Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Serum ACE2 activity is significantly correlated with SBP in stroke-alert patients and healthy young adults, but not AIS patients. Correlation graphs of ACE2 activity and SBP among stroke-alert patients (a) and healthy young adults (b) as compared to stroke patients (c). Young adult blood plasma samples in panel (b) were from a biorepository established by Wegman et al., which were obtained from research participants undergoing baseline measurements. (d) Correlation graph of ACE activity and mRS at discharge from hospital among AIS patients. ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; mRS: modified Rankin score; RFU: relative fluorescence unit; SBP: systolic blood pressure.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Clinical Proteomics, Modification, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Activity of ACE2 and ACE in serum is altered following stroke. For human serum, bar graphs are means ± SEM and represent enzyme activity levels of ACE2 (a) and ACE (c) from control, stroke-alert, or AIS patients at an average of 3.6 hours and again at 3 days after stroke. Individual differences and means ± SEM in ACE2 (b) and ACE (d) are shown. * P <0.05 versus control and † P <0.05 versus stroke-alert. ‡ P <0.05 versus AIS <6 hours. ACE: angiotensin converting enzyme; ACE2: angiotensin converting enzyme 2; AIS: acute ischemic stroke; RFU: relative fluorescence unit.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay, Control, Fluorescence

Journal: Journal of the Renin-Angiotensin-Aldosterone System: JRAAS

Article Title: Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke

doi: 10.1177/1470320316661060

Figure Lengend Snippet: Predictors of acute ischemic stroke by multiple linear regression analysis.

Article Snippet: Reaction Km and Vmax were determined using control samples and recombinant human ACE2 (R&D Systems, Inc., #933-ZN-010) as a positive control, and all samples were run in duplicate.

Techniques: Activity Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: Molecular models for HDP-2P (gold) complexes with ACE2 (red), and RBD (turquoise) obtained by protein docking. A Structures of HDP-2P complexed with ACE2 (left panel) and S-protein RBD (right panel) determined using Frodock. The central (control) panel shows re-docking of ACE2 to RBD, which results in a structure that resembles published X-ray structures; B A Rosetta-optimized binding interface between HDP-2P and RBD intersects the ACE2-RBD binding surface, which could prevent RBD from interacting with ACE2; C A different Rosetta-optimized binding interface between HDP-2P and ACE2 where HDP-2P docks away from the ACE2-RBD binding surface, implying no direct interplay between viral protein and PLA 2

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Control, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: PLA 2 s exhibit antiviral activity against viruses containing mutations and interfere with different stages of the SARS-CoV-2 replication cycle. a CPE inhibition assay. Vero E6 cells were infected with SARS-CoV-2 strain PMVL-19 or PMVL-20 at 100 TCID 50 in the presence of different concentrations of HDP-2 for 72 h. CPE inhibition was then measured by colorimetric assay with MTT. b Time-of-drug-addition assay. Vero E6 cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01, and virus yield in the infected cell supernatants was quantified by qRT-PCR 18 h after infection. c 293T/ACE2 were infected with different pseudo-SARS-CoV-2-GFP either in the presence of vehicle (PBS, Control) or HDP-2 (10 µg/ml). Representative fluorescent microscopy images of 293T/ACE2 cells infected with the pseudo-SARS-CoV-2 and treated with HDP-2. HDP-2 treatment led to a decrease in the entry of pseudoviruses, which was manifested in a decrease in the number of GFP-positive cells compared to the control. Scale bars, 100 µm. d Infectivity of pseudo-SARS-CoV-2 particles on 293T/ACE2 cells was quantified by measuring GFP fluorescence. Wuhan, B.1.1.7 and B.1.351 are the Wuhan reference strain, the lineage B.1.1.7 (United Kingdom) and the lineage B.1.351 (South Africa), respectively. Significant difference was determined using a Student’s t test: *p ˂0.05; **p ˂0.01; ***p ˂0.001. All results are shown as mean ± SD of n = 3 or 5 biologically independent samples. RFU relative fluorescence units

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Activity Assay, Inhibition, Infection, Colorimetric Assay, Virus, Quantitative RT-PCR, Control, Microscopy, Fluorescence

Journal: Cellular and Molecular Life Sciences

Article Title: Snake venom phospholipase A 2 s exhibit strong virucidal activity against SARS-CoV-2 and inhibit the viral spike glycoprotein interaction with ACE2

doi: 10.1007/s00018-021-03985-6

Figure Lengend Snippet: HDP-2P reduces the binding of an anti-ACE2 antibody and RBD of glycoprotein S to ACE2 receptor at 293T/ACE2 cells. a , b Inhibition of anti-ACE2 antibody binding to 293T/ACE2 cells by HDP-2P. 293T/ACE2 cells were incubated with (100 µg/ml) or without (control) HDP-2P for 30 min and stained using human phycoerythrin (PE) conjugated anti-mouse ACE2 antibody. MFI mean fluorescence intensity. c , d Cells were incubated with PBS (control) or HDP-2P. The RBD protein fused with human Fc was then added for 1 h. After washing, the binding of RBD was detected using a DyLight 650-conjugated secondary anti-human Fc antibody. e Representative RBD binding profile in control and HDP-2P-treated cells. The cell populations binding high (RBD hi ) and low (RBD lo ) amount of RBD are shown. The percentage of positive cells was determined using flow cytometry analysis. Significant difference was determined using a Student’s t test: *p ˂0.05, ** p ˂0.01. Results are mean ± SD and are representative of at least three independent determinations

Article Snippet: Recombinant human ACE2 (Cusabio, Houston, TX, USA) was immobilized on 3D carboxymethyl dextran hydrogel-coated sensor slides.

Techniques: Binding Assay, Inhibition, Incubation, Control, Staining, Fluorescence, Flow Cytometry

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: The impaired ANG1 signaling and angiogenesis in diabetic wound healing. ( A ) A comparison of the wound healing process between normal mice and diabetic mice. ( B ) Quantitative analysis showing the dynamic changes in wound areas during the healing process in normal and diabetic mice. n = 6. ( C ) Representative immunostaining images of CD34, a marker of vascular endothelial cells, in normal and diabetic wounds at 14 or 28 days post-wound creation. W, wound central areas and E, wound edges. ( D ) Quantitative analysis of CD34 + vessel-like structures in the wound edge area of diabetic wounds compared to normal wounds. n = 6. ( E ) The results of q-PCR revealing the differential expression levels of various molecules regulating angiogenesis on days 7, 14, 21, and 28 during normal and diabetic wound healing. Notably, the expression levels of ANG1 were significantly lower throughout the diabetic wound healing process compared to normal healing. All data are presented as mean ± SD. Scale bar: 100 μm in C and 20 μm in the magnified figure in C. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Comparison, Immunostaining, Marker, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: Construction and characterization of engineered MSCs stably expressing ANG1 (MSC ANG1 ). ( A ) Schematic illustration of the lentiviral plasmids carrying GFP, or both ANG1 and GFP. ( B ) Representative immunostaining images of GFP showing the presence of GFP in MSCs infected with GFP (MSC GFP ) and ANG1 (MSC ANG1 ) lentivirus. ( C ) Quantitative analysis of GFP intensity in MSC GFP and MSC ANG1 . n = 6. ( D ) Statistical analysis of GFP intensity in the diabetic wounds engrafted with MSC GFP and MSC ANG1 for 7 days. ( E ) Representative images of GFP immunostaining showing the survival of MSC GFP or MSC ANG1 engrafted onto the diabetic wounds for 7 days. The areas in the white boxes were enlarged and shown in the right panel. ( F ) WB showed the expression levels of ANG1 in MSC ANG1 and MSC GFP , as well as the levels in CM of MSC ANG1 and MSC GFP . Full-length blots are presented in Fig. ( G ) WB showed the expression levels of ANG1 in CM of MSC ANG1 cultured for 12 h, 24 h, 48 h, and 72 h, respectively. Full-length blots are presented in Fig. . Scale bar: 50 μm in B, 100 μm in left panel of D and 20 μm in right panel of D. All data are presented as mean ± SD

