Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Wholemount immunostaining of cerebral cortex slices for VE-cadherin and ALK1 from P31 control and Alk1 iΔAEC mice (25µg tamoxifen on P2). (B) Wholemount immunostaining for CD31 and ALK1 in cerebral cortex slices of P10 control and Alk1 iΔAEC mice expressing Efnb2 H2B-GFP (50µg tamoxifen on P2). A and V label arteries and veins, respectively. Dashed outlines in ALK1 images indicate artery position.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Immunostaining, Control, Expressing

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Photograph of P33 Alk1 iΔAEC mouse with blood on nose (arrowhead). (B) Photograph of blood present (arrowhead) in cage housing P81 Alk1 iΔAEC mice. (C) Gross pathological images demonstrating multi-focal hemorrhages (arrowheads) present in Alk1 iΔAEC mice brains but absent in control at P7. All mice administered 25µg tamoxifen on P2.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: Kaplan-Meier analysis showed that time to moribundity in Alk1 iΔAEC mice administered 100µg tamoxifen on P2 and P3 (red triangles) was significantly faster (median 23 days) than control (black circles) and Alk1 iΔAEC mice administered 25µg tamoxifen on P2 (N = 45-80 mice per condition).

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) MICROFIL casting of nasal cavity and cranial exterior of control (P16) and Alk1 iΔAEC (P19) mice revealed enlarged and tortuous vessels in Alk1 iΔAEC mouse (100µg tamoxifen on P2 & P3). Black and white arrowheads indicate the facial artery/vein pair and a tangle of blood vessels in the distal nasal vestibule, respectively. Mouse schematic created in BioRender.com. (B) Wholemount immunostaining for CD31 and Rosa26 Ai75 Cre reporter signal in nasal mucosa of P21 control ( Bmx-Cre ERT2 ; Alk1 fx/+ ) and Alk1 iΔAEC mice (25µg tamoxifen on P2). Blue dashed line indicates the most dorsal crease of the nasal cavity. Caudal (C), rostral (R), dorsal (D), and ventral (V) directions are indicated.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control, Immunostaining

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) MICROFIL casting of the brain demonstrating tortuous vessels in control and Alk1 iΔAEC mice at P24 (100µg tamoxifen on P2 & P3). (B) Wholemount immunostaining for CD31 in cerebral cortex slices of P31 control and Alk1 iΔAEC mice (75µg tamoxifen on P2).

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Control, Immunostaining

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Wholemount immunostaining for CD31 and α-smooth muscle actin (αSMA) in nasal mucosa of P103 control and Alk1 iΔAEC mice (100µg tamoxifen per g body weight on P13 & P14). (B) Photomicrographs of wholemount immunostaining for CD31 and αSMA in cerebral cortex slices of P31 control and Alk1 iΔAEC mice (75µg tamoxifen on P2). Red arrows indicate gaps in smooth muscle coverage. Blue arrow indicates regions of misaligned smooth muscle cells. Brain schematic created in BioRender.com.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Immunostaining, Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Wholemount immunostaining for CD31 and expression of Efnb2 H2B-GFP arterial endothelial reporter in nasal mucosa of P13 control and Alk1 iΔAEC mice (100µg tamoxifen per g body weight on P13 & P14). Red boxed indicates location of inset images. Black dashed line indicates position of artery. (B) Wholemount immunostaining for CD31 and expression of Rosa26 Ai75 Cre recombinase reporter and Efnb2 H2B-GFP arterial endothelial reporter in nasal mucosa of P13 control and Alk1 iΔAEC mice (25µg tamoxifen on P2). Red boxed indicates location of inset images. (C) Wholemount immunostaining for CD31 and expression of Efnb2 H2B-GFP arterial endothelial reporter in cerebral cortex slices of P13 control and Alk1 iΔAEC mice (25µg tamoxifen on P2). Mouse schematic created in BioRender.com. A and V label arteries and veins, respectively.

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Immunostaining, Expressing, Control

Journal: bioRxiv

Article Title: Arterial endothelial deletion of Alk1 causes epistaxis and cerebral microhemorrhage with aberrant arteries and defective smooth muscle coverage

doi: 10.1101/2024.11.25.622742

Figure Lengend Snippet: (A) Schematic demonstrating location of pial vessel imaging during in vivo arterial tone assay. (B) Representative images of pial vasculature in live P13 control and Alk1 iΔAEC mice (100µg tamoxifen on P2 & P3) visualized through an open cranial window. aCSF, artificial cerebrospinal fluid. (C) Quantification of changes in acetylcholine-induced vasodilation between control and Alk1 iΔAEC mice (unpaired Student’s t-test). (D) Quantification of changes in arterial tone between control and Alk1 iΔAEC mice (Mann-Whitney U test).

