyte shape and diminished association between pericytes and endothelial cells in ITGA8 iPCKO mice, highlighted by arrowheads. G–J) Quantification of tdTomato+ pericyte coverage on vessels G), vascular area H), vessel diameter I) and PDGFRβ+ cells number J) at 7 days in ITGA8 iPCKO mice and control mice ( n = 6 mice, p = 0.0014, 0.4052, 0.0066, 0.7621). K) Lectin perfusion into infarct area vessels was determined. Yellow color (green / red double‐staining) indicates blood perfused vessels. Scale bar: 50 µm. Quantification of vessel perfusion % = Lectin+CD31+ area/total CD31+area*100 ( n = 6 mice, p < 0.0001). L) Dextran‐2000 kDa leaks out of blood vessels in ITGA8 iPCKO mice subjected to ischemia at 7 days. Scale bar: 20 µm. Yellow boxes correspond to higher magnification images in right panels. Scale bar: 10 µm. Arrowheads show tracer hotspots in ITGA8 iPCKO mice. Quantification of vessel permeability in brain sections of ITGA8 iPCKO mice and ITGA8 fl/fl mice ( n = 6 mice, p = 0.0030). M–O) Immunostaining of CD31 and Claudin5 M), Occludin N), and ZO1 O) in the brain sections at 7 days. Scale bar: 25 µm. Tight junctions’ MFI on the microvessels were quantified in ITGA8 iPCKO and ITGA8 fl/fl mice ( n = 6 mice, p = 0.0062, 0.0058, 0.3632). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), * p < 0.05; ** p < 0.01; **** p < 0.0001. Intergroup comparisons were analyzed using unpaired 2‐tailed Student t‐ test.

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Control, Immunostaining, Staining, Double Staining, Permeability

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Rescue of BBB Function via ITGA8 Overexpression in Pericytes Post‐Stroke. A) Experimental time: Tail vein injection of AAV‐PR vector to overexpress ITGA8 , MCAO surgery and functional analysis, including neural and BBB function assessment. B) Immunoblots showing the ITGA8 level in AAV‐PR‐con and AAV‐PR‐ ITGA8 group subjected to ischemia at 7 days. The ITGA8 expression levels were quantified ( n = 4 mice, p = 0.0004). C) MAP2 immunostaining showing the brain infarct size, the infarct area was indicated by the yellow broken line. Scale bar: 1000 µm. The infarct size of AAV‐PR‐con and AAV‐PR‐ ITGA8 group mice were quantified ( n = 6 mice, p = 0.0097). D) Statistical analysis of the neurological deficit score in AAV‐PR‐con and AAV‐PR‐ ITGA8 group mice ( n = 8 mice, p = 0.0033). E) Kaplan–Meier survival analysis of AAV‐PR‐con and AAV‐PR‐ ITGA8 group mice post‐MCAO (> 95%, 20 days). Values are expressed as percentage of surviving mice ( n = 15 mice, p = 0.0172). F) Immunostaining of brain sections for CD31 and tdTomato showed the morphogenesis and localization of pericytes in AAV‐PR‐con and AAV‐PR‐ ITGA8 group mice. Scale bar: 20 µm. Right panels are magnified views of boxes indicated in left panels. Scale bar: 10 µm. The arrowheads showing the elongated process of pericytes and increased interconnectivity between neighboring pericytes. G,H) Quantification of pericyte coverage G) and vessel diameter H) in AAV‐PR‐con and AAV‐PR‐ ITGA8 group mice ( n = 6 mice, p < 0.0001, 0.0035). I) Lectin perfusion into infarct area vessels was determined. Yellow color (green / red double‐staining) indicates blood perfused vessels. Scale bar: 50 µm. The vessel perfusion in infarct area was quantified ( n = 6 mice, p = 0.0016). J) Reduced leakage of dextran‐2000 kDa from vessels in AAV‐PR‐ ITGA8 group mice mice. Scale bar: 40 µm. The vessel permeability was quantified ( n = 6 mice, p = 0.0028). K–M) Immunostaining of CD31 and Claudin5 K), Occludin (), ZO1 M) in the brain sections at 26 days. Scale bar: 40 µm. The tight junctions’ MFI on the microvessels were quantified in AAV‐PR‐Con and AAV‐PR‐ ITGA8 group mice ( n = 6 mice, p = 0.0034, 0.0152, 0.9093). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Intergroup comparisons were analyzed using unpaired 2‐tailed Student t‐ test.

Article Snippet: The following mouse strains were used: ITGA8 flox/flox (The Jackson laboratory, stock 015840); Pdgfrb ‐P2A‐CreERT2 (The Jackson laboratory, stock 029684); B6/JGpt‐H11em1Cin (CAG‐LoxP‐ZsGreen‐Stop‐LoxP‐tdTomato) (H11‐tdTomato‐GFP) (GemPharmatech Co. Ltd, stock T006163); Pdgfrb ‐iCre (BIOCYTOGEN, stock 110129); ITGA8 ‐P2A‐iCre (GemPharmatech co. Ltd, Chengdu, China).

Techniques: Over Expression, Injection, Plasmid Preparation, Functional Assay, Western Blot, Expressing, Immunostaining, Double Staining, Permeability