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Stable Transfection, Expressing, Immunostaining, Infection, Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted the survival, tubulogenesis and Akt activation in HUVECs. ( A ) CCK8 assay showing the total number of HUVECs treated with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. n = 6. ( B ) Flow cytometry of PI and Annexin-V in HUVECs treated with Ctrl, MSC GFP -CM or MSC ANG1 -CM for 48 h. ( C ) Statistical analysis of apoptosis (PI + /Annexin-V + ) in HUVECs treated with Ctrl, MSC GFP -CM or MSC ANG1 -CM for 48 h. n = 3. ( D ) Representative images of Calcein-AM and PI staining, illustrating HUVECs treated with Ctrl, MSC GFP -CM, or MSC ANG1 -CM for 48 h. ( E and F ) Statistical analysis of the mean fluorescence intensity (MFI) for Calcein-AM ( E ) and PI ( F ). n = 6. ( G ) Representative images showing the tube formation of HUVECs treated with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 4 h. ( H ) Quantitative analysis of the length of tubes in HUVECs. n = 9. ( I ) Representative WB images of p-Tie2, Tie2, p-Akt and Akt expression levels in HUVECs after treatment with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. β-Actin was the internal control. Full-length blots are presented in Fig. S3. ( J ) Semi-quantitative analysis of the phosphorylation of Tie2 (p-Tie2/Tie2) and Akt (p-Akt/Akt) in HUVECs. n = 9. Scale bar: 100 μm in C. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Activation Assay, CCK-8 Assay, Flow Cytometry, Staining, Fluorescence, Expressing, Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted VE-Cadherin expression, vascular integrity, and inhibited Src activation. ( A ) Schematic diagram illustrating the evaluation of endothelial barrier integrity. ( B ) Statistical analysis of the fluorescence intensity of FITC-Dextran that permeated through the monolayer of HUVECs treated with Ctrl, MSC GFP -CM, or MSC ANG1 -CM for 48 h. n = 3. ( C ) Representative immunostaining images of VE-Cadherin in HUVECs following treatment with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. ( D ) Quantitative analysis of VE-Cadherin fluorescence intensity in HUVECs post-treatment. n = 9. ( E ) Representative WB images depicting the expression levels of p-Src, Src, and VE-Cadherin in HUVECs after treatment with Ctrl, MSC GFP -CM, and MSC ANG1 -CM for 48 h. β-Actin served as the internal control. Full-length blots are presented in Fig. S3. ( F ) Semi-quantitative analysis of Src phosphorylation levels (p-Src/Src) and VE-Cadherin expression in HUVECs. n = 9. Scale bar: 50 μm in C. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Expressing, Activation Assay, Fluorescence, Immunostaining, Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted angiogenesis and activated Tie2/Akt pathway in diabetic wound healing. ( A ) Representative images of double staining for CD34 and αSMA in the diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. W, the center area of wounds, and E, the transitional area of wounds. The area in the dashed box was enlarged and shown in the insets. Arrows indicated the vessel-like structure positive for both CD34 and αSMA. ( B ) Quantitative analysis of the vessel-like structures positive for CD34, or CD34 and αSMA. n = 6. ( C ) Representative WB image revealing the expression levels of CD34 in diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. β-Actin was set as the internal control. Full-length blots are presented in Fig. S3. ( D ) Semi-quantitative analysis of CD34 expression in diabetic wounds. n = 9. ( E ) Representative WB images of p-Tie2, Tie2, p-Akt and Akt expression levels in diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. β-Actin was set as the internal control. Full-length blots are presented in Fig. S3. ( F ) Semi-quantitative analysis of the phosphorylation of Tie2 (p-Tie2/Tie2) and Akt (p-Akt/Akt) in the diabetic wound healing. n = 9. Scale bar: 100 μm in A, 20 μm in the insert panel of A. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Double Staining, Control, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 promoted the VE-Cadherin expression and inhibited Src activation in diabetic wound healing. ( A ) Representative images of double staining for CD34 and VE-Cadherin in the diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. W, the center area of wounds, and E, the transitional area of wounds. The area in the dashed box was enlarged and shown in the insets. Arrows indicated the vessel-like structures positive for both CD34 and VE-Cadherin. ( B ) Quantitative analysis of vessel-like structure positive for both CD34 and VE-Cadherin in diabetic wounds. n = 6. ( C ) Representative WB images showing the expression levels of p-Src, Src VE-Cadherin in diabetic wounds treated with control, MSC GFP , and MSC ANG1 for 14 days. β-Actin served as the internal reference. Full-length blots are presented in Fig. S3. ( D ) Semi-quantitative analysis of Src activation (p-Src/Src) and VE-Cadherin expression levels in the diabetic wounds. n = 9. Scale bar: 100 μm in A, 20 μm in the insert panel of A. All data are presented as mean ± SD. ⁎ p < 0.05, ⁎⁎ p < 0.01, ⁎⁎⁎ p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Expressing, Activation Assay, Double Staining, Control

