VEGF-A inhibits agonist-mediated Ca2+ responses and activation of IKCa channels in mouse resistance artery endothelial cells

Abstract

Key points: Prolonged exposure to vascular endothelial growth factor A (VEGF-A) inhibits agonist-mediated endothelial cell Ca²⁺ release and subsequent activation of intermediate conductance Ca²⁺ -activated K⁺ (IK_Ca ) channels, which underpins vasodilatation as a result of endothelium-dependent hyperpolarization (EDH) in mouse resistance arteries. Signalling via mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) downstream of VEGF-A was required to attenuate endothelial cell Ca²⁺ responses and the EDH-vasodilatation mediated by IK_Ca activation. VEGF-A exposure did not modify vasodilatation as a result of the direct activation of IK_Ca channels, nor the pattern of expression of inositol 1,4,5-trisphosphate receptor 1 within endothelial cells of resistance arteries. These results indicate a novel role for VEGF-A in resistance arteries and suggest a new avenue for investigation into the role of VEGF-A in cardiovascular diseases.

Abstract: Vascular endothelial growth factor A (VEGF-A) is a potent permeability and angiogenic factor that is also associated with the remodelling of the microvasculature. Elevated VEGF-A levels are linked to a significant increase in the risk of cardiovascular dysfunction, although it is unclear how VEGF-A has a detrimental, disease-related effect. Small resistance arteries are central determinants of peripheral resistance and endothelium-dependent hyperpolarization (EDH) is the predominant mechanism by which these arteries vasodilate. Using isolated, pressurized resistance arteries, we demonstrate that VEGF-A acts via VEGF receptor-2 (R2) to inhibit both endothelial cell (EC) Ca²⁺ release and the associated EDH vasodilatation mediated by intermediate conductance Ca²⁺ -activated K⁺ (IK_Ca ) channels. Importantly, VEGF-A had no direct effect against IK_Ca channels. Instead, the inhibition was crucially reliant on the downstream activation of the mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). The distribution of EC inositol 1,4,5-trisphosphate (IP₃ ) receptor-1 (R1) was not affected by exposure to VEGF-A and we propose an inhibition of IP₃ R1 through the MEK pathway, probably via ERK1/2. Inhibition of EC Ca²⁺ via VEGFR2 has profound implications for EDH-mediated dilatation of resistance arteries and could provide a mechanism by which elevated VEGF-A contributes towards cardiovascular dysfunction.

Keywords: MEK; VEGF-A; endothelial cell calcium; endothelium-derived hyperpolarizing factor; vasodilation.

Figure 1. VEGF‐A receptor expression in mouse mesenteric resistance arteries

A, Pecam‐1 and Acta2 expression in intact arteries and isolated EC tubes. B, Flt1, Flk1 and Nrp1 expression (n = 4 animals). Eyes from a single animal were used as a positive control for the VEGF‐A receptors. C, arteries were pre‐constricted with PE. When pumped into the lumen of arteries, neither vehicle, nor VEGF‐A dilated the artery within 15 min (n = 3–4). ^* P < 0.05 compared to intact arteries.

Figure 2. VEGF‐A inhibits EDH‐mediated vasodilatation evoked by SLIGRL

Arteries were perfused with either vehicle or VEGF‐A for 60 min. A, vasodilatation to SLIGRL (n = 7–8). B, vasodilatation to SLIGRL in the presence of l‐NAME (L‐N) (n = 5–8). C, vasodilatation to DEA NONOate (n = 7–9). D, vasodilatation to NS309 (n = 3–12). TR, TRAM‐34; Ap, apamin. ^* P < 0.05 compared to vehicle. ^# P < 0.05 compared to vehicle + L‐N.

Figure 3. VEGF‐A inhibits IKCa and SKCa‐dependent EDH vasodilatation

Arteries were perfused with either vehicle or 1 nm VEGF‐Afor 60 min. All experiments were performed in the presence of l‐NAME. Vehicle and VEGF‐A are from Fig. 2. A, vasodilatation to SLIGRL with the addition of TRAM‐34 (TR) (n = 5–13). B, vasodilatation with the addition of apamin (Ap) (n = 3–13). C, SLIGRL‐mediated vasodilatation in the presence of TRAM‐34 and apamin (n = 3–13). D, vasodilatation to 10 μm SLIGRL in the presence of IK_Ca and SK_Ca inhibitors. ^* P < 0.05 compared to VEGF‐A. ^# P < 0.05 compared to vehicle + TR.

Figure 4. VEGF‐A pre‐exposure inhibits propagation of EC Ca²⁺

Pressurized arteries were luminally perfused with vehicle or 1 nm VEGF‐A for 60 min. Baseline Ca²⁺ was recorded for 1 min, SLIGRL was then added and remained in the bath for the duration of the recording. A, following perfusion of vehicle, 3 and 10 μm SLIGRL stimulated a clear increase in Ca²⁺ waves along the length of the cell. B, following perfusion of VEGF‐A, 3 μm SLIGRL stimulated less frequent, localized Ca²⁺ events, whereas 10 μm SLIGRL increased Ca²⁺ event frequency and Ca²⁺ waves propagated along the cell. Data are shown as line scans (corresponding to white lines in A and B) with fluorescence traces referring to subcellular regions of interest (coloured boxes in A and B). Black trace refers to whole‐cell recording. ^*Dip in intensity as a result of a movement artefact. Representative of five or six independent experiments.

Figure 5. VEGF‐A inhibits SLIGRL‐mediated Ca²⁺ events and wave propagation

A, arteries were stimulated with 3 μm SLIGRL. Regions of interests were positioned 20 μm apart in active ECs: upper: vehicle perfused with trace to right showing a propagating wave; lower: 1 nm VEGF‐A perfused with a local event shown to the right. Representative of multiple cells within five or six independent experiments. B–D, summary of Ca²⁺ events. Following luminal perfusion with 1 nm VEGF‐A for 60 min, fewer ECs respond to 3 μm SLIGRL (B) and, of the active cells, there were fewer Ca²⁺ events (C). D, VEGF‐A treated arteries produced fewer propagating Ca²⁺ events compared to the vehicle‐treated arteries. n = 5–6; ^* P < 0.05 compared to vehicle.

Figure 6. VEGF‐A inhibits SLIGRL‐mediated EC Ca²⁺ events and vasodilatation via ERK1/2 signalling

Vehicle and VEGF‐A are from Fig. 5. Arteries were perfused with either vehicle or 1 nm VEGF‐A with and without inhibitors for 60 min. A, ZM323881 and UO126 prevented VEGF‐A induced inhibition of SLIGRL‐mediated vasodilatation (n = 3–12). B–D, quantification of EC Ca²⁺ activities in arteries treated with ZM323881 or UO126 (n = 3–6). ^* P < 0.05 compared to vehicle.

Figure 7. Distribution of IP₃R1 in ECs is not modified by VEGF‐A

A, Itpr1‐3 expression in intact arteries and EC tubes (n = 4). B, pressurized arteries were perfused with either vehicle or 1 nm VEGF‐A for 60 min. Immunolabelling showed the IP₃R1 distribution in ECs (horizontal nuclei). The distribution of ZO‐1 appears to be unaltered by VEGF‐A in arteries. Representative of three arteries per treatment.