canf 4–23  (Bachem)

Journal: Human Molecular Genetics

Article Title: The biological impact of blood pressure-associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle

doi: 10.1093/hmg/ddx375

Figure Lengend Snippet: The BP-elevating variant at NPR3 locus is associated with increased human VSMCs proliferation. VSMCs in the absence and presence of C-type natriuretic peptide (CNP; 100 nmol/l) and the NPR-C specific agonist (cANF 4–23 ; 100 nmol/l) for 24 h were analysed for proliferation rate. Cell proliferation over the 24-h period was calculated as (OD450 at 24 h−OD450 at baseline)/OD450 at baseline. ‘E’ indicates the BP-elevating allele, ‘L’ denotes the BP-lowering allele. ( A ) Differences in proliferation between untreated cells of different genotypes. Representative images of DAPI stained cells of the three different genotypes at baseline and 24 h. Scale bars=200µm. Mean±SEM, * P <0.05. ( B ) Differences in proliferation between CNP or cANF 4–23 treated cells of different genotypes, and ( C ) Inhibitory effects of CNP and cANF 4–23 on proliferation of cells of different genotypes, calculated by (OD450 at 24 h of untreated cells−OD450 at 24 h of CNP-treated or cANF 4–23 -treated cells)/OD450 at 24 h of untreated cells. Mean±SEM, * P <0.05.

Article Snippet: After overnight incubation, VSMCs were serum starved for 24 h then transferred to 15% FBS DMEM with or without 100 nmol/l CNP (Sigma-Aldrich) or cANF 4–23 (Bachem).

Techniques: Variant Assay, Staining

Journal: Human Molecular Genetics

Article Title: The biological impact of blood pressure-associated genetic variants in the natriuretic peptide receptor C gene on human vascular smooth muscle

doi: 10.1093/hmg/ddx375

Figure Lengend Snippet: The BP-elevating allele at NPR3 locus increases Ang II-induced intracellular calcium changes in human VSMCs. Human VSMCs were incubated with or without C-type natriuretic peptide (CNP; 1 μmol/l) and the NPR-C specific agonist cANF 4–23 (1 μmol/l), and then exposed to Ang II (10 or 100 nmol/l). ‘E’ indicates the BP-elevating allele, ‘L’ denotes the BP-lowering allele. ( A ) Representative images of intracellular calcium changes among genotypes under stimulation of 100 nmol/l Ang II. ( B ) Differences between genotypes in intracellular calcium changes in response to Ang II in the absence of CNP or cANF 4–23 . Differences between genotypes in intracellular calcium level changes in response to Ang II ( C ) and drug inhibitory effect ( D ) in the presence of CNP or cANF 4–23 . Mean±SEM, * P <0.05, ** P <0.01.

Article Snippet: After overnight incubation, VSMCs were serum starved for 24 h then transferred to 15% FBS DMEM with or without 100 nmol/l CNP (Sigma-Aldrich) or cANF 4–23 (Bachem).

Techniques: Incubation

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Description of the LNA probe and primer sequences used to quantify gene expression

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Comparison of natriuretic peptide receptor 2 (Npr2) and Npr3 expression in non-diseased (grade 0/I) and diseased (grade III to IV) cartilage . Tissues were taken from donors aged 55 to 60 and 80 to 85 years. Paraffin-embedded sections from a single donor aged 60 years were stained with Npr2 or Npr3 antibodies (green) and examined by immunofluoresence microscopy ( A ). Nuclei (blue) were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar represents 10 μM. Negative controls showed no staining (not shown). Inset shows Npr2 (110 kDa) and Npr3 expression (60 kDa) by western blot analysis from the same donor. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques: Comparison, Expressing, Staining, Microscopy, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Effect of pharmacological agents that influence natriuretic peptide and 3,5-cyclic guanosine monophosphate (cGMP) signalling . Constructs were cultured under free-swelling conditions with 0 or 10 ng/ml IL-1β and/or 100 nM C-type natriuretic peptide (CNP) or 1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one ODQ (5 μM), P19 (0.5 μM) and cANF 4-23 (1 μM) on nitric oxide (NO) release (A), prostaglandin E 2 (PGE 2 ) production (B), glycosaminoglycan (GAG) synthesis (C) and total 3,5-cyclic guanosine monophosphate (cGMP) content (D) for 48 hours ( n = 9 to 25). Asterisks indicate significant comparisons between untreated control samples with IL-1β and/or IL-1 + CNP and/or IL-1β + CNP + cANF 4-23 and/or IL-1β + CNP + P19. All other comparisons (not indicated) were not significant.

