Soluble fms-like tyrosine kinase-1 and angiotensin2 target calcitonin gene-related peptide family peptides in maternal vascular smooth muscle cells in pregnancy†

Abstract

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and adrenomedullin2 (ADM2) are hypotensive peptides that belong to CALCB family of peptides. Goal of this study was to identify the effect of fms-like tyrosine kinase (sFLT-1) and angiotensin2 (Ang2) on the function of these peptides in OA smooth muscle cells (OASMC) and assess the sensitivity of OA for these peptides in preeclampsia (PE) and normotensive pregnancy.

Methods: Peptide function was assessed by Cyclic adenosine monophosphate (cAMP) assays and wire myograph; mRNA expression by Polymerase chain reaction (PCR) and protein-protein interaction by proximity ligation assay and co-immunoprecipitation.

Findings: All three peptides increased cAMP synthesis in the order of efficacy CALCB > ADM = ADM2 and vascular endothelial growth factor (VEGF) mRNA in OASMC (P < 0.05); sFLT-1 mediated decrease in cAMP synthesis (P < 0.05) is differentially rescued by all three CALCB family peptides in OASMC (P < 0.005); sFLT-1 decreased receptor activity-modifying protein (RAMP)1 and RAMP2 mRNA expression (P < 0.05); Ang2 decreased the expression of calcitonin-receptor-like receptor and RAMP1 mRNA and desensitized CALCB and ADM2 receptors in OASMC (P < 0.05); sFLT-1 increased RAMP1and Ang2 type 1 receptor (AT1R) interaction in OASMC which is inhibited in presence of all three peptides; and all three peptides relax OA in PE with enhanced ADM2 response (P < 0.05).

Conclusion: sFLT-1 and Ang2 impair OASMC mediated functional responses of CALCB family peptides which can be inhibited by respective peptide treatment. The sensitivity of OA for CALCB, ADM, and ADM2-mediated relaxation is retained in PE.

Keywords: angiotensin 2; cAMP; calcitonin gene-related peptides; pregnancy; sFLT-1; vascular smooth muscle cells.

Figure 1

Stimulation of cAMP production and VEGF-A mRNA transcription by CALCB, ADM, and ADM2 in OASMC. (A) All three peptides (10⁻⁸ M) increase the synthesis of cAMP in the order of efficacy CALCB > ADM = ADM2 compared with the cells treated with IBMX alone (Control). Intracellular cAMP was quantified using Enzyme linked immunoassay (EIA) kit. Forskoline was used as a positive control. Data are presented as mean ± SEM for three replicates. Bars with different letters differ significantly among the groups (P ≤ 0.05); (B) Effect of CALCB, ADM, and ADM2 treatment (10⁻⁸ M) on the expression of VGEF-A mRNA by qPCR. The bar graph represents mRNA levels of VEGF-A in OASMC treated with different concentrations of sFLT-1 in presence or absence of sFLT-1(10 ng/ml) for 24 h (N = 3). Bars with different letters differ significantly among the groups (P ≤ 0.05), and (C) Effect of sFLT-1 treatment for 24 h on cAMP synthesis in presence or absence of CALCB, ADM, and ADM2 in OASMC. Intracellular cAMP was quantified using EIA. As shown, sFLT-1 treatment decreases the synthesis of cAMP in OASMC and all three peptides differentially inhibit the sFLT-1 induced decreases in cAMP levels in OASMC. Data are presented as mean ± SEM for three replicates and bars with different letters indicate significant difference between the groups (P ≤ 0.05).

Figure 2

Effect of sFLT-1 and Ang2 treatment on the mRNA expression of CALCB, ADM, and ADM2 receptor system in OASMC. (A) Effect of sFLT-1 on the expression of CALCRL, RAMP1, and RAMP2 mRNA in OASMC. The bar graph represents mRNA levels of RAMP1 and RAMP2 in OASMC treated with different concentrations of sFLT-1 for 24 h (N = 3). As shown, sFLT-1 decreases the mRNA expression of both RAMP1 and RAMP2 in OASMC. The mRNA levels were normalized to the mRNA expression of GAPDH. Data are presented as mean ± SEM. Asterisk indicates P < 0.05 compared with the control. (B) Effect of Ang2 on the expression of CALCRL, RAMP1, and RAMP2 mRNA in OASMC. The bar graph represents mRNA levels of RAMP1 and RAMP2 in OASMC treated with Ang2 (10⁻⁷ M) for 24 h (N = 3). As shown, Ang2 decreases the mRNA expression of CALCRL and RAMP2 with no significant effect on the expression of RAMP1 in OASMC. The mRNA levels were normalized to the mRNA expression of GAPDH. Data are presented as mean ± SEM. Asterisk indicates P < 0.05 compared with the control.