Journal: Stem Cell Research & Therapy

Article Title: Activation of angiopoietin-1 signaling with engineering mesenchymal stem cells promoted efficient angiogenesis in diabetic wound healing

doi: 10.1186/s13287-025-04207-7

Figure Lengend Snippet: MSC ANG1 treatment promoted diabetic wound healing process. ( A ) Representative images displaying the dynamic changes in skin wounds of diabetic mice treated with control, MSC GFP , and MSC ANG1 . ( B ) MSC ANG1 treatment resulted in a substantial reduction in wound area in diabetic mice models at various time points compared to control and MSC GFP treatment. The percentage of wound healing area was calculated as 100% × (initial wound area - wound area at different time points) / initial wound area. n = 6. ( C ) Hematoxylin and Eosin (H&E) staining of diabetic wounds transplanted with control, MSC GFP , and MSC ANG1 for 14 and 28 days, respectively. ( D ) Representative immunostaining images of K14 in diabetic wounds transplanted with control, MSC GFP , and MSC ANG1 for 14 and 28 days, respectively. The epithelial gap (EG) signifies the area of the wound that remained uncovered by the K14-positive epidermis. ( F ) Representative images of double immunostaining for K14 and K17 in diabetic wounds transplanted with control, MSC GFP , and MSC ANG1 for 14 and 28 days, respectively. The area within the dashed box was magnified and presented in the right panel. Arrows indicate hair follicles positive for both K14 and K17. ( F ) Statistical analysis indicating that MSC ANG1 -treated diabetic wounds depicted a notably reduced EG compared to the control or MSC GFP -treated wounds. n = 6. ( G ) Statistical analysis illustrating that MSC ANG1 and MSC GFP -treated wounds exhibited significantly increased dermal thickness (THK) of the regenerated tissues compared to the control. THK was defined as the dermal thickness in the central area of the regenerated tissues. n = 6. ( H ) Statistical analysis demonstrating that MSC ANG1 -treated diabetic wounds displayed significantly more hair follicles positive for both K14 and K17 compared to the control and MSC GFP -treated wounds. n = 6. Scale bar: 1 mm in C and D, 100 μm in the left three panel of E and 20 μm in the right panel of E. All data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were washed with PBS, followed by incubation with D/F containing 50% of CM or Ctrl for 48 h. HUVECs cultured with D/F containing 200 ng/mL ANG1 (HY-P74412A, MCE, USA) were set as positive controls.

