anti hresistin  (R&D Systems)

Journal: PLOS One

Article Title: Human resistin is critical to activation of the NLRP3 inflammasome in macrophages

doi: 10.1371/journal.pone.0337682

Figure Lengend Snippet: ( A ) Schematic presentation of proposed pathway. ( B ) Human macrophages were cultured in 6-well plates under the conditions shown for 15 minutes before being washed and lysed for western blot analysis with antibodies to phospho-tyrosine (p-Y) and NLRP3 (image is representative of 6 repeats). ( C ) Human macrophages were cultured under the conditions shown for 12 hours. PBS was used as a control. Cells were washed, lysed, and assayed for their ability to cleave a fluorescent caspase-1 substrate, YVAD-AFC. Values were normalized to PBS controls. All conditions were run in duplicate wells, and two independent experiments were performed. Error bars represent the mean ± SD (n = 4). **** p < 0.001 versus PBS. ( D ) Hypoxia upregulates BTK and NLRP3 colocalization in C57BL/6 WT mice but not in RELMα KO mice. Immunofluorescence images of NLRP3 and BTK in lung tissues of mice kept under normoxic or hypoxic conditions for 4 days. Lung sections were stained with anti-NLRP3 (red) and BTK (green). The arrowheads point to cells positively stained for BTK and NLRP3 (yellow). The upper images are shown at higher magnification (400×); the lower panels display separate channels. Representative photograph of n = 6 mice per group. ( E ) Human resistin colocalized with BTK and NLRP3 in patients with PH. Immunofluorescence images of lung tissue slices from PH patients. Sections were stained with anti-hResistin (red) and co-stained with anti-BTK (green) and anti-NLRP3 (cyan) antibodies. The arrowheads point to cells positively stained for hResistin, BTK, and NLRP3. Separate channels are displayed in the lower panels. Original magnification: 100 × , 200 × , and 400 × . 4DHx, 4-day hypoxic; ab, antibody; BTK, Bruton’s tyrosine kinase; Con, control; Hres, human resistin; Ib, ibrutinib; KO, knockout; NLRP3, nucleotide-binding domain–like receptor protein 3; Nx, normoxic; PBS, phosphate-buffered saline; PH, pulmonary hypertension; RELMα, resistin-like molecule alpha; WT, wild-type. Created in BioRender. Lam, W. (2026) https://BioRender.com/1zhhut0 .

Article Snippet: After deparaffinization of tissue, rehydration, and antigen retrieval, sections were treated with anti-NLRP3 (Abcam, ab214185) and anti-Mac2 (Cedarlane, CL8942LE), anti-MPO (R&D Systems, AF3667), anti-CD79b (Abcam, ab134147), anti-BTK (Sigma-Aldrich, SAB4502936), anti-hResistin (R&D Systems, AF1359), or anti-RELMα (R&D Systems, MAB1523) antibodies overnight at 4°C and then with Alexa Fluor 488-donkey anti-mouse IgG (Jackson ImmunoResearch, 715-545-150) and Cy3-donkey anti-rabbit IgG (Jackson ImmunoResearch, 711-166-152) for double fluorescence staining.

Techniques: Cell Culture, Western Blot, Control, Immunofluorescence, Staining, Knock-Out, Binding Assay, Saline

Journal: Frontiers in Immunology

Article Title: Single-cell RNA sequencing unraveled immune-related expression heterogeneity and lymphoid cell development dysregulation in childhood asthma

doi: 10.3389/fimmu.2025.1606650

Figure Lengend Snippet: Cell communication pattern for asthma patients and healthy controls, serum resistin expression level and its PPI network. (A) The number of cell-cell interactions and interaction strength for four sample groups. (B) The annexin signaling pathway network of three sample groups. The annexin signaling pathway network in all cell types is shown for healthy controls, Asthma 1 and Asthma 3. The line thickness represents the signal strength, e.g. CD14 + monocytes have an annexin strength of 0.00006 in control, 0.0009 in asthma 1, and 0.0007 in asthma 3, respectively. The signal strength was estimated with the gene expression data of ligand-receptor pair. (C) The resistin signaling pathway network of three sample groups. The resistin signaling pathway network in all cell types is shown for healthy controls, Asthma 2 and Asthma 3. The line thickness represents the signal strength, e.g. CD14 + monocytes have a resistin strength of 0.0005 in control, 0.0025 in asthma 2, and 0.006 in asthma 3, respectively. The signal strength was estimated with the gene expression data of ligand-receptor pair. (D) The expression of RETN gene in two sample groups. The level of serum resistin between asthma patients and healthy controls (*** p < 0.001). (E) Protein–protein interaction network of RETN, its directly interacting genes and their neighbors. RETN is marked with red color and SQSTM1, HSPA5, and A2M are marked with light green color. RETN has a network degree of 8. SQSTM1, HSPA5, and A2M have a network degree of 109, 44, and 37, respectively. Network degree represents the number of neighbors (genes) connected to a hub, which is a core metric for measuring node importance and connection strength in complex network analysis.

