AM₁-receptor-dependent protection by intermedin of human vascular and cardiac non-vascular cells from ischaemia-reperfusion injury

Abstract

Intermedin (IMD) protects rodent heart and vasculature from oxidative stress and ischaemia. Less is known about distribution of IMD and its receptors and the potential for similar protection in man. Expression of IMD and receptor components were studied in human aortic endothelium cells (HAECs), smooth muscle cells (HASMCs), cardiac microvascular endothelium cells (HMVECs) and fibroblasts (v-HCFs). Receptor subtype involvement in protection by IMD against injury by hydrogen peroxide (H₂O₂, 1 mmol l⁻¹) and simulated ischaemia and reperfusion were investigated using receptor component-specific siRNAs. IMD and CRLR, RAMP1, RAMP2 and RAMP3 were expressed in all cell types.When cells were treated with 1 nmol l⁻¹ IMD during exposure to 1 mmol l⁻¹ H₂O₂ for 4 h, viability was greater vs. H2O2 alone (P<0.05 for all cell types). Viabilities under 6 h simulated ischaemia differed (P<0.05) in the absence and presence of 1 nmol l⁻¹ IMD: HAECs 63% and 85%; HMVECs 51% and 68%; v-HCFs 42% and 96%. IMD 1 nmol l⁻¹ present throughout ischaemia (3 h) and reperfusion (1 h) attenuated injury (P<0.05): viabilities were 95%, 74% and 82% for HAECs, HMVECs and v-HCFs, respectively, relative to those in the absence of IMD (62%, 35%, 32%, respectively). When IMD 1 nmol l⁻¹ was present during reperfusion only, protection was still evident (P<0.05, 79%, 55%, 48%, respectively). Cytoskeletal disruption and protein carbonyl formation followed similar patterns. Pre-treatment (4 days) of HAECs with CRLR or RAMP2, but not RAMP1 or RAMP3, siRNAs abolished protection by IMD (1 nmol l⁻¹) against ischaemia-reperfusion injury. IMD protects human vascular and cardiac non-vascular cells from oxidative stress and ischaemia-reperfusion,predominantly via AM1 receptors.

Figure 1. Relative IMD (filled column) and AM (open column) mRNA expression normalised to GAPDH (A) or β-actin (B) in human aortic endothelial cells (HAECs) and smooth muscle cells (HASMCs), human coronary artery microvascular endothelial cells (HMVECs) and ventricular-human cardiac fibroblasts (v-HCFs) in basal conditions

Results (means ± SEM) from n = 3 independent cell sources (or n = 3 individual cultures for v-HCFs) with each source (or culture) run in quadruplicate are presented as percentage expression compared to IMD expression in HAECs. Analysis (2-way ANOVA): *P < 0.05 vs. IMD in HAEC and #P < 0.05 vs. IMD in same cell type.

Figure 2. Cellular IMD vs. AM protein distribution

A, indirect immunofluorescence staining to detect the presence and location of IMD protein (green: signal enhanced in IMD alone micrographs) in the cell-types investigated in Fig. 1. Actin counter-staining (red) of the same cells facilitates normal cellular structure identification. B, for comparison AM (green) is shown. Scale bar: 10 μm.

Figure 3. Relative mRNA expression of the calcitonin receptor-like receptor (CRLR) receptor components in the cell-types investigated in Fig. 1 under basal conditions

Results, normalised against GAPDH, are presented as relative to CRLR for each RAMP. n = 3 cell sources (or individual cultures for v-HCFs) run in triplicate: *P < 0.05 vs. CRLR and #P < 0.05 vs. other RAMPs. In data not shown similar results were found when β-actin was employed as the house-keeping gene.

Figure 4

A, indirect immunofluorescence staining to detect the presence and location of CRLR receptor components (green) and actin (red) protein in HAECs and v-HCFs. RAMP1 and RAMP3 signals have been intensified to allow distribution to be seen in micrographs. Scale bar: 10 μm. B and C, indirect immunofluorescence staining images (using fixed fluorescence and image capture settings and no image intensification) were analysed for n≥ 30 cells (obtained from n≥ 3 microscopic fields) for n = 3–4 independent cultures for each condition, normalised for actin and presented (means ± SEM) as relative to CRLR for each RAMP. *P < 0.05 vs. CRLR.

Figure 5

A, phase contrast images showing the effect of 4 h exposure to 1 mmol l⁻¹ H₂O₂ and/or 1 nmol l⁻¹ IMD to endothelial, smooth muscle and fibroblast cells. Scale bar 100 μm. B, counts of viable cells for the cell-types and treatment conditions in Fig. 5A. n = 4 cell sources (or individual cultures for v-HCFs) run in quadruplicate. *P < 0.01 vs. untreated control and #P < 0.01 vs. H₂O₂ alone for that cell-type. C, indirect immunofluorescence staining of cells exposed to 1 mmol l⁻¹ H₂O₂ (actin, red). With IMD addition a degree of normal cellular structure is maintained, represented by HAECs in the larger micrograph. Scale bar: 10 μm. D, protein carbonyl formation (an indicator of oxidative stress) in HAECs in response to the treatment conditions employed in Fig. 5A. n = 3 cell sources *P < 0.05 vs. control and #P < 0.05 vs. H₂O₂ alone. Representative blot shows, in order, both derivatised and non-derivatised signal for each treatment condition.

Figure 6

Dose-dependent effect of IMD (at concentrations ≤10⁻⁹ mol l⁻¹; grey columns) on cell viability under simulated ischaemia (4 h) for those cell types investigated in Fig. 1. n = 3 cell sources (or individual cultures for v-HCFs) run in duplicate. *P < 0.05 vs. normoxia alone (open column) and #P < 0.05 vs. Ischaemia without IMD (black filled column).

Figure 7

A, viable cell counts for cells exposed to normoxia (4 h), ischaemia (4 h) or ischaemia (3 h)–reperfusion (1 h; IR) without/with co-incubation with 10⁻⁹ mol l⁻¹ IMD throughout the 4 h period or only during the 1 h of reperfusion. n = 4 cell sources (or individual cultures for v-HCFs) run in quadruplicate. *P < 0.01 vs. untreated control, †P < 0.05 vs. ischaemia alone, and #P < 0.01 vs. non-IMD counterpart. Representative phase contrast images showing the effect of exposure of endothelial cells and fibroblasts to IR and IMD are also included. Scale bar 100 μm. B, indirect immunofluorescence staining of cells exposed to ischaemia or IR (actin, red). With IMD addition during reperfusion alone a degree of normal cellular structure is maintained. Scale bar: 10 μm. C, protein carbonyl formation in HAECs in response to normoxia, ischaemia and IR without/with co-incubation with 10⁻⁹ mol l⁻¹ IMD. n = 3 individual cultures *P < 0.05 vs. control and #P < 0.05 vs. non-IMD treated counterpart. Representative blot showing in order both derivatised and non-derivatised signal for each treatment condition.

Figure 8

A and B, relative mRNA expression of IMD normalised against GAPDH (A; similar results were found using β-actin as a housekeeping gene – not shown) or protein level normalised against β-actin (B) are presented as relative to normoxia. n = 3 HMVECs sources run in triplicate: #P < 0.05 vs. normoxia. C–F, viable cell counts for cells which have had the CRLR receptor components knocked-down before exposure to normoxia, ischaemia and ischaemia–reperfusion without/with co-incubation with 10⁻⁹ mol l⁻¹ IMD. n = 3 HAEC sources run in triplicate. *P < 0.05 vs. untreated control, †P < 0.05 vs. non knock-down counterpart, and #P < 0.05 vs. non-IMD counter part. G, blots obtained when membrane protein lysates were immunoprecipitated (IP) with RAMP2 and western blotted with CRLR antibody (and vice versa) to confirm co-precipitation of molecules. The heavy chain of the IP antibody also transfers to the blot and is employed as a loading control. Treatment conditions included no pre-treatment or pre-treatment with either random (non-targeting siRNA) or siRNA directed against CRLR or RAMP2.