enpp1  (R&D Systems)

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: Impact of high glucose on extracellular pyrophosphate metabolism. Aortic smooth muscle cells were cultured for one month in media containing either low (1 g/L) or high (4.5 g/L) glucose. A Measurement of extracellular pyrophosphate levels. B Extracellular pyrophosphate-to-ATP ratio. C , D Analysis of the gene expression of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1, eNPP1, and TNAP, from isolated total RNA. (E) Immunoblot analysis of proteins associated with extracellular pyrophosphate metabolism. F , G Quantification of protein levels via ELISA, highlighting significant differences. The data are shown as the mean ± SEM, with data derived from 4 independent experiments, each containing 4 replicate plates. Statistical significance was determined via Student’s t test, with asterisks denoting significance levels: * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Expressing, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: High glucose levels impair the pyrophosphate-to-phosphate ratio. Aortic smooth muscle cells were incubated for one month in medium containing 1 g/L or 4.5 g/L glucose. A Autoradiograph displaying representative products from the hydrolysis of ATP (1 µmol/L ATP, 10 µCi/mL [γ 32 Pi]ATP) incubated with or without recombinant eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) or eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) enzymes. Enzymatic hydrolysis generated radiolabeled 32 PPi (32-pyrophosphate) and 32 Pi (32-phosphate), which, alongside unreacted [γ 32 Pi]ATP, were separated by thin-layer chromatography (TLC), as detailed in the section. B Representative time course of ATP hydrolysis showing the products released over time. C Synthesis of the pyrophosphate 32 PPi via hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) in the absence or presence of 100 µmol/L SBI245 (a specific TNAP inhibitor) or inorganic pyrophosphatase (PPase). D The pyrophosphate-to-phosphate ( 32 PPi/ 32 Pi) ratio was quantified following hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) under various conditions: in the absence of inhibitors (Control), in the presence of an ectonucleoside triphosphate diphosphohydrolase (eNTPD) inhibitor (INH, 200 µmol/L), or with the recombinant enzymes eNPP1 and eNTPD1 (100 ng/mL). Experiments were conducted in media containing either physiological (1 g/L) or elevated (4.5 g/L) glucose concentrations. D) Synthesis of 32 PPi by hydrolysis of [γ 32 Pi]ATP (10 µCi/mL and 1 µmol/L ATP). E Hydrolysis of 32 PPi (10 µCi/mL and 5 µmol/L PPi). The results are shown as the mean ± SEM (4 independent experiments with 4 plates per experiment). Student’s t test ( E, F ) or one-way ANOVA with Tukey’s post hoc test ( C, D ) was used for statistical analysis. Asterisks indicate a statistically significant difference compared with the control group: * P < 0.05; *** P < 0.001. ### Indicates a value of P < 0.001 compared with the control group (1 g/L)

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Autoradiography, Recombinant, Generated, Thin Layer Chromatography, Control

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: STZ-treated rats exhibit impaired extracellular pyrophosphate metabolism in the aortic wall. A A representative time course of ATP hydrolysis was conducted using a 1 µmol/L ATP solution containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The products of hydrolysis, 32 PPi (32-pyrophosphate), 32 Pi (32-phosphate), and [γ- 32 P]ATP-, were separated and quantified via thin layer chromatography, as outlined in the section. B The synthesis of pyrophosphate (PPi) was analyzed by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The reactions were carried out in the absence or presence of either a specific TNAP inhibitor (SBI-425) or inorganic pyrophosphatase (PPase). C The ratio of 32 PPi to 32 Pi generated by ATP hydrolysis was calculated to assess the efficiency and specificity of pyrophosphate synthesis. D The synthesis of 32 PPi was evaluated by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP. E The release of 32 Pi was measured following the hydrolysis of 5 µmol/L pyrophosphate, which contained 10 µCi/mL 32 PPi as a radiotracer. F Quantification of protein levels via ELISA. G , H Total RNA was isolated from rat aortas to evaluate the expression levels of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), and tissue-nonspecific alkaline phosphatase (TNAP) (panel G . Additionally, the expression of calcification-related proteins, such as matrix Gla protein (MGP) and osteopontin (OPN), was assessed (panel H). The data are shown as the mean ± SEM and represent data from 12–16 independent aortas. Statistical analyses were performed via Student’s t test. Asterisks indicate a significant difference with *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Thin Layer Chromatography, Generated, Enzyme-linked Immunosorbent Assay, Isolation, Expressing