Article Snippet: We followed a human resistin ELISA kit (Signalway Antibody, Maryland, USA, Catalog No: EK2351) protocol to analyze peripheral blood serums.

Techniques: Expressing, Control, Gene Expression

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: The ECS regulates resistin expression in CB1R-positive PBMCs. (A) CB1R and resistin expression in human arteries. Immunofluorescence for CB1R and resistin in human atheromatous artery. Green, CB1R; red, resistin; gray, CD68; sytox blue, nuclei. Scale bars, 10 μm. (B and C) Resistin expression in human PBMCs. Human PBMCs were treated with 10 μM 2-AG, a ligand of the ECS, with (or without) 0.1, 1, and 10 μM SR141716, a CB1R antagonist. Cells were harvested, and real-time PCR was performed for resistin (* P < 0.01, vehicle versus 2-AG, 2-AG + V versus 2-AG + SR141716; n = 3; hereafter, n represents the number of biological replicates). n.s., not significant; V, vehicle; SR, SR141716. (D to F) CB1R-positive cell sorting and resistin expression in CB1R-positve cells. (D) FACS for CB1-positive cells and purities of the isolated cells confirmed by FACS analysis. FITC, fluorescein isothiocyanate. SSC-A, side scatter area; FSC-A, foward scatter area. Resistin expression in sorted CB1R-positive cells, as determined by real-time PCR (E) and Western blot assay (F). N indicates the negative control. (G and H) Resistin expressions following treatment with 10 μM 2-AG with or without 1 μM SR141716 and 10 μM AM251 (a CB1R antagonist) in CB1R-positive cells, as determined by real-time PCR. AM630, a CB2R antagonist (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4). 251, AM251; 630, AM630. (I and J) Western blot analysis of p38 phosphorylation in CB1R-positive cells treated with 10 μM 2-AG, 1 μM SR141716, and 10 μM AM251. (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4). pp38, phospho-p38. (K and L) ChIP assay for the resistin promoter showed that the increased expression of resistin induced by 2-AG was induced by the enhanced binding of Sp1 on the promoter region of resistin. The increased binding activity of Sp1 induced by 2-AG was reversed by treatment with 1 μM SR141716, 10 μM AM251, and 5 μM SB203580 (a p38 inhibitor) (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716, AM251 or SB203580; n = 4). SB, SB203580; IgG, immunoglobulin G. (M) Real-time PCR for resistin in CB1R-positive cells treated with 10 μM 2-AG, 1 μM SR141716, 10 μM AM251, 5 μM SB203580, and mithramycin. Moreover, mithramycin, a Sp1 inhibitor, reversed the increased expression of resistin induced by 2-AG treatment. (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716, AM251, SB203580, or mithramycin; n = 4). (N) Real-time PCR for resistin in CB1R-negative and -positive cells treated with 10 μM 2-AG, 1 μM SR141716, 10 μM AM251, and 5 μM SB203580. Resistin expression was affected only in CB1R-positive fraction (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716, AM251 or SB203580; n = 3).

Article Snippet: We used primary antibodies against CB1R (Abcam), human resistin, and CD68 (Santa Cruz Biotechnology), followed by secondary antibodies.

Techniques: Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, FACS, Isolation, Western Blot, Negative Control, Phospho-proteomics, Binding Assay, Activity Assay

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: The ECS regulates inflammation in vivo by the infiltration of resistin-secreting CB1R-positive cells. (A and B) Schematic figures of 2 in vivo models. (A) To generate humanized NOG mice, the NOG mice were irradiated, and 1 × 10 5 human CD34-positive cells were transplanted through tail vein injection. (B) Humanized resistin mice were generated after inserting human resistin gene into the monocyte of resistin knockout (KO) mice. (C) Hematoxylin and eosin staining of visceral adipose tissues. CLSs are indicated by arrows. (D) The FACS of PBMCs into several fractions according to the expression of CD14 and CD16. The CD14 ++ CD16 − fraction of cells, considered as classical subsets of human monocytes, was mostly CB1R-positive cells. (E) In the CD14 ++ CD16 − fraction of cells having CB1R, 2-AG treatment significantly increased resistin expression, which was reversed by CB1R antagonists, SR141716 or AM251 (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4). (F to J) Visceral adipose tissues of humanized NOG mice (F) and humanized resistin mice (K). Mice were fed with a normal diet and a high-fat diet with or without intraperitoneally injected SR141716 for 8 weeks. Green, CB1R; red, resistin; 4′,6-diamidino-2-phenylindole, nuclei. Scale bars, 10 μm. Real-time PCR (G, H, L, and M) and Western blot analysis of CB1R and resistin in visceral adipose tissues of humanized NOG mice and humanized resistin mice (I, J, N, and O) (* P < 0.05, ND versus HFD; ** P < 0.05, HFD versus HFD + SR; n = 4). ND, normal chow diet; HFD, high-fat diet. (P to R) Proinflammatory gene expressions by real-time PCR in visceral adipose tissues. TNF-α, IL-1β, and IL-6 in humanized NOG mice and humanized resistin mice, respectively (* P < 0.05, ND versus HFD; ** P < 0.05, HFD versus HFD + SR; n = 4).