Techniques: Control, Staining, Immunostaining, Double Immunostaining

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 1 Schematic Illustration. a Preparation of rhACE2-loaded electrospun nanofiber patch by hyaluronan (HA) micro-sol electrospun and the mechanism of formation of core-shell structure. b Myocardial infarction mice model established by precise ligation of the left anterior descending (LAD) coronary artery along with the illustration of rhACE2 patch implantation. c In situ rhACE2 patch niche degrading angiotensin II (AngII) into a cardioprotective heptapeptide, Ang1–7, which counter the AngII/AT1R mediated effects by inhibiting cardiac fibrosis and cardiomyocyte apoptosis. PLLA poly(L-lactic acid), rhACE2 recombinant human angiotensin-converting enzyme 2, AT1R angiotensin II receptor type 1, PKC protein kinase C, MAPK mitogen-activated protein kinase, ECM extracellular matrix.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: Ligation, In Situ, Recombinant

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 2 Characterization of the rhACE2 patch. The general view, SEM and TEM micrographs demonstrating the core-shell structure (a), dynamic light scattering (b), water contact angle (c) and stress-strain curve (d) from PLLA-HA/ACE2 electrospun nanofibers. Scale bars represent 1 cm in general view, 20 μm in SEM and 1 μm in TEM.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques:

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 3 Biocompatibility tests in vitro. a Representative live/dead fluorescence stained by Calcein AM (green) and Propidium Iodide (red) at day 3. Scale bars represented 50 and 20 μm for zoomed pictures. b Live and dead cell count per HPF of control, PLLA, PLLA-HA/ACE2 groups. n = 4/group. c CCK-8 cell viability quantification results in different groups after 1, 2, or 3 days. n = 4/group. Data were represented as the mean ± SEM and analyzed for statistical significance using One-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: In Vitro, Staining, Cell Counting, Control, CCK-8 Assay, Comparison

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 5 rhACE2 patch sustained-release activity test. a In vitro releasing curve of PLLA-HA/ACE2 fibrous membranes. b Releasing buffer collected at specified time points during release study was added into culture media of NRCMs undergone 6 h hypoxia. Representative immunofluorescence image of TUNEL (red) and nuclear visualized by DAPI (blue). Scale bars represented 50 μm. c Statistical analysis of TUNEL- positive cell counts. n = 3/group. Data were represented as the mean ± SEM and analyzed for statistical significance using One-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference; **P < 0.01 compared to the control group.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: Activity Assay, In Vitro, TUNEL Assay, Comparison, Control

Journal: NPJ Regenerative medicine

Article Title: Myocardial fibrosis reversion via rhACE2-electrospun fibrous patch for ventricular remodeling prevention.

doi: 10.1038/s41536-021-00154-y

Figure Lengend Snippet: Fig. 6 rhACE2 patch preserve left ventricular function after acute myocardial infarction in vivo. a The animal research timeline design with images represented the acute MI model and successful implantation of rhACE2 patch. Echo, echocardiography. b Representative M-mode parasternal long axis view of left ventricle echocardiographic images of different groups at day 28 after LAD coronary artery ligation. Statistical analysis of left ventricular ejection fraction (c), shortening fraction (d), heart weight/body weight ratio (e), LV end-diastolic diameter (f), LV end- systolic diameter (g) and heart weight/tibial length (h) determined by echocardiography obtained from PLLA, intramyocardial injection (IM) and PLLA-HA/ACE2 treatment group at day 7, day 14 and day 28 after operation. n = 5/group. Data were represented as the mean ± SEM and analyzed for statistical significance using two-way ANOVA followed by Tukey’s multiple comparison test; NS no significant difference, *P < 0.05, **P < 0.01.

Article Snippet: Human ACE2 (18-652) recombinant protein (rhACE2, #85054) was purchased from Cell Signaling Technology (USA). rhACE2 contains necessary enzymatic activity associated protein structure and its effectiveness in mice and rats had been previously tested7, so it was selected for both our cell and animal studies.

