Journal: Journal of cellular and molecular medicine

Article Title: Sodium Hyaluronate-PDGF Repairs Cartilage and Subchondral Bone Microenvironment via HIF-1α-VEGF-Notch and SDF-1-CXCR4 Inhibition in Osteoarthritis.

doi: 10.1111/jcmm.70515

Figure Lengend Snippet: FIGURE 6 | Effects of PDGF-BB and SH-PDGF on NFATc1, CXCR4, ALK1, and ALK5 expression in OA in vivo (5 rats in each group). (A and E) Effects of PDGF-BB and SH-PDGF on NFATc1 expression in subchondral bone determined by IF. (B and F) Effects of PDGF-BB and SH-PDGF on CXCR4 expression in cartilage, as shown by IF. (C and G) Effects of PDGF-BB and SH-PDGF on ALK1 expression in cartilage shown by IF. (D and H) Effects of PDGF-BB and SH-PDGF on ALK5 expression in cartilage, as shown by IF. ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The following primary antibodies were used: EMCN (1:500, rabbit, Proteintech), CD31 (1:500, rabbit, Proteintech), TRAP (1:200, rabbit, Proteintech), pimonidazole (1:50, mouse, Hypoxyprobe), HIF- 1α (1:500, rabbit, Proteintech), VEGF (1:300, rabbit, Proteintech), Notch1 (1:200, rabbit, Proteintech), Smad2 (1:500, rabbit, Proteintech), p- Smad2 (1:200, rabbit, Proteintech), Smad3 (1:500, rabbit, Proteintech), p- Smad3 (1:200, rabbit, Proteintech), NFATc1 (1:300, rabbit, Proteintech), SDF- 1 (1:300, rabbit, Proteintech), CXCR4 (1:200, rabbit, Proteintech), ALK1 (1:500, rabbit, Proteintech) and ALK5 (1:500, rabbit, Proteintech).

Techniques: Expressing, In Vivo

Journal: Theranostics

Article Title: FBLN7 KO attenuates age-related cardiac fibrosis by promoting TGFBR3/ALK1/Smad1 signaling and inhibiting the profibrotic phenotypes of cardiac fibroblasts

doi: 10.7150/thno.116477

Figure Lengend Snippet: FBLN7 modulates the impaired profibrotic abilities of senescent cardiac fibroblasts through the ALK1-Smad1/5/9 pathway. A-B) Representative western blot images displaying the levels of p-Smad2, Smad2, p-Smad1/5/9, and Smad1/5/9 proteins in cardiac fibroblasts with FBLN7 silencing (siFBLN7) (A), FBLN7 overexpression (adFBLN7) (B), and corresponding control cardiac fibroblasts (siNC or adNC) following treatment with D-gal or PBS. Quantifications of band intensity are presented on the right. C-D) Representative images illustrating the nuclear translocation of p-Smad1/5/9 (red) (C) and p-Smad2 (red) (D) in siFBLN7 and siNC cardiac fibroblasts after treatment with D-gal or PBS, labeled with Vimentin (green). E) Representative western blot images showing the protein levels of p-Smad2, Smad2, p-Smad1/5/9 and Smad1/5/9 in D-gal treated cardiac fibroblasts transfected with siNC, siFBLN7, or siFBLN7 plus ML347 treatment. Quantifications are displayed on the right. F) Representative western blot images showing the protein levels of Collagen I and III, ALK1, α-SMA and P21 in D-gal treated cardiac fibroblasts transfected with siNC, siFBLN7, or siFBLN7 plus ML347 treatment. Quantifications are displayed on the right. Error bars represent the standard error of the mean (SEM). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: When indicated, CFs were pretreated with 250 nM ALK1 inhibitor (ML347, MCE, Shanghai, China) or 0.01% DMSO (Sigma-Aldrich) for 2 h prior to a 48-hour treatment with D-gal.

Techniques: Western Blot, Over Expression, Control, Translocation Assay, Labeling, Transfection

Journal: Theranostics

Article Title: FBLN7 KO attenuates age-related cardiac fibrosis by promoting TGFBR3/ALK1/Smad1 signaling and inhibiting the profibrotic phenotypes of cardiac fibroblasts