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques: Construct, Cell Culture, Control

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Effect of dynamic compression and pharmacological agents that influence the natriuretic peptide receptor 2 (Npr2) or Npr3 pathways . Absolute values for nitric oxide (NO) release ( A ), prostaglandin E 2 (PGE 2 ) production ( B ), glycosaminoglycan (GAG) synthesis ( C ) and total 3,5-cyclic guanosine monophosphate (cGMP) content ( D ). Chondrocyte/agarose constructs were cultured in the presence and absence of 0 or 10 ng/ml IL-1β and/or 100 nM C-type natriuretic peptide CNP or 0.5 μM P19 or 1 μM cANF 4-23 for 48 hours. Error bars represent the mean and standard error of the mean (SEM) values of between eight and thirteen replicates from four separate experiments. In addition, the corresponding normalised strained values, presented as a percentage change of the unstrained controls, with SEM values are shown in brackets. Asterisks indicate significant comparisons in unstrained (black bars) and strained (white bars) constructs for the multiple treatment conditions. All other comparisons (not indicated) were not significant.

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques: Construct, Cell Culture

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Effect of C-type natriuretic peptide (CNP) and dynamic compression on natriuretic peptide receptors (Nprs) and CNP levels . Npr2 ( A ), Npr3 ( B ) CNP ( C ) and CNP concentration ( D ), aggrecan ( E ) and type II collagen gene expression ( F ). Constructs were cultured in the presence and absence of 0 or 10 ng/ml IL-1β and/or 100 nM CNP or 0.5 μM P19 or 1 μM cANF 4-23 6 hours. Error bars represent the mean and standard error of the mean (SEM) values of eight to thirteen replicates from four separate experiments. Asterisks indicate significant comparisons in unstrained (black bars) and strained (white bars) constructs for the multiple treatment conditions. All other comparisons (not indicated) were not significant. For CNP concentration, numbers in brackets indicates the percentage change from unstrained control values.

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques: Concentration Assay, Gene Expression, Construct, Cell Culture, Control

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Effect of dynamic compression and pharmacological agents that influence the natriuretic peptide receptor 2 (Npr2) or Npr3 pathways . Absolute values for nitric oxide (NO) release ( A ), prostaglandin E 2 (PGE 2 ) production ( B ) and glycosaminoglycan (GAG) synthesis ( C ). Constructs were cultured in the presence and absence of 0.5 μM P19 or 1 μM cANF 4-23 for 48 hours. Error bars represent the mean and standard error of the mean (SEM) values of eight replicates from three separate experiments. In addition, the corresponding normalised strained values, presented as a percentage change of the unstrained controls, with SEM values are shown in brackets. Asterisks indicate significant comparisons in unstrained (black bars) and strained (white bars) constructs for the multiple treatment conditions. All other comparisons (not indicated) were not significant.

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques: Construct, Cell Culture

Journal: Arthritis Research & Therapy

Article Title: Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model

doi: 10.1186/ar4253

Figure Lengend Snippet: Proposed signalling interactions between C-type natriuretic peptide (CNP) and mechanical loading in chondrocytes . CNP primarily activates natriuretic peptide (Npr)2 which underlies guanylyl cyclase (GC) activity and mediates several cell-signalling effects through the synthesis of 3,5-cyclic guanosine monophosphate (cGMP) and membrane-bound cyclic GMP-dependent protein kinase II (PKGII). It has been reported that the Gi/o binding domain in Npr3 activates extracellular-signal-regulated kinase 1/2 (ERK 1/2) or the phospholipase C (PLC) and inosital triphosphate (IP 3 ) pathway via adenylate cyclase/cAMP inhibition. PLC activation and IP 3 generation is well known to contribute to Ca 2+ release from intracellular stores. This pathway supports cartilage homeostasis by mediating the anabolic effects involved in mechanotransduction. Furthermore, mechanical signals stimulate Npr expression and CNP levels, which mediates anabolic effects involving extracellular matrix (ECM) synthesis. In contrast, IL-1β stimulates catabolic activities via inducible nitric oxide synthase (iNOS) and nitric oxide (NO)/sGC/cGMP levels. The IL-1β-induced catabolic response could be inhibited by mechanosensitive CNP or Ca 2+ release through stretch-activated ion channels or integrin-mediated signals involving F-actin/cytoskeletal reorganisation.

Article Snippet: Constructs were cultured in 1 ml of defined media supplemented with 100 n M CNP in the presence and absence of 10 ng/ml IL-1β and/or 5 μM specific sGC antagonist (1H-(1,2,4)oxadiazolo-(4, 3-a)quinoxalin-1-one) (ODQ) and/or 1 μM selective Npr3 agonist cANF 4-23 (C-Atrial Natriuretic Factor) (Bachem AG, Bubendorf, Switzerland) and/or 0.5 μM selective Npr2 antagonist cyclic gly-24-ser (P19, Gentaur Molecular Products, Whetstone, UK) for 48 hours [ , ].

Techniques: Activity Assay, Membrane, Binding Assay, Inhibition, Activation Assay, Expressing