Techniques: In Vivo, Ligation, Injection, Comparison

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Antibody Table

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Sequencing, Affinity Purification

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Shedding of ACE2 in insulinoma cells. The 832/13 cells were cotransfected with 0.5 μg ACE2 expression plasmids per well and the indicated amounts of the ADAM17 expression plasmid pAd17 or pcDNA3.1(−) in two experiments, with each treatment given to duplicate wells. A–C, Western blots of cellular ACE2, ACE2 released into the cell culture medium, and cellular ADAM17, respectively. D–F, Densitometric analyses of band intensities observed in the Western blots for cellular ACE2, shed ACE2, and cellular ADAM17, respectively. Bands were normalized relative to the average intensity of bands of interest on each blot, which was set at a value of 100. *,***, P < .05, P < .001 vs 0 μg pAd17; ###, P < .001 vs 0.5 μg pAd17 for post hoc contrasts of means after an ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Expressing, Plasmid Preparation, Western Blot, Cell Culture

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Enzymatic activity of shed ACE2. A, Maintenance medium for 832/13 cells and select culture broths from the experiments outlined in were tested for hydrolytic activity against the ACE2 substrate Mca-APK(Dnp) in the absence and presence of the ACE2-specific inhibitor DX600. B, The ACE2 activities of 832/13 maintenance medium stored at 4°C, after an overnight incubation at 37°C, and after an overnight incubation with untransfected 832/13 cells at 37°C, were compared by measuring six aliquots of each. C, The ACE2 activities of the cell culture media from the experiments outlined in were determined. **,***, P < .01, P < .001 vs 0 μg pAd17; #, ###, P < .05, P < .001 vs 0.5 μg pAd17 for post hoc contrasts of means after an ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay, Incubation, Cell Culture

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Overexpressed ADAM17 affects ACE2 levels. A, ACE2 and ADAM17 mRNA concentrations were determined from four experiments in which 832/13 cells were transfected with 0.5 μg/well mACE2/pcDNA3.1 and cotransfected with 2 μg/well of pAd17, pAd17E406A (an expression plasmid for inactive ADAM17), or pcDNA3.1(−). B–F, In six experiments, 832/13 cells were transfected with 4, 20, 100, or 500 ng mACE2/pcDNA3.1 per well and cotransfected with 2 μg/well of pAd17, pAd17E406A, or pcDNA3.1(−). We measured the activity (SensoLyte activity) with a SensoLyte ADAM17 activity kit (B), the activity by a more specific ADAM17 assay (C), the shed ACE2 activity (D), and the cellular ACE2 activity (E) compared with the average ACE2 activity level from five wild-type mice. We calculated the ratio of total shed ACE2 activity to total cellular ACE2 (F). G, The total cellular ACE2 and ADAM17 activities were determined at the beginning and end of a 24-hour incubation period for 832/13 cells that were cotransfected four times with 25 ng mACE2/pcDNA3.1 and 2 μg pAd17 per well. H, 832/13 cells were individually transfected with 25 ng/well mACE2/pcDNA3.1, 2 μg/well pAd17, and 2 μg/well pAd17E406A. The next day, cells were trypsinized and mixed for coculture of ACE2-expressing cells and cells overexpressing either active or inactive ADAM17. We measured ADAM17 activity, cellular ACE2 activity, and shed ACE2 activity. I, The control strengths of the amount of the ACE2 expression plasmid mACE2/pcDNA3.1 on cellular and shed ACE2 are determined as the slopes of the indicated regression lines for the natural logarithms of ACE2 activities plotted against the natural logarithms of the amount of mACE2/pcDNA3.1. Data were analyzed by an ANOVA (A–F) and t tests (H). Due to the large range in SDs for ACE2 activity measurements occurring with the different levels of the ACE2 expression plasmids, separate ANOVAs for the ACE2 parameters were conducted for each level of mACE2/pcDNA3.1(−) (D–F). *, **, ***, P < .05, P < .01, P < .001 vs. pAd17E406A.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Transfection, Expressing, Plasmid Preparation, Activity Assay, Incubation