doi: 10.7150/thno.116477

Figure Lengend Snippet: TGFBR3 is essential for the differential transduction of TGF-β signals mediated by FBLN7. A) Endogenous co-immunoprecipitation (Co-IP) of FBLN7 and TGFBR3. Immunoprecipitation (IP) was performed with TGFBR3, followed by immunoblotting (IB) with FBLN7 in senescent cardiac fibroblasts (CFs). B) Representative confocal images demonstrating the co-localization of FBLN7 and TGFBR3 in senescent CFs. C) Reciprocal Co-IP of FBLN7 and TGFBR3. IP was conducted with GFP-tag and IB with Flag-TGFBR3 in 293T cells (left panel). Conversely, IP was performed with Flag-tag and IB with GFP-FBLN7 in 293T cells (right panel). D) Representative confocal images illustrating the co-localization of Myc-FBLN7 and Flag-TGFBR3 in 293T cells. E) Results of molecular docking between FBLN7 and TGFBR3. a) The optimal docking conformation of FBLN7 and TGFBR3 reveals a stable complex, with FBLN7 highlighted in blue and TGFBR3 in yellow. b) The white semi-transparent structure represents the entire molecular framework, providing a global view of the docking region. The black box in the center indicates the binding site, emphasizing critical areas on the binding interface. c) The Docking Zoom Map displays key amino acid residues at the binding interface and their interactions. F-G) Representative western blot images showing TGFBR3 and ALK1 protein levels in CFs with FBLN7 silencing (F) and overexpression (G) after treatment with D-gal or PBS. Quantifications of band intensity are presented on the right. H) Representative western blot images illustrating protein levels of FBLN7 and TGFBR3 in D-gal-treated CFs infected with either Null, adenovirus encoding FBLN7 (adFBLN7), or adFBLN7 plus Flag-TGFBR3. Quantifications are displayed on the right. I) Representative western blot images illustrating protein levels of p-Smad1/5/9, Smad1/5/9, p-Smad2 and Smad2 in D-gal-treated CFs infected with either Null, adFBLN7, or adFBLN7 plus Flag-TGFBR3. Quantifications are displayed on the bottom. J) Representative western blot images illustrating protein levels of Collagen I and III, ALK1, α-SMA, and P21 in D-gal-treated CFs infected with either Null, adFBLN7, or adFBLN7 plus Flag-TGFBR3. Quantifications are displayed around the imge. Error bars represent the standard error of the mean (SEM). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: When indicated, CFs were pretreated with 250 nM ALK1 inhibitor (ML347, MCE, Shanghai, China) or 0.01% DMSO (Sigma-Aldrich) for 2 h prior to a 48-hour treatment with D-gal.

Techniques: Transduction, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, FLAG-tag, Binding Assay, Over Expression, Infection

Journal: Theranostics

Article Title: FBLN7 KO attenuates age-related cardiac fibrosis by promoting TGFBR3/ALK1/Smad1 signaling and inhibiting the profibrotic phenotypes of cardiac fibroblasts

doi: 10.7150/thno.116477

Figure Lengend Snippet: GRO rescues the impaired profibrotic phenotypes of senescent cardiac fibroblasts and prevents age-related cardiac fibrosis and diastolic dysfunction. A) Representative Western blot images displaying protein levels of Collagen I and III, TGFBR3, ALK1, α-SMA, and P21 in senescent CFs infected with adenovirus encoding FBLN7 (adFBLN7) and treated with DMSO or various concentrations of GRO (50, 100, and 200 μM). Quantifications are presented on the right. *: adNC+D-gal+DMSO vs. adNC+PBS+DMSO, &: adFBLN7+D-gal+DMSO vs . adNC+D-gal+DMSO, $: adFBLN7+D-gal+GRO50 vs. adFBLN7+D-gal+DMSO, #: adFBLN7+D-gal+GRO100 vs. adFBLN7+D-gal+DMSO, %: adFBLN7+D-gal+GRO200 vs. adFBLN7+D-gal+DMSO. B) Schedule for FBLN7 overexpression and GRO supplementation in mice. C) Gross appearances of mice and their hearts in each group. D) Representative echocardiography images of the two groups of mice. Upper: B-mode; middle: PW Doppler mode; lower: tissue Doppler mode. Echocardiographic analysis of interventricular septal thickness in diastole (IVSd) and systole (IVSs), left ventricular posterior wall thickness in diastole (LVPWd) and systole (LVPWs), left ventricular mass, the ratio of early to late diastolic filling velocities (E/A), and the ratio of early diastolic filling velocity to left atrial pressure (E/e'), left ventricular ejection fraction (LVEF) and fractional shortening (FS%) are shown at the bottom. E) Representative images of H&E staining of hearts from aging mice (18 months old) injected with AAV-FBLN7 and administered 40 mg/kg/d of GRO or solvent control via gavage. F) Representative WGA staining images of the two groups of mice, with quantification shown on the right. G) Representative micrographs of Masson staining in heart sections from the AAV-FBLN7-18M-GRO and AAV-FBLN7-18M-CTL mice, displaying three different views: upper: perivascular; middle: global; lower: interstitial. Quantifications of fibrotic areas are presented on the right. H) FBLN7 exerts its reversing effect on the impaired profibrotic phenotypes of senescent CFs by downregulating the TGFBR3/ALK1/Smad1 pathway, which may contribute to the pathogenesis of age-related cardiac fibrosis. GRO exerts antifibrotic effects possibly through interaction with FBLN7. Error bars represent the standard error of the mean (SEM). Statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: When indicated, CFs were pretreated with 250 nM ALK1 inhibitor (ML347, MCE, Shanghai, China) or 0.01% DMSO (Sigma-Aldrich) for 2 h prior to a 48-hour treatment with D-gal.