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet: Avidity, potency, homology modeling, and off-target binding assays for hENPP1mAb (A) Dissociation constant of hENPP1mAb calculated with flow cytometry to determine concentration-dependent binding of hENPP1mAb to HEK cells overexpressing human ENPP1. Calculated K D value (measure of avidity) shown ( n = 3). (B) Potency curve of hENPP1mAb in inhibiting human ENPP1 catalytic activity in a concentration-dependent manner. Calculated IC50 shown ( n = 3). (C and D) Homology modeling and protein-protein docking interactions show the (C) docked model of hENPP1mAb and human ENPP1. The antibody chains (CDR of light and heavy chains) occlude the enzyme active site (dark gray surface) (human ENPP1 antigen in blue, Fab segment of hENPP1mAb in green, CDRs color coded as shown). (D) Magnified image demonstrating several CDRs of hENPP1mAb inserting into human ENPP1 catalytic domain. (E) Flow cytometry to determine binding of hENPP1mAb to other members of human ENPP family and to other phosphatases ( n = 5/group). (F–H) Retrogenix membrane array screening by immunoblotting to determine binding of hENPP1mAb to 6,101 human plasma membrane proteins and 396 human heterodimers expressed on HEK cells. (F) hENPP1mAb shows a significant specific interaction with human ENPP1 isoforms (red) (plasma membrane isoforms upper and middle, and tethered secreted form, lower). Note hENPP1mAb also shows binding to IGHG that may serve as IgG receptors. (G) Rituximab is used as a positive control to determine any non-specific binding of a mAb and demonstrates no binding to human ENPP1 and binding to IGHG proteins. (H) PBS is used as a negative control for the entire assay and demonstrates signal against the IGHG proteins as well. Immunoblotting demonstrates representative images of n = 3. Data are represented as mean ± SEM.

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: Binding Assay, Flow Cytometry, Concentration Assay, Activity Assay, Membrane, Western Blot, Positive Control, Negative Control

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet: Species reactivity of hENPP1mAb and development of humanized ENPP1 mice (A) Flow cytometry to determine binding of hENPP1mAb against mouse, rat, pig, monkey, and human ENPP1 overexpressed in HEK cell line. HEK cells expressing eGFP used as a negative control ( n = 3 for eGFP and monkey, n = 5 in mouse, rat, pig, and human). (B) Schematic representation of generation of the humanized ENPP1 mouse. Using CRISPR-Cas9, human ENPP1CDS with a PolyA signal at the 3′ end is inserted to replace the 1st exon of murine ENPP1 gene. (C) Agarose gel electrophoresis of RT-PCR products of heart tissue from humanized ENPP1 mice or wild-type C57BL/6J mice ( n = 3 animals/group). Discriminatory PCR primers are used to distinguish murine and human ENPP1 expression. (D) qPCR demonstrating ENPP1 gene expression in the injured region of the heart compared with uninjured region at 7 days after MI ( n = 3 animals/group). (E) qPCR on infarcted heart of humanized ENPP1 mouse at day 7 post MI demonstrating the absence of murine ENPP1 and expression of human ENPP1 in the infarcted region ( n = 3 animals/group). (F) Immunostaining for ENPP1 (green, arrowheads) and cardiac troponin I (red) in the injured regions at day 7 after MI. Magnified images demonstrate cells in the infarcted region of the inset expressing human ENPP1 (arrowheads). Note that ENPP1 expression is present in troponin-negative regions. Data are represented as mean ± SEM. ∗ p < 0.05, Statistical significance was determined using Student’s t test, 2 tailed.