Article Snippet: We used primary antibodies against CB1R (Abcam), human resistin, and CD68 (Santa Cruz Biotechnology), followed by secondary antibodies.

Techniques: In Vivo, Irradiation, Injection, Generated, Knock-Out, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: Migration of CB1R-positive cells induced by 2-AG and different 2-AG levels in adipose tissues. (A and B) 2-AG levels in visceral and subcutaneous adipose tissues of humanized NOG mice and humanized resistin mice measured by LC-MS/MS (* P < 0.05, ND versus HFD; ** P < 0.05, HFD versus HFD + SR; n = 4). (C to E) CB1R-positive cells and CB1R-negative cells were added to the transwell insets, and 2-AG was added at 1 and 10 μM concentrations to the lower compartment. Incubation was carried out for the indicated periods of time (* P < 0.05, V versus 2-AG 1 μM; ** P < 0.05, V versus 2-AG 10 μM; n = 4). (D) Representative figure of each group in the transwell migration assay. (E) Cells were pretreatment with vehicle, 1 μM SR141716, and 10 μM AM251; they were then added to the transwell inserts. The migration of the cells from the upper to lower compartment was measured (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4).

Article Snippet: We used primary antibodies against CB1R (Abcam), human resistin, and CD68 (Santa Cruz Biotechnology), followed by secondary antibodies.

Techniques: Migration, Liquid Chromatography with Mass Spectroscopy, Incubation, Transwell Migration Assay

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: Insulin resistance facilitated by resistin may be associated with mitochondrial dysfunction induced by resistin. (A) Electron microscopic image of resistin treated human myoblast and hepatocellular carcinoma cells (HepG2) treated with resistin (50 ng/ml) for 3 h. It shows mitochondrial swelling, decreased matrix density, and distorted mitochondrial cristae in both cell types. Arrows indicate mitochondria. Scale bar, 100 nm. (B to D) The extent of the infiltrated cells secreting resistin was measured in target tissues such as the skeletal muscles, liver, and the heart. The high-fat diet-fed group showed increased number of resistin-positive cells in each organ, which was reversed by treatment with SR141716. Arrowheads indicate the cells that are positive for CB1R and resistin. Scare bars for immunofluorescence staining, 10 μm (for low power field) and 2.5 μm (for high power field). (E) Electron microscopy demonstrated that a high-fat diet changed the morphology of mitochondria into the swollen form, as seen in in vitro experiments. This change was reversed by treatment with SR141716. (F and G) Double immunofluorescence staining of human tissues such as the heart and liver between non-DM (normal) versus obese-DM (DM, diabetes mellitus) subject. Obese-DM subject showed a strong coexpression of infiltrated CB1R + resistin + monocytes (white arrows). Scare bars for immunofluorescence staining, 25 μm (for low power field) and 10 μm (for high power field). (H) Expression levels of resistin were measured in tissues of humanized resistin mouse models. Real-time PCR resistin in the bone marrow, spleen, visceral and subcutaneous fat of humanized resistin mice shows that high-fat diet group increased resistin expression that was reversed by SR141716 treatment (* P < 0.05, ND versus HFD, HFD versus HFD + SR; n.s., not significant; n = 4).

Article Snippet: We used primary antibodies against CB1R (Abcam), human resistin, and CD68 (Santa Cruz Biotechnology), followed by secondary antibodies.

Techniques: Muscles, Immunofluorescence, Staining, Electron Microscopy, In Vitro, Double Immunofluorescence Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: Schematic figure of CB1R-positive cells producing resistin. (A) CB1R-positive cells that secrete resistin may serve as mediators that elucidate the discrepancy between in mice and in humans in terms of the role of resistin in inducing insulin resistance. Mouse resistin is produced from adipose tissues; in contrast, human resistin is secreted by monocytes and macrophages. Owing to pathophysiological differences between mice and humans, we utilized animal models, such as humanized NOG mice and humanized resistin mice. (B) In both mouse models, a high-fat diet, which mimics the status of human obesity, increased the level of 2-AG in the visceral adipose tissue. Increased level of endocannabinoid ligands recruits the CB1R-positive cells into the target tissues, such as the adipose tissue, liver, and skeletal muscle. Mobilized CB1R-positive cells secrete resistin, which induces inflammation and insulin resistance in those tissues. (C) Resistin expression in CB1R-positive cells is regulated through the p38–Sp1 pathway.

Article Snippet: We used primary antibodies against CB1R (Abcam), human resistin, and CD68 (Santa Cruz Biotechnology), followed by secondary antibodies.

Techniques: Produced, Expressing

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: The ECS regulates resistin expression in CB1R-positive PBMCs. (A) CB1R and resistin expression in human arteries. Immunofluorescence for CB1R and resistin in human atheromatous artery. Green, CB1R; red, resistin; gray, CD68; sytox blue, nuclei. Scale bars, 10 μm. (B and C) Resistin expression in human PBMCs. Human PBMCs were treated with 10 μM 2-AG, a ligand of the ECS, with (or without) 0.1, 1, and 10 μM SR141716, a CB1R antagonist. Cells were harvested, and real-time PCR was performed for resistin (* P < 0.01, vehicle versus 2-AG, 2-AG + V versus 2-AG + SR141716; n = 3; hereafter, n represents the number of biological replicates). n.s., not significant; V, vehicle; SR, SR141716. (D to F) CB1R-positive cell sorting and resistin expression in CB1R-positve cells. (D) FACS for CB1-positive cells and purities of the isolated cells confirmed by FACS analysis. FITC, fluorescein isothiocyanate. SSC-A, side scatter area; FSC-A, foward scatter area. Resistin expression in sorted CB1R-positive cells, as determined by real-time PCR (E) and Western blot assay (F). N indicates the negative control. (G and H) Resistin expressions following treatment with 10 μM 2-AG with or without 1 μM SR141716 and 10 μM AM251 (a CB1R antagonist) in CB1R-positive cells, as determined by real-time PCR. AM630, a CB2R antagonist (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4). 251, AM251; 630, AM630. (I and J) Western blot analysis of p38 phosphorylation in CB1R-positive cells treated with 10 μM 2-AG, 1 μM SR141716, and 10 μM AM251. (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4). pp38, phospho-p38. (K and L) ChIP assay for the resistin promoter showed that the increased expression of resistin induced by 2-AG was induced by the enhanced binding of Sp1 on the promoter region of resistin. The increased binding activity of Sp1 induced by 2-AG was reversed by treatment with 1 μM SR141716, 10 μM AM251, and 5 μM SB203580 (a p38 inhibitor) (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716, AM251 or SB203580; n = 4). SB, SB203580; IgG, immunoglobulin G. (M) Real-time PCR for resistin in CB1R-positive cells treated with 10 μM 2-AG, 1 μM SR141716, 10 μM AM251, 5 μM SB203580, and mithramycin. Moreover, mithramycin, a Sp1 inhibitor, reversed the increased expression of resistin induced by 2-AG treatment. (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716, AM251, SB203580, or mithramycin; n = 4). (N) Real-time PCR for resistin in CB1R-negative and -positive cells treated with 10 μM 2-AG, 1 μM SR141716, 10 μM AM251, and 5 μM SB203580. Resistin expression was affected only in CB1R-positive fraction (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716, AM251 or SB203580; n = 3).

Article Snippet: Primary antibodies against human resistin (Santa Cruz Biotechnology), phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK), JNK, phospho-extracellular signal-regulated kinase (ERK), ERK (Cell Signaling Technology), and β-actin (Sigma-Aldrich) were used.

Techniques: Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, FACS, Isolation, Western Blot, Negative Control, Phospho-proteomics, Binding Assay, Activity Assay

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: The ECS regulates inflammation in vivo by the infiltration of resistin-secreting CB1R-positive cells. (A and B) Schematic figures of 2 in vivo models. (A) To generate humanized NOG mice, the NOG mice were irradiated, and 1 × 10 5 human CD34-positive cells were transplanted through tail vein injection. (B) Humanized resistin mice were generated after inserting human resistin gene into the monocyte of resistin knockout (KO) mice. (C) Hematoxylin and eosin staining of visceral adipose tissues. CLSs are indicated by arrows. (D) The FACS of PBMCs into several fractions according to the expression of CD14 and CD16. The CD14 ++ CD16 − fraction of cells, considered as classical subsets of human monocytes, was mostly CB1R-positive cells. (E) In the CD14 ++ CD16 − fraction of cells having CB1R, 2-AG treatment significantly increased resistin expression, which was reversed by CB1R antagonists, SR141716 or AM251 (* P < 0.01, vehicle versus 2-AG; ** P < 0.01, 2-AG + V versus 2-AG + SR141716 or AM251; n = 4). (F to J) Visceral adipose tissues of humanized NOG mice (F) and humanized resistin mice (K). Mice were fed with a normal diet and a high-fat diet with or without intraperitoneally injected SR141716 for 8 weeks. Green, CB1R; red, resistin; 4′,6-diamidino-2-phenylindole, nuclei. Scale bars, 10 μm. Real-time PCR (G, H, L, and M) and Western blot analysis of CB1R and resistin in visceral adipose tissues of humanized NOG mice and humanized resistin mice (I, J, N, and O) (* P < 0.05, ND versus HFD; ** P < 0.05, HFD versus HFD + SR; n = 4). ND, normal chow diet; HFD, high-fat diet. (P to R) Proinflammatory gene expressions by real-time PCR in visceral adipose tissues. TNF-α, IL-1β, and IL-6 in humanized NOG mice and humanized resistin mice, respectively (* P < 0.05, ND versus HFD; ** P < 0.05, HFD versus HFD + SR; n = 4).

Article Snippet: Primary antibodies against human resistin (Santa Cruz Biotechnology), phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK), JNK, phospho-extracellular signal-regulated kinase (ERK), ERK (Cell Signaling Technology), and β-actin (Sigma-Aldrich) were used.

Techniques: In Vivo, Irradiation, Injection, Generated, Knock-Out, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Research

Article Title: Resistin Regulates Inflammation and Insulin Resistance in Humans via the Endocannabinoid System

doi: 10.34133/research.0326

Figure Lengend Snippet: Schematic figure of CB1R-positive cells producing resistin. (A) CB1R-positive cells that secrete resistin may serve as mediators that elucidate the discrepancy between in mice and in humans in terms of the role of resistin in inducing insulin resistance. Mouse resistin is produced from adipose tissues; in contrast, human resistin is secreted by monocytes and macrophages. Owing to pathophysiological differences between mice and humans, we utilized animal models, such as humanized NOG mice and humanized resistin mice. (B) In both mouse models, a high-fat diet, which mimics the status of human obesity, increased the level of 2-AG in the visceral adipose tissue. Increased level of endocannabinoid ligands recruits the CB1R-positive cells into the target tissues, such as the adipose tissue, liver, and skeletal muscle. Mobilized CB1R-positive cells secrete resistin, which induces inflammation and insulin resistance in those tissues. (C) Resistin expression in CB1R-positive cells is regulated through the p38–Sp1 pathway.

Article Snippet: Primary antibodies against human resistin (Santa Cruz Biotechnology), phospho-p38, p38, phospho-c-Jun N-terminal kinase (JNK), JNK, phospho-extracellular signal-regulated kinase (ERK), ERK (Cell Signaling Technology), and β-actin (Sigma-Aldrich) were used.

Techniques: Produced, Expressing

Journal: Journal of biomedical science

Article Title: Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

doi: 10.1186/s12929-023-00947-x

Figure Lengend Snippet: Fig. 1 Reversible loss of SpeB is associated with tissue pathology and inflammation. A Distribution of SpeB+ and SpeB− GAS clones directly isolated from NSTI patient tissue biopsies (n = 23). B Percentage of SpeB+ and SpeB− GAS clones after the passage in THY media (p1, passage 1; p2, passage 2). Representative analysis of 2006 GAS patient isolate is shown. C–G Correlation analysis of bacterial load (left panel; n = 81 biopsies) or percentage of SpeB− clones (right panel; n = 23 biopsies) with the presence of HMGB1 (C), IL-8 (D), infiltrating neutrophils (E), and resistin (F) in patient biopsies. Correlation was determined using Spearman test. Semiquantitative acquired computerized image analyses (ACIA) of immuno-histochemical staining were performed as described in the methods section. The cell area was defined by the hematoxylin counterstaining, and the results are presented as percent positively stained area × mean intensity of positive staining

Article Snippet: The following antibodies were used for immunohistochemistry: anti-human HMGB1 (clone EPR3507; Abcam), anti-human IL-8 (clone NAP-1; Invitrogen), anti-human resistin (clone 184,305; R&D systems), and anti-human neutrophilelastase (clone NP57; DAKO).