Figure 3

Acute effect of sFLT-1 and Ang2 pretreatment on CALCB, ADM, and ADM2 mediated cAMP synthesis in OASMC. (A) Effect of sFLT-1 on desensitization of CALCB, ADM, and ADM2 specific receptor for cAMP generation in OASMC. Cells were pretreated with or without sFLT-1 (10 ng/ml) for 30 min followed by treatment with or without peptide (10⁻⁸ M) for 5 min. Intracellular cAMP was quantified using EIA kit. As shown, all three peptides, CALCB (a), ADM (b), and ADM2(c) retain their effects on cAMP synthesis in OASMC pre-incubated with sFLT-1 compared with the cells without any pretreatment (Control). Data are presented as mean ± SEM for three replicates and bars with different letters differ significantly among the groups (P ≤ 0.05). (B) Effect of Ang2 on desensitization of CALCB, ADM, and ADM2 specific receptor for cAMP generation in OASMC. Cells were pretreated with or without Ang2 (10⁻⁷ M) for 30 min followed by treatment with or without peptide (10⁻⁸ M) for 5 min. Intracellular cAMP was quantified using EIA kit. As shown, CALCB (a) and ADM2 (c) mediated cAMP synthesis is blocked, while ADM (b) effects are retained in OASMC pre-incubated with Ang2 compared with the cells without any pretreatment (Control). Data are presented as mean ± SEM for three replicates and bars with different letters differ significantly among the groups (P ≤ 0.05).

Figure 4

Association of AT1R with RAMPs in OASMC: (A) Proximity ligation assay showing representative images of the associations between AT1R with RAMP1 and RAMP2. The fluorescent red signal is indicative of protein–protein interaction between the two target proteins. The red spots were quantified and normalized with the blue nuclear staining. DAPI was used for nuclear staining and absence of probe served as the control (Magnification = 20X). The bar graph shows the number of red spots/nuclei (n = 3 replicate experiments); (B) Co-immunoprecipitation (Co-IP) of Ang2 receptor type 1 (AT1R) with RAMP1 and RAMP2 protein in OASMC treated with sFLT-1 in presence or absence of peptides. Cells were pretreated with or without sFLT-1 (10 ng/ml), peptides (10⁻⁸ M), or sFLT-1 (10 ng/ml) with the three peptides for 24 h. This was followed by cell lysis and immuno-precipitation (IP) with RAMP1 or RAMP2 antibody followed by elution using anti-AT1R agarose slurry. Western blot analysis was performed by probing with antibodies for AT1R or their respective input samples. Figure shows AT1R co-immuno-precipitates with both RAMP1 (a) and RAMP2 (b) protein in OASMC. Bar graph below shows the relative fold change in the band densities normalized with the band density of the respective input samples. Different letters indicate significant difference between the groups P < 0.05. Data are presented as mean ± SEM of three replicate experiments. IP (immunoprecipitation), AT1R (Ang2 type-1 receptor), R1 (RAMP1), and R2 (RAMP2)

Figure 5

Relaxation responses of omental arteries (OA) to CALCB, ADM, ADM2, and Bradykinin. (A) Relaxation induced by CALCB in OA from pregnant women with normotensive (n = 10) and pre-eclamptic pregnancy (PE; n = 5). Dose response curves show no significant difference in the relaxation by CALCB in OA from PE (n = 10) compared with normotensive group (n = 7). (B) Relaxation induced by ADM in OA from pregnant women with normotensive (n = 10) and PE (n = 7). The dose–response curves indicate no significant difference in response to ADM between the two groups. (C) Relaxation induced by ADM2 in OA from pregnant women with normotensive (n = 10) and PE (n = 7). Figure shows enhanced relaxation to ADM2 in OA from PE (n = 10) compared with normotensive group (n = 7), and (D) Relaxation induced by Bradykinin (BK) in OA from pregnant women with normotensive (n = 10) and PE (n = 7). Figure shows enhanced relaxation to Bradykinin in OA from PE (n = 10) compared with that in the normotensive group (n = 7). Results are presented as mean ± SEM. Different letters indicate significant differences between groups (P < 0.05).

Figure 6

Flow chart showing potential involvement of CALCB family peptides, Ang2 and sFLT-1 system in maternal circulation during normal pregnancy and pregnancy complicated by PE. In normal (normotensive) pregnancy, maternal vasculature is relaxed due to increase in NO system, balanced AT1R and AT2R levels, and increased expression and function of CALCB family peptides. In PE, decreases in NO system and elevated sFLT-1 levels are accompanied with down regulation of ligand-receptor system of CALCB family of hypotensive peptides resulting in vasoconstriction. AT1R (Ang2 type-1 receptor), AT2R (Ang2 type-1 receptor), and sFLT-1.