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: Flow Cytometry, Binding Assay, Expressing, Negative Control, CRISPR, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Immunostaining

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet: hENPP1mAb attenuates post-infarct cardiac dysfunction in humanized ENPP1 animals (A) Strategy for hENPP1mAb administration in humanized ENPP1 animals subjected to MI. (B) Western blotting for ENPP1 in wild-type mice hearts at 3, 7, and 14 days following MI. (C) Quantitative densitometry of ENPP1 level ( n = 3). (D) Extracellular ATP hydrolytic activity in injured and uninjured hearts of animals treated with IgG or hENPP1mAb ( n = 4 animals/group). (E) B (top) and M-mode (below) echocardiogram demonstrating superior contractile function in hENPP1mAb-treated animals. Diastolic (green line) and systolic internal dimensions (yellow line) in hearts of hENPP1mAb/IgG-treated animals. (F) Ejection fraction, fractional shortening, and left ventricular (LV) chamber size in systole (LVIDs) and diastole (LVIDd) in IgG or hENPP1mAb-treated animals at 1, 2, and 4 weeks following MI ( n = 21/IgG and n = 19/hENPP1mAb). (G) Pie chart illustrating the fraction of animals with mild, moderate, and severe reduction in EF at 4 weeks after injury following IgG or hENPP1mAb administration. (H) 4D gated cardiac CT showing transverse and coronal views of the heart of IgG or hENPP1mAb-injected animals at day 14 post MI (arrowheads point to the thin wall post infarct scar that is decreased in hENPP1mAb-injected groups). (I) Ejection fraction measurement by gated cardiac CT ( n = 7 animals/group). (J) Myocardial strain analysis of cardiac segments in longitudinal axis at day 7 post MI in IgG versus hENPP1mAb-treated animals. Heatmap demonstrating wall strain generated with deeper color corresponding to greater contractile force. (K) Myocardial deformation measurements to demonstrate strain forces generated at various cardiac segments between IgG and hENPP1mAb-treated animals. GLS, global longitudinal strain); Post, posterior base; Post. Mid; Post. Apex; Ant., Anterior apex; Ant. Mid and Ant. base. ( n = 9 animals/group). Data are expressed as mean ± SEM. ∗∗ p < 0.01, ∗ p < 0.05, ns: not significant. Statistical significance was determined using ordinary one-way ANOVA with Tukey’s multiple comparison test (C and D), unpaired multiple t test (F), or Student’s t test, 2 tailed (I and K).

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: Western Blot, Activity Assay, Injection, Generated, Comparison

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet: Humanized ENPP1 animals treated with hENPP1mAb after MI exhibit histologic evidence of superior cardiac repair (A) Masson trichrome staining to demonstrate scar size as a fraction of LV surface area measured 4 weeks after injury at the apex and mid ventricle in IgG or hENPP1mAb-injected humanized ENPP1 animals. (B) Quantitation of scar surface area ( n = 15/IgG and 22/hENPP1mAb) and (C) pie chart illustrating the fraction of animals with mild, moderate, and severe fibrosis following IgG or hENPP1mAb administration. (D) Hematoxylin/eosin staining to demonstrate the thickness of infarcted wall at 4 weeks after MI in IgG or hENPP1mAb-injected animals with quantification of wall thickness ( n = 11/IgG and n = 12/hENPP1mAb). (E) Heart weight (HW), body weight (BW), and HW/BW ratio in IgG versus hENPP1mAb-treated animals ( n = 21/IgG and n = 19/hENPP1mAb). (F) Immunostaining for cardiac troponin and wheat germ agglutinin to determine myocyte surface area and quantification (surrogate for cardiac muscle hypertrophy) 4 weeks after MI in IgG or hENPP1mAb-injected animals ( n = 10/IgG and n = 9/hENPP1mAb). (G) Staining for endothelial cells (CD31) to determine capillary formation (arrowheads) 4 weeks after MI in IgG or hENPP1mAb-treated animals and quantification of capillary formation ( n = 13/IgG and n = 16/hENPP1mAb). Data are represented as mean ± SEM. ∗∗ p < 0.01, ∗ p < 0.05, ns: not significant. Statistical significance was determined using Student’s t test, 2 tailed.

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: Staining, Injection, Quantitation Assay, Immunostaining

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet: Single-nuclei RNA sequencing of hearts of humanized ENPP1 animals treated with IgG or hENPP1mAb and harvested at 7 days following MI (A) Uniform manifold approximation and projection (UMAP) demonstrating different phenotypes of cell clusters in the infarcted heart and (B) distribution of cells from IgG and hENPP1mAb-treated animals across these clusters ( n = 3 animals/group). (C) Fraction of different cell populations in IgG versus hENPP1mAb-injected animals. (D) Gene ontology analysis of main pathways differentially downregulated in cardiac fibroblasts in hENPP1mAb-treated animals versus IgG control animals. (E) UMAP demonstrating subclustering of fibroblast population across IgG and hENPP1mAb groups and (F) distribution of fibroblasts of IgG versus hENPP1mAb groups across these fibroblast subclusters. (G) Fraction of fibroblasts in IgG or hENPP1mAb-treated groups contributing to the fibroblast subclusters with cluster 0 contributed by IgG-injected group and cluster 1 by the hENPP1mAb group. ( p < 0.05 in cluster 0, p < 0.01 in cluster 1, and no significance in cluster 2). (H) Expression of ECM and myofibroblast genes (Col1a1, Postn, and Acta2) across these fibroblast subclusters with abundant expression of ECM genes and myofibroblast marker Postn in subcluster 0 compared to subcluster 1. (I) Dot plot demonstrating distribution of abundantly expressed genes representing the fibroblast subclusters (Note: myofibroblast and ECM genes are abundant in subcluster 0 compared to subcluster 1). (J) Dot blot representing expression of myofibroblast and ECM genes in the entire cardiac fibroblast population of IgG versus hENPP1mAb animals. Data are expressed as mean ± SEM. ∗∗ p < 0.01. Statistical significance was determined using Student’s t test, 2 tailed.

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: RNA Sequencing Assay, Injection, Control, Expressing, Marker, Dot Blot

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet: A single dose of hENPP1mAb administered in humanized ENPP1/Tg32 animals after MI is sufficient to significantly rescue post-infarct cardiac function (A) Genetic strategy of generating humanized ENPP1/Tg32 animals and (B) determining the effects of a single dose of hENPP1mAb administered after MI. (C) B mode (top) and M mode (below) echocardiogram demonstrating cardiac contractile function and chamber dilatation in IgG versus hENPP1mAb-injected animals. Green line points to cardiac dimensions in diastole and yellow lines point to dimensions in systole. (D) Ejection fraction, fractional shortening, and LV dimensions in systole (LVIds) and diastole (LVIDd) at 1, 2, and 4 weeks after MI following a single shot of hENPP1mAb or IgG after MI ( n = 11/IgG and n = 12/hENPP1mAb). (E) Heart weight, body weight, and heart weight/body weight ratios of hearts harvested at 4 weeks in animals receiving a single dose of hENPP1mAb or IgG after MI ( n = 11/IgG and n = 12/hENPP1mAb). (F) Masson trichrome staining to demonstrate fibrosis at 4 weeks post MI in animals receiving a single dose of hENPP1mAb or IgG and quantification of fibrosis ( n = 9/IgG and n = 11/hENPP1mAb). Data represented as mean ± SEM, ∗∗ p < 0.01, ∗ p < 0.05, ns: not significant. Statistical significance was determined using unpaired multiple t test (D) or Student’s t test, 2 tailed (E and F).

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: Injection, Staining

Journal: Cell Reports Medicine

Article Title: A humanized monoclonal antibody targeting an ectonucleotidase rescues cardiac metabolism and heart function after myocardial infarction

doi: 10.1016/j.xcrm.2024.101795

Figure Lengend Snippet:

Article Snippet: ENPP1 Human Recombinant , Prospec , ENZ-729.

Techniques: Purification, Recombinant, Staining, RNA Sequencing Assay, Clone Assay, Software