Techniques: Western Blot, Infection, Over Expression, Staining, Injection, Solvent, 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 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: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) UMAP plot of retinal cells from P6. (B) UMAP plot of retinal cells from P6 Alk1 control and Alk1 ECKO retinas. (C) UMAP plot of Pecam1 and Cldn5 from P6 Alk1 control and Alk1 ECKO retinal cells. (Pecam1: light green, Cldn5: blue) (D) UMAP plot of ECs from P6 Alk1 control and Alk1 ECKO retinas. (E) UMAP plot of EC sub-clusters from P6 Alk1 control and Alk1 ECKO retinas. (F) Dot plot of expression level and frequency among cell clusters of selected genes in P6 ECs. Color scale: green, high expression; yellow, low expression.(G) Gene set enrichment analysis of differentially expressed genes (DEGs) comparing the AVM cluster to Alk1 f/f retinal ECs. (H) Volcano plot of DEGs between the AVM cluster in Alk1 ECKO ECs and ECs from Alk1 control at P6.

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) PIEZO1 mRNA expression by qPCR in purified mouse retinas from P6 mice injected with 100 µg TAM at P4 (n=5). Error bars: SEM. **P-value < 0.01, Mann– Whitney U test. (B) IB4 (magenta) and PIEZO1 (white) staining of retinal flat mounts from postnatal day (P) 6 Alk1 f/f Cdh5 Cre ERT2 and Alk1 f/f pups. (C) PIEZO1 mRNA expression by qPCR in HUVECs transfected with Ctrl or ALK1 siRNA (n=3) (D) Western blot analysis HUVECs transfected with Ctrl or ALK1 siRNA after 72 hr. (E) Quantifications of PIEZO1 levels normalized to β-ACTIN (n=4) (F) Immunostaining of healthy skin adjacent (ADJ) to telangiectasias and telangiectatic lesions (TE lesions) from patients with HHT type 2 for PIEZO1 (green), CD31 (magenta), and ENG (white). (G) Quantification of PIEZO1 staining (n=8; 4 patients, with 2 images selected per patient for analysis). Error bars: SEM. *P-value < 0.05, Mann–Whitney U test. Scale bars: 500 μm (B) and 50 μm (F).

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Expressing, Purification, Injection, MANN-WHITNEY, Staining, Transfection, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) Schematic representation of the experimental strategy for Alk1 and Piezo1 deletion in mice from P4 to P6. (B) IB4 staining of retinal flat mounts from Alk1 f/f controls, Alk1 f/f Mfsd2a Cre ERT2 , Alk1 f/f Piezo1 f/+ Mfsd2a Cre ERT2 , and Alk1 f/f Piezo1 f/f Mfsd2a Cre ERT2 P6 mice. (C) Quantification of AVM number. n=10-12 mice per group. One-way ANOVA with Sidak’s multiple comparisons test. (D) Quantification of AVM vessel diameter (n=10-12). One-way ANOVA with Sidak’s multiple comparisons test. (E and F) Vascular labeling with latex dye (blue) in the retinas and gastrointestinal (GI) tract of Alk1 f/f and Alk1 f/f Cdh5 Cre ERT2 , Alk1 f/f Piezo1 f/+ Cdh5 Cre ERT2 , and Alk1 f/f Piezo1 f/f Cdh5 Cre ERT2 P6 pups. Black arrows indicate AVMs. (G) Experimental strategy to assess the effects of PIEZO1 inhibitor in Alk1 deleted retinas. Arrowheads indicate the time course of TAM (100 μg) and GsMTx4 (1 mg/kg) or vehicle administration. (H) IB4 staining of P6 retinal flat mounts from Alk1 f/f Cdh5 Cre ERT2 mice injected with GsMTx4 or vehicle at P4 and P5. ‘A’ indicates arteries, ‘V’ indicates veins, and arrows denote AVMs. (I) Quantification of AVM count. Each dot represents one retina (n=10). Mann–Whitney U test. Error bars: SEM. *P-value < 0.05, **P-value < 0.01, *** P-value < 0.001, ns: nonsignificant, Scale bars: 500 μm (B and H), 200 μm (E), 10 mm (F)