Article Snippet: Antibodies and fluorescent probes used in this study include: (1) rat anti-CD31 (BD Pharmingen 555370; 1:500 dilution), (2) mouse anti-α-smooth muscle actin-Cy3-conjugated antibody (Sigma, C6198/F3777; 1:2000 dilution), (3) mouse anti-α-smooth muscle actin-Alexa Fluor 488-conjugated antibody (eBioscience,53-9760-82; 1:2000 dilution), (4) rat anti-VE-cadherin (BD Pharmingen, 555289, 1:200 dilution), (5) goat anti-ALK1 (R&D Systems, 1:200 dilution), (6) donkey anti-rat Alexa Fluor 488 (Invitrogen, A-11006, 1:1000 dilution), (7) donkey anti-rat Alexa Fluor 555 (Invitrogen, A21434, 1:1000 dilution), and (8) donkey anti-rat Alexa Fluor 647 (Jackson Immunoresearch Labs, 712-605-150, 1:500 dilution).

Techniques: Imaging, In Vivo, Control, MANN-WHITNEY

Journal: The European Respiratory Journal

Article Title: Regulating the cell shift of endothelial cell-like myofibroblasts in pulmonary fibrosis

doi: 10.1183/13993003.01799-2022

Figure Lengend Snippet: Berbamine induces FoxA2 to regulate transforming growth factor (TGF)β signalling and control endothelial cell (EC)-like myofibroblast differentiation. a) Expression of activin receptor-like kinase (ALK)5 in tdTomato + VE-cadherin + cells isolated from the lungs of SM22a cre Rosa tdTomato mice and treated with berbamine (20 µM). b) Gene expression in tdTomato + VE-cadherin + cells transfected with matrix Gla protein (MGP) siRNA in combination with berbamine treatment with or without overexpression of ALK5 (n=5). c) Expression of FoxA2 in tdTomato + VE-cadherin + cells isolated from the lungs of SM22a cre Rosa tdTomato mice and treated with berbamine (20 µM). d) Immunoblotting with densitometry using tdTomato + VE-cadherin + cell lysates treated with berbamine (Bbm) with or without overexpression of FoxA2 (cytomegalovirus promoter (CMV)-FoxA2) (n=3). e) Expression of endothelial markers in tdTomato + VE-cadherin + cells treated with berbamine in combination with FoxA2 overexpression or FoxA2 knockdown (FoxA2 siRNA) (n=6). f) Immunoblotting with densitometry of the lungs of VE-cadherin cre Mgp flox/flox mice, Sm22a cre Mgp flox/flox mice and control mice after treatment of berbamine (Bbm) (n=6). g) Quantification of VE-cadherin-positive and platelet-derived growth factor receptor (PDGFR)α-positive (VE-cadherin + PDGFRα + ) cells and VE-cadherin negative and PDGFRα positive (VE-cadherin − PDGFRα + ) cells after immunostaining of the lungs of bleomycin-injected wild-type mice, where gene expression was examined by real-time PCR (n=8). h) Immunostaining of ALK5 and FoxA2 of human pulmonary fibrosis (n=3). Scale bars=100 µm. a) and c) were analysed for statistical significance by unpaired two-tailed t-test. b), e) and g) were analysed for statistical significance by ANOVA with post hoc Tukey's test. The bounds of the boxes are upper and lower quartiles with data points; the line in the box is median; error bars are maximal and minimal values. SCR: scrambled siRNA; Ctr: control; Fn1: fibronectin 1. **: p<0.001, ***: p<0.0001.

Article Snippet: Blots were incubated with specific antibodies to BMP-1 and latent TGFβ1-binding protein-1 (LTBP-1) (ab205394 and ab78294; Abcam), Flag (F3165; Sigma-Aldrich), ALK5 (MAB5871; R&D Systems), Col3a1 and Fn1 (NB600 and NBP1-91258; Novus Biologicals). β-Actin (A2228; Sigma-Aldrich) was used as a loading control.

Techniques: Control, Expressing, Isolation, Gene Expression, Transfection, Over Expression, Western Blot, Knockdown, Derivative Assay, Immunostaining, Injection, Real-time Polymerase Chain Reaction, Two Tailed Test