Techniques: Clone Assay, Isolation, Staining

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 1. Expression of Hresistin/RELMα during right ventricular dysfunction. A, Immunofluorescence images of RV tissue slices from monocrotaline (MCT)-treated rats and normal controls. Sections were stained with anti-RELMα (red), co-stained with anti-myosin (green), and counterstained with DAPI. Original magnification: ×200. The bottom panels show the outlined area from the 2-week post-MCT time point at higher magnification (×400). The arrowheads point to the myosin (green)-labeled cardiomyocytes positively stained for RELMα (red), whereas the arrows point to the myosin-negative RELMα-positive immune cells that infiltrated into the myocardial interstitium. Representative images are from 5 individual RV samples per group. B, Hresistin detection in human RV tissue (n=5 subjects per group). Confocal images show Hresistin (green signal) staining in the RV heart biopsy of non-PH control subjects and patients with scleroderma (SSc)-associated pulmonary arterial hypertension. Light micrograph of fluorescence images to show structure. Signals of light micrograph (showing tissue structure) and fluorescence images (of Hresistin and DAPI staining) are digitally merged and the boxed area is enlarged and displayed in the right panel. The arrowheads point to the Hresistin protein signals in cardiomyocytes whereas the arrows point to the Hresistin-positive staining in the infiltrating immune cell. C, Quantitative analysis of data in (A). Percentage of areas positive for RELMα in rat RV tissues was determined by the histogram tool with Adobe Photoshop software. Data are presented as mean±SEM (n=5 animals per group). *P<0.05 vs normal group. D, Quantitative real-time PCR analysis of RELMα genes in the right heart and left heart tissues of MCT-injected rats (n=5 animals per group; the dots in the graphs represent single individuals) with cardiac hypertrophy (2 weeks post-MCT) and failure (4 weeks post-MCT). E, Quantitative analysis of data in (B). The Hresistin-positive (+) cells were counted and expressed as numbers per high-power field (hpf). Data are presented as mean±SEM (n=5 subjects per group). *P<0.05. DAPI indicates 4’6-diamidino-2-phenyl-indole; Hresistin, human resistin; PCR, polymerase chain reaction; PH, pulmonary arterial hypertension; RELMα, resistin-like molecule-α; and RV, right ventricular.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Control, Fluorescence, Software, Real-time Polymerase Chain Reaction, Injection, Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 2. Generation of cardiac-specific Hresistin transgenic mice. A and B, Images present the nucleotide sequence for the Hresistin (hRETN) construct (A) and a schematic representation of the αMHC-hRETN transgene structure (B). C, Immunofluorescence images of heart tissue samples from hRETN cardiac- overexpressing humanized mice and their corresponding littermate controls. Sections were stained with anti-myosin (green), co-stained with anti-FLAG (red), and counterstained with DAPI to validate the expression of the FLAG-tagged hRETN protein. Separate channels are displayed in middle and right panels, and digitally merged in left panels. Original magnification: ×200. Boxed areas are shown at higher magnification (×400) in the lower panels. Images are representative of 3 individual heart samples. D, Genotyping by PCR analysis of genomic DNA. Amplification of a 363-bp product encoding the myc-RETN epitope region of the transgene indicates that the humanized animals carry the knock-in hRETN gene in hearts. A 535-bp product was specifically amplified from animals carrying the tTA transgene. The 494-bp 18 S housekeeping gene served as a control. DAPI indicates 4’6-diamidino-2-phenyl-indole; MHC, myosin heavy chain; PCR, polymerase chain reaction; and tTA, tetracycline trans-activator.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Transgenic Assay, Sequencing, Construct, Immunofluorescence, Staining, Expressing, DNA Amplification, Knock-In, Amplification, Control, Polymerase Chain Reaction

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 3. Cardiac dysfunction and remodeling in humanized mice that overexpress MHC-hRETN. A, Pooled data of force-frequency (left panel) and intracellular Ca2+ transient-frequency (right panel) relationships in trabecular muscles from RV of wild-type and cardiac-specific MHC-hRETN-overexpressing mice (in force-frequency test: n=6 and 5 for control and overexpressing mice, respectively; in Ca2+ transient-frequency test: n=4 animals per group; the 2 assays used samples from different animals). *P<0.05, **P<0.01. B and C, Changes in phosphorylation of PKA (B) and AMPK (C) in cardiac tissue from cardiac-specific MHC-hRETN-overexpressing mice and control littermates were determined by western blotting. Left panels show representative immunoblots. Right panels show quantitative analysis of expression. Data are shown as mean±SEM (n = 6 animals per group). *P<0.05 vs control littermates. P- indicates phosphorylated protein; t- indicates total protein. D, Wheat germ agglutinin (WGA) cell boundary staining in the Hresistin-overexpressing myocytes. Left panels: heart tissue samples show cell nuclei (blue) and cell boundary (green). Magnification: ×400. Right panels: quantification of cell surface area based on histologic analysis of cardiomyocytes. Data represent means±SEM (n=5 animals per group). *P<0.05 vs littermate control group. E, Masson’s trichrome staining of heart tissue samples from MHC-Hresistin (RETN) humanized and littermate control groups. Magnification: ×200. Representative images (left panels) and quantitative analysis (right panels) were presented. Pooled data for quantification of fibrosis from 5 randomly selected histological fields at a magnification of ×400 on each slide. Data are shown as mean±SEM (n=5 animals per group). *P<0.05 vs control group. AMPK indicates AMPK-activated protein kinase; DAPI, 4’6-diamidino-2-phenyl-indole; hRETN, human resistin; MHC, myosin heavy chain; and PKA, protein kinase A.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Muscles, Control, Phospho-proteomics, Western Blot, Expressing, Staining