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Staining, Labeling, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) Western blot analysis of HUVECs transfected with control, ALK1 , PIEZO1 , and ALK1 + PIEZO1 siRNAs, followed by 45 min of flow exposure. (B-D) Quantification of P-VEGFR2/VEGFR2, P-ERK5/ERK5 and ERK5/β-ACTIN. One-way ANOVA with Sidak’s multiple comparisons test. (E) ERK5 staining for siCon , siALK1 and siALK1 + siPIEZO1 transfected HUVECs under static conditions and after 16 hr of flow exposure. ERK5 (green) DAPI (blue) (F) Quantification of nuclear ERK5 translocation for siCon , siALK1 and siALK1 + siPIEZO1 transfected HUVECs under static conditions and following 16 hr of flow exposure. *P<0.05, ***P<0.001, ns: nonsignificant, One-way ANOVA with Sidak’s multiple comparisons test. (G) p62 immunostaining in siCon , siALK1 and siALK1 + siPIEZO1 transfected HUVECs under static conditions and following 16 hr of flow exposure. (H) Quantification of p62 expression in siCon , siALK1 and siALK1 + siPIEZO1 transfected HUVECs under static conditions and after 16 hr of flow exposure. One-way ANOVA with Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ns: nonsignificant, Scale bars: 25 μm (E and G)

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Western Blot, Transfection, Control, Staining, Translocation Assay, Immunostaining, Expressing

Journal: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) KLF4 (white) and IB4 (green) staining of retinal flat mounts from P6 Alk1 f/f , Alk1 f/f Mfsd2a Cre ERT2 and Alk1 f/f Piezo1 f/f Mfsd2a Cre ERT2 pups. (B) KLF4 immunostaining in siCon , siALK1 and siALK1 + siPIEZO1 transfected HUVECs under static conditions and after 16 hr of flow exposure. KLF4 (magenta), DAPI (blue) (C) Quantification of nuclear KLF4 in HUVECs transfected with siCon, si ALK1 , or si ALK1 +si PIEZO1 under static conditions and following 16 hr of flow exposure. One-way ANOVA with Sidak’s multiple comparisons test. (D) Western blot analysis of HUVECs transfected with control, ALK1 , PIEZO1 , and ALK1 + PIEZO1 siRNAs, followed by 16 hr of flow exposure. (E and F) Quantification of KLF4/β-ACTIN and P-AKT/AKT. One-way ANOVA with Sidak’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001, ns: nonsignificant, Scale bars: 250 μm (A) and 25 μm (B)

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Staining, Immunostaining, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) Area of retinal hypoxia 90 min after pimonidazole injection in Alk1 f/f , Alk1 f/f Cdh5 Cre ERT2 and Alk1 f/f Piezo1 f/f Cdh5 Cre ERT2 P6 pups. Hypoxyprobe (green), IB4 (magenta) (B) Immunostaining of HIF1A (white) and IB4 (green) in retinal flat mounts from P6 Alk1 f/f , Alk1 f/f Mfsd2a Cre ERT2 and Alk1 f/f Piezo1 f/f Mfsd2a Cre ERT2 mice. Scale bars: 500 μm (A and B),

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Injection, Immunostaining

Journal: bioRxiv

Article Title: PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations

doi: 10.1101/2024.11.27.625696

Figure Lengend Snippet: (A) Labeling for EdU (green), ERG (white), and CD31 (magenta) in the vascular plexus of retinas from P6 Alk1 f/f and Alk1 f/f Mfsd2a Cre ERT2 and Alk1 f/f Piezo1 f/f Mfsd2a Cre ERT2 mice. (B) Quantification of the number of ERG and EdU double positive nuclei per vascular area. Mann–Whitney U test. (C) Labeling for EdU (green) and Hoechst33342 (blue) in HUVECs transfected with control, ALK1 , PIEZO1 , and ALK1 + PIEZO1 siRNAs. (D) Quantification of the number of EdU and Hoechst33342 double positive nuclei (n = 5). Two-way ANOVA with Tukey’s multiple comparisons test. Error bars: SEM. **P-value < 0.01, ***P<0.001, ns: nonsignificant, Scale bars: 500 μm (A), 25 μm (C)

Article Snippet: Depletion of ALK1 or PIEZO1 was achieved by transfecting 20 pmol of small interfering RNA (siRNA) against ALK1 (Qiagen, mixture of 2 siRNAs: S102659972 and S102758392) or PIEZO1 (Invitrogen, mixture of 2 siRNAs: S18893 and S18892) using Lipofectamine RNAiMax (Invitrogen).

Techniques: Labeling, MANN-WHITNEY, Transfection, Control