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Effect of ADAM17-mediated ACE2 shedding on ACE2 protein stability. The 832/13 cells that were cotransfected in four experiments with 100 μg/well mACE2/pcDNA3.1 and 2 μg/well of either pAd17 or pAd17E406A were treated with cycloheximide. The total ACE2 activity (A) and ADAM17 activity (B) were measured for up to 21 hours. Exponential decay curves were fitted to the data between the 0- and 8-hour time points. The ratio of total shed ACE2 to total cellular ACE2 activity was also determined (C). *, ***, P < .05, P < .001 vs pAd17E406A for post hoc contrasts after an ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Determination of the control strength of ADAM17 on ACE2 activity. 832/13 cells were cotransfected with 25 ng/well mACE2/pcDNA3.1 and 2 μg/well of a mix of pAd17 and pAd17E406A. The pAd17 content in the mix were 0, 0.4, 0.8, 1.2, 1.6, and 2 μg/well in four experiments and 0, 0.05, 0.1, 0.2, 0,4, and 2 μg/well in eight experiments. ADAM17 activities (A) and ACE2 activities (B) were measured. C, For estimation of transfection efficiencies, green fluorescent 832/13 cells transfected with 2 μg pEGFP-C3 and 25 ng mACE/pcDNA3.1 were determined as in the upper right panel compared with control cells in the upper left panel. To estimate cotransfection efficiency, cells cotransfected with a plasmid encoding the red fluorescent tdTomato and pAd17 (lower left panel) were compared to cells cotransfected with plasmids for eGFP and tdTomato expression (lower right panel). For the different amounts of pAd17 used in the transfection experiments, shed ACE2 (D) and cellular ACE2 (E) were plotted against the estimated ADAM17 activity of the transfected cells, ie, corrected for the transfection efficiency. Curves describing saturation kinetics were fitted to the data. F, Based on the model fits, the control strengths were calculated and plotted as a function of the ADAM17 activity of transfected cells.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay, Transfection, Cotransfection, Plasmid Preparation, Expressing

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: ACE2 and ADAM17 in mouse pancreatic islet cells. A, Dispersed islet cells were assessed for red tdTomato fluorescence. The lower panel shows a cell subpopulation with high red fluorescence that is sorted from the non-red cells. B, Quantitative RT-PCR was performed to measure ACE2, ADAM17, insulin 2, glucagon, and somatostatin mRNA for sorted red and non-red islet cells from four female Tom +/− , Ins2-Cre +/− mice. C, ACE2 activity was determined for sorted red and non-red islet cells from two female and one male Tom +/− , Ins2-Cre +/− mice. *, **, P < .05, P < .01 vs non-red cells as determined by paired t tests.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Fluorescence, Quantitative RT-PCR, Activity Assay

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Expression of ACE2 and ADAM17 in diabetes. RNA from pancreatic islets was isolated from db/db and db/m mice. There were four mice in each group, except for only three db/db mice at 12 weeks of age. The concentrations of ACE2 mRNA (A), ADAM17 mRNA (B), and insulin 2 mRNA (C) were determined. Protein extracts from pancreatic islets were isolated from db/db and db/m mice with four mice in each group. ACE2 activities (D) and ADAM17 activities (E) were measured. F, The hydrolytic activity against the ADAM17 substrate in the absence of the ADAM17 inhibitor, DPC-333, was increased in db/db mice. *, ***, P < .05, P < .001 vs db/m mice in the same age group for post hoc contrasts of means after a two-way ANOVA.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Expressing, Isolation, Activity Assay

Journal: Endocrinology

Article Title: Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice

doi: 10.1210/en.2015-1556

Figure Lengend Snippet: Effects of inhibiting ADAM17 activity on ACE2 expression. 832/13 cells were transfected in four experiments with 100 ng/well mACE2/pcDNA3.1 and cotransfected with 2 μg/well of pAd17 or pAd17E406A. After transfection, cells were incubated in the absence or presence of 1 μM DPC-333. We determined the cellular ACE2 activity (A) and calculated the ratio of total shed ACE2 activity to total cellular ACE2 (B). *, **, ***, P < .05, P < .01, P < .001 vs no DPC-333; ###, P < .001 vs pAd17 in the absence of DPC-333 for post hoc contrasts of means after an ANOVA. Pancreatic islets were isolated from C57BL/6 mice aged 4–12 months. Pools of islets from four mice each were generated for a total of three pools from male mice and two pools from female mice. Aliquots of the islets were incubated for 24 hours in RPMI 1640 medium without serum. We determined the cellular ACE2 activity (C) and calculated the ratio of total shed ACE2 activity to total cellular ACE2 (D). There were no significant differences for post hoc contrasts of means in the absence and presence of DPC-333 after an ANOVA, but the main effect for sex was significant at P < .001 for cellular ACE2 activity.

Article Snippet: A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene.

Techniques: Activity Assay, Expressing, Transfection, Incubation, Isolation, Generated

Journal: Allergy

Article Title: Development and preclinical evaluation of virus‐like particle vaccine against COVID‐19 infection

doi: 10.1111/all.15091

Figure Lengend Snippet: Immunoprotective activity of the VLP vaccine in K18‐hACE2 transgenic mice. K18‐hACE2 transgenic mice ( n = 10/group) were subcutaneously immunized with 2 µg (low dose; LD) or 8 µg (high dose; HD) of the VLP vaccine on days 0 and 14. Two weeks after booster injection, (A) RBD‐specific IgG, IgG1, IgG2c antibody titers were determined by ELISA and (B) neutralizing antibody titers against the authentic Wuhan strain and the B.1.1.7. Alpha variant were determined. Groups were compared by one‐way ANOVA with Dunnett's multiple comparisons test. * P < .05, ** P < .01, *** P < .001, **** P < .0001. On day 21 after booster, mice were challenged intranasally on 3 consecutive days with 50 µl of 1 × 10 5 pfu/mouse of SARS‐CoV‐2 (Wuhan strain). Lungs were collected 7 days after last virus instillation. (C) Infectious virus loads in lung homogenates were assessed by qRT‐PCR against the nucleocapsid (NC1 and NC2). Bars represent the mean virus load ( n = 10/group) as 1/ct values. Comparisons were performed by unpaired Student's t test; * P < .05, ** P < .01, *** P < .001, **** P < .0001. (D) Histological micrographs showing healthy (first column), placebo (second column), low‐dose vaccine (third column), and high‐dose vaccine (fourth column) groups. (A–D) Hematoxylin‐eosin (H&E), areas marked green shows inflamed parts of the lungs; (E–L) H&E, 20×; M‐P, Gomori Trichrome (GT), 40×. a, alveoli; b, bronchiole; v, blood vessel; blue arrow, protein debris; red arrow, hyaline membrane. (E) Histomorphometric measurements. The descriptive statistics were presented as median and interquartile range in all graphs except for inflamed area percent (mean ± SD). Statistical significance ( P < .05): a, compared to healthy group; b, compared to placebo group; c, compared to low‐dose vaccine group, d, compared to high‐dose vaccine group. Nonparametric variables were compared between groups using Kruskal‐Wallis test. Pairwise comparisons were made with Dunn's test. Parametric variables were compared in multiple groups using one‐way analysis of variance. Pairwise comparisons were performed with the Tukey test

Article Snippet: Carboxyl modified latex beads (2 mg of 4% (w/v), Thermo Fisher Scientific) were coated with 5 μg recombinant hACE2 (ProSci) or anti–IL‐1β in PBS and blocked in 5% BSA in PBS.

Techniques: Activity Assay, Transgenic Assay, Injection, Enzyme-linked Immunosorbent Assay, Variant Assay, Virus, Quantitative RT-PCR, Membrane