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 4. Hresistin activates HMGB1 signaling in the RV during RV r dysfunction. A, Immunofluorescence images of hypertrophic RV (RV-H, 2 weeks post-MCT induction) and failing RV (RV-F, 4 weeks post-MCT induction) from MCT-treated rats. Sections were stained with anti-HMGB1 antibody (green), costained with anti-myosin (red), and counterstained with DAPI (blue). Representative photographs of 4 individual animals per group. Original magnification: ×200. Boxed region in the RV-H group is shown at higher magnification to the right (×400). Further magnification (×1000) of the 2 framed areas are shown in the upper and lower panels to illustrate the HMGB1-positive, myosin-negative cells that infiltrated the myocardial interstitium. B and C, Quantitative analysis of data in A. Percentage of area positive for HMGB1 signal (HMGB1+) in rat right heart determined with Adobe Photoshop software (B) and the number (No.) of HMGB1-positive cells counted (C) on 5 randomly chosen RV fields in each animal at 200-fold magnification. Data are presented as mean±SEM (n=4 animals per group). *P<0.05, **P<0.01 vs normal (non-MCT-treated) rats. D, Immunofluorescence images of RV tissue from wild-type (WT) mice on post-hypoxia day 4 and from MHC-hRETN humanized mice. Some hypoxic (Hx) mice received daily intraperitoneal injections of the HMGB1-specific inhibitor ethyl pyruvate (EP, 50 mg/kg) for 4 days. Representative photographs of 4 individual animals per group. Original magnification: ×200. In the MHC-hRETN group, the corresponding co-staining for HMGB1 (green) with myosin (red) is presented in the lower panel, and the boxed region in it is shown at higher magnification (×400) on the left. E and F, Quantitative analysis of data in D. Percentage of area positive for HMGB1 signal in mouse right hearts was determined (E), and HMGB1- positive cells (per observed field) were counted (F). Data are presented as mean±SEM (n=5 animals per group for the control [normal WT] group and n=4 animals per group for the other 3 groups [Hx, Hx+EP, and MHC-hRETN]; the dots in the graphs represent single individuals). *P<0.05, **P<0.01 (increase) vs normal WT mice; †P<0.05 (decrease) vs the hypoxia (Hx)-only group. DAPI indicates 4’6-diamidino-2- phenyl-indole; EP, ethyl pyruvate; HMGB1, high mobility group box 1; hRETN, human resistin; hx, hypoxia; MCT, monocrotaline; MHC, myosin heavy chain; RV-F, failing right ventricle; and RV-H, hypertrophic right ventricle.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Immunofluorescence, Staining, Software, Control

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 5. Hresistin/HMGB1 signaling axis upregulates Ki67 expression in RV. A, Sections of RV tissues during hypertrophy or failure (as described in above panel A) were stained with anti-Ki67 antibody (green) and counterstained with DAPI (blue). Representative photographs of 4 individual animals per group. Magnification: ×200. B, Quantitative analysis of data from A. Ki67-positive (Ki67+) cells were counted on 5 randomly chosen fields of RV sections in each animal at ×200 magnification. Data are presented as mean±SEM (n=5 animals per group for the normal control group and n=4 animals per group for the groups of RV-H and RV-F; the dots in the graphs represent single individuals). *P<0.05 vs normal (non-MCT-treated) rats. C, Immunofluorescence images of RV tissue from WT mice exposed to 4 days of hypoxia with or without the HMGB1 inhibitor EP and from cardiac-specific MHC-hRETN-overexpressing mice. The boxed region in the fourth panel is shown at higher magnification (×400) on the right with costaining for Ki67 (green) and myosin (red). Representative photographs of 5 individual animals per group. Original magnification: ×200. D, Quantitative analysis of data in C. The Ki67-positive cells were counted (per observed field). Data are presented as mean±SEM (n=5 animals per group for the control [normal WT] group and n = 4 animals per group for the other 3 groups [Hx, Hx+EP, and MHC-hRETN]). *P<0.05, **P<0.01. E, In the RV tissues of the MHC-hRETN-overexpressing mice, Ki67 (red, middle panels) was further costained with the markers (green, left panels) of macrophages (F4/80), neutrophils (MPO), or fibroblasts (Vimentin). Images were merged in the right panels. The boxed areas were further enlarged in the far right panels showing the double positive cells. Representative photographs of 4 individual animals per group. Original magnification: ×400. DAPI indicates 4’6-diamidino-2-phenyl-indole; EP, ethyl pyruvate; HMGB1, high mobility group box 1; h-RETN, human resistin; Hx, hypoxia; MCT, monocrotaline; MHC, myosin heavy chain; MPO, myeloperoxidase; RV-F, failing right ventricle; RV-H, hypertrophic right ventricle; and WT, wild type.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Expressing, Staining, Control, Immunofluorescence

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 6. Anti-Hresistin human antibody ameliorates RV dysfunction in rats with PH. The anti-Hresistin antibody (Ab) or the isotype-matched control IgG1 (Con IgG) at 4 mg/kg were administered intraperitoneally twice a week in the hypoxia-induced PH rats. A and B, Echocardiographic analysis of right ventricular (RV) wall thickness and pulmonary artery blood velocity in Ab-treated hypoxic rats. RV wall thickness external diameter (RV-WTED) was measured as the distance from the free wall to the interventricular septum (millimeter) in the parasternal long-axis view using M-mode (A). Data are expressed as a percentage of the value of normoxic control mice. The anti-Hresistin Ab treatment also lengthened pulmonary artery acceleration time (PAT). Results of pulsed wave Doppler measurement of PAT are shown in B. PAT values were normalized by pulmonary ejection time (PET). Data are expressed as means±SEM (n=6 animals per group). *P<0.05, **P<0.01 vs hypoxia (no Ab) group. Representative echocardiographic images are shown in the right panels. C, Immunoprecipitation analysis of the binding of rat RELMα to the human therapeutic Ab targeting Hresistin. The protein-Ab binding was detected by western blotting with the anti-Hresistin antibody from R&D (AF1359). Recombinant rat RELMα protein was loaded as the positive control. D and E, Analysis of RV hypertrophy and hemodynamics in the hypoxia (Hx)-induced rat PH model. We measured the RV systolic pressure (RVSP) (D) and Fulton index (ratio of RV weight/ LV+S weight) (E). Data are presented as means±SEM (normal no Ab: n=6, normal con Ab: n=6, hypoxia no Ab: n=5, hypoxia con Ab: n=6, hypoxia Ab: n=5; n refers to the number of animals per group). **P<0.01 vs Hx-treated group without Ab treatment. F, Results of treatment with the human antibody targeting Hresistin to improve the survival rate among rats with monocrotaline-induced PH. *P<0.05 by log rank test; n=6 animals in each group. LV+S indicates left ventricle plus septum; MCT, monocrotaline; PH, pulmonary arterial hypertension; and RELMα, resistin-like molecule-α.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Control, Immunoprecipitation, Binding Assay, Western Blot, Recombinant, Positive Control

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 7. Hresistin/HMGB1 signaling axis induction of cardiac dysfunction and hypertrophy. A, Representative immunoblots of the protein levels of RELMα, HMGB1, and the hypertrophy markers for atrial natriuretic peptide (ANP) and myosin heavy chain-β (β-MHC) in RV showing anti-Hresistin Ab inhibition of the hypoxia-induced expression of HMGB1 and RV hypertrophy markers in the RV of PH rats in vivo. B, Quantitative analysis of data in A. n=4 rats per group, one-way ANOVA with Tukey post hoc analysis for multiple group comparisons. C, Immunoblots showing anti-Hresistin Ab prevention of the Hresistin- induced expression of HMGB1 and RV hypertrophy markers in the neonatal rat cardiomyocytes (NRCMs) in vitro. The primary cultured NRVMs were pretreated with 3 μg/mL Con IgG or the anti-Hresistin Ab followed by transduction of MOI-300 GFP-tagged adeno- associated virus (AAV) expressing the Hresistin (hRETN). Empty AAV vector (null) served as the negative control. Representative immunoblots are shown (n=4 per group). D, Quantitative analysis of data in C. Data are expressed as means±SEM (n=4 per group). *P<0.05, **P<0.01, ***P<0.001. E through G, Representative and quantitative WB images and quantitative analysis of the HMGB1 inhibitor ethyl pyruvate (EP) attenuation of the Hresistin-induced cardiac hypertrophy in NRCMs in vitro. The primary NRVMs were pretreated with 5 μmol/L EP followed by MOI-300 AAV-hRETN transduction. Representative WB images (E) and quantitative analysis (F) of ANP and β-MHC protein are displayed (n=4 per group). Quantification of the cell surface area of NRCMs is shown in G (n=13 per group). *P<0.05, **P<0.01. Ab indicates antibody; GFP, green fluorescent protein; HMGB1, high mobility group box 1; Hresistin, human resistin; Hx, hypoxia; NRVM, neonatal rat cardiomyocyte; PH, pulmonary arterial hypertension; RELMα, resistin-like molecule-α; RV, right ventricle; and WB, western blot.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: Western Blot, Inhibition, Expressing, In Vivo, In Vitro, Cell Culture, Transduction, Virus, Plasmid Preparation, Negative Control

Journal: Journal of the American Heart Association

Article Title: Human Resistin Induces Cardiac Dysfunction in Pulmonary Hypertension

doi: 10.1161/jaha.122.027621

Figure Lengend Snippet: Figure 8. Schematic illustration of Hresistin-induced cardiac inflammation and dysfunction. During pulmonary arterial hypertension (PH) development, Hresistin activates the damage-associated molecular pattern (DAMP), signaling triggering of inflammation in the right ventricle (RV) and contributing to RV dysfunction pathogenesis. Targeting the Hresistin signaling cascade may constitute a novel therapeutic approach to RV dysfunction and other related cardiac diseases in humans. HMGB1 indicates high mobility group box 1; Hresistin, human resistin; LV, left ventricle; and RELMα, resistin-like molecule-α.

Article Snippet: Protein– antibody binding was detected by western blotting with anti- Hresistin antibody (AF1359, R&D Systems, Minneapolis, MN).

Techniques: