edhu 1  (Novus Biologicals)

Journal: Redox Biology

Article Title: Oxidation of hemoglobin to ferryl hemoglobin contributes to remodeling of the artery wall in abdominal aortic aneurysm

doi: 10.1016/j.redox.2025.103908

Figure Lengend Snippet: Circulating ferryl hemoglobin is detected in the blood of patients diagnosed with ruptured abdominal aortic aneurysms, and accumulation of ferryl hemoglobin-positive neutrophils and macrophages in human hemorrhaged aortic aneurysm. ( A ) We developed an enzyme-linked immunosorbent assay utilizing monoclonal antibodies against human ferryl hemoglobin (ferrylHb) to measure the concentration of ferrylHb in the serum of patients who were diagnosed with ruptured abdominal aortic aneurysm (AAA) and underwent open vascular surgery as well as in healthy volunteers. Results are shown as mean values. Statistical significance was determined using an unpaired t -test: ∗∗P < 0.01 ( n = 26/healthy donors; n = 40/AAA patients). ( B ) Macroscopic views of a hemorrhaged abdominal aorta with an AAA and a healthy aorta harvested from deceased organ donors are shown. ( C ) Hematoxylin-eosin (H&E), ferrylHb immunohistochemical (IHC), myeloperoxidase (MPO), naphthol AS-D chloroacetate esterase (NASD), and CD163 of abdominal aorta tissues are shown ( n = 5). Quantitative analysis of ferrylHb, MPO, NASD, and CD163 staining of tissue sections was performed using ImageJ software ( n = 5). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 (unpaired t -test). ND, not detectable. ( D ) Images from the hemorrhagic AAA sections were stained with Hoechst 33,258 to visualize DNA (blue), an anti-ferrylHb antibody coupled with Alexa Fluor 488 to identify ferrylHb (green), and either anti-MPO or anti-carboxypeptidase M (CPM) antibodies conjugated with Alexa Fluor 647 to detect MPO or CPM, respectively (red). ( E and F ) Images from the healthy aorta sections and the hemorrhagic AAA sections were stained with Hoechst 33,258 to visualize DNA (blue), an anti-ferrylHb antibody coupled with Alexa Fluor 488 to identify ferrylHb (green), an anti-CD163 antibody coupled with Alexa Fluor 647 to identify CD163 (red), and either anti-MPO or anti-CD68 antibodies conjugated with Alexa Fluor 568 to detect MPO or CD68, respectively (yellow). All images were captured using a Leica TCS SP8 gated STED-CW nanoscopic system and subsequently deconvolved with Huygens Professional software to enhance image clarity and resolution. The images presented are representative of the findings from five separate samples ( n = 5). Scale bars shown in the images represent 10 μm, 20 μm or 50 μm. Super-resolution images (2D techniques) confirmed the co-localization of ferrylHb and CD163 receptor. Representative image, colocalization rate is the average value ± SD ( n = 4).

Article Snippet: The scavenger receptor CD163 or MPO were stained using a rabbit anti-CD163 primary antibody (Novus Biologicals, NB110-40686) or rabbit anti-MPO primary antibody (Thermo Scientific, RB373A), followed by an Alexa Fluor 488 F (ab’)2 fragment of goat anti-rabbit IgG (H + L) secondary antibody (Cat. No. A11070, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Bioprocessing, Concentration Assay, Immunohistochemical staining, Staining, Software

Journal: Redox Biology

Article Title: Oxidation of hemoglobin to ferryl hemoglobin contributes to remodeling of the artery wall in abdominal aortic aneurysm

doi: 10.1016/j.redox.2025.103908

Figure Lengend Snippet: Hemoglobin uptake by neutrophils and macrophages in hemorrhaged abdominal aortic aneurysms in mice. ( A ) Mice were infused with AngII for 4 weeks and abdominal aortas were harvested for histological analysis. H&E and Verhoeff's elastin staining (EVG) of mouse AAA sections are shown. Cell number expressed as relative to the control ( n = 7–8), ∗∗P < 0.01 (unpaired t -test). Quantitative analysis of elastin degradation grade ( n = 5–7). ( B ) Hb IHC staining of mouse AAA sections. Red dashed circles indicate intracellular Hb. High-magnification insets on the right highlight neutrophils exhibiting multilobed nuclei with intracellular cytoplasmic Hb staining. Quantitative analysis of Hb staining of tissue sections was performed using ImageJ software ( n = 5). ∗∗P < 0.01 (unpaired t -test). ND, not detectable. ( C ) H&E, EVG, Masson's trichrome, and Hb staining in advanced aortic aneurysm tissues. Red dashed circles inset on the right highlight intracellular Hb staining and round large single nucleus characteristics of macrophages. Lower panels provide higher magnification images with the arrow indicating neutrophil extracellular traps (NETs). ( D ) Mouse AAA (AngII + HFD) sections were stained with Hoechst 33,258 to visualize DNA (blue) and anti-MPO antibody conjugated with Alexa Fluor 647 to detect MPO (red). Images were captured using a Leica TCS SP8 gated STED-CW nanoscopic system and subsequently deconvolved with Huygens Professional software to enhance image clarity and resolution. Scale bars shown in the images represent 20 μm. The arrow indicates NETs. ( E ) FerrylHb and CD163-positive cells were detected by immunofluorescence staining in AAA (HFD + AngII) and in healthy aortas (STD + Saline) of mice. ( F ) Human neutrophils (NEUs) were isolated from the blood of healthy donors and subsequently treated with ferrylHb with or without anti-CD163 pretreatment for 16 h. The cells were then stained with Hoechst 33,258 for DNA (blue), an anti-ferrylHb antibody with Alexa Fluor 488 secondary antibody for ferrylHb (green), and CD163 antibody for the scavenger receptor (red). Images were acquired using a Leica TCS SP8 gated STED-CW nanoscopy and deconvolved using Huygens Professional software. Scale bars represent 5 μm. Pixel intensity for red color and green color were quantified using ImageJ software ( n = 5). ∗∗P < 0.01, ∗∗∗P < 0.001 (unpaired t -test).

Article Snippet: The scavenger receptor CD163 or MPO were stained using a rabbit anti-CD163 primary antibody (Novus Biologicals, NB110-40686) or rabbit anti-MPO primary antibody (Thermo Scientific, RB373A), followed by an Alexa Fluor 488 F (ab’)2 fragment of goat anti-rabbit IgG (H + L) secondary antibody (Cat. No. A11070, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Staining, Control, Immunohistochemistry, Software, Immunofluorescence, Saline, Isolation

Journal: Redox Biology

Article Title: Oxidation of hemoglobin to ferryl hemoglobin contributes to remodeling of the artery wall in abdominal aortic aneurysm

doi: 10.1016/j.redox.2025.103908

Figure Lengend Snippet: Ferryl hemoglobin - induced NETosis occur via PAD4. ( A ) Human neutrophils (NEUs) collected from healthy donors were stimulated under the following conditions: 10 μg/ml 12-myristate 13-acetate (PMA), 10 μg/ml PMA combined with 10 μmol/l Hb, 10 μmol/l Hb alone, or 10 μmol/l ferrylHb alone. NEUs were cultured on coverslips. Cells were stained with Hoechst 33,258 to visualize DNA (blue), an anti-MPO antibody conjugated with Alexa Fluor 647 to detect MPO (red), and an anti-ferrylHb antibody conjugated with Alexa Fluor 488 to identify ferrylHb (green). Imaging was performed using Leica TCS SP8 gated STED-CW nanoscopy, and images were subsequently deconvolved using Huygens Professional software. ( B ) The concentrations of various redox states of Hb were determined by analyzing the visible spectra of the samples. The presence of different Hb redox states was calculated as a percentage. ( C ) Hb was analyzed using an anti-Hb antibody. The Western blots show the presence of Hb and ferrylHb in NEUs. A quantitative analysis of Western blots was performed ( n = 3); ∗P < 0.05 (unpaired t -test). ( D ) Western blot analysis was performed to assess CD163 and extracellular MPO levels in supernatants. NEUs treated with peptidylarginine deiminase 4 (PAD4) inhibitor GSK484. For each lane, 37.5 μl of supernatant was loaded. Quantification of CD163 and MPO expression is presented as mean ± SEM ( n = 3 per group). Statistical significance was determined by paired t -test, with ∗P < 0.05 indicating significance. ( E ) NEU-elastase release was quantified using an ELISA. The absorbance was measured at 450 nm using a microplate reader. The concentrations of NEU-elastase were expressed as ng/ml. Statistical significance was determined using an unpaired t -test: ∗∗P < 0.01 ( n = 3/group). ( F ) Cells were stained with Hoechst 33,258 to visualize DNA (blue), an anti-CD163 antibody conjugated with Alexa Fluor 647 to detect CD163 (red), and an anti-ferrylHb antibody conjugated with Alexa Fluor 488 to identify ferrylHb (green). Imaging was performed using Leica TCS SP8 gated STED-CW nanoscopy, and images were subsequently deconvolved using Huygens Professional software. Scale bars shown in the images represent 5 μm. ( G ) Super-resolution images (2D techniques) confirmed the co-localization of ferrylHb and CD163 receptor. Representative image, colocalization rate is the average value ± SD, n = 3. ( H ) Western blot analysis was performed to assess CD163 expression in macrophages (MAC). MAC were treated with GSK484; for each lane, 20 μg of proteins were loaded. CD163 expression is presented as mean ± SEM ( n = 3 per group). Statistical significance was evaluated using a paired t -test, with ∗P < 0.05 considered significant.

Article Snippet: The scavenger receptor CD163 or MPO were stained using a rabbit anti-CD163 primary antibody (Novus Biologicals, NB110-40686) or rabbit anti-MPO primary antibody (Thermo Scientific, RB373A), followed by an Alexa Fluor 488 F (ab’)2 fragment of goat anti-rabbit IgG (H + L) secondary antibody (Cat. No. A11070, Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Cell Culture, Staining, Imaging, Software, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay