Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro

Abstract

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five G_i protein-coupled receptors (SST_1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST₁ and SST₅ receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST₁ and SST₅ receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.

Keywords: Akt; MAPK; MYPT1; RhoA/Rock; angiogenesis; cAMP; endothelial permeability; somatostatin receptors.

Figure 1

SST receptor gene (SSTR) expression and signalling in HUVECs. (A) The expression of SSTR mRNAs in HUVECs. The expected PCR fragment sizes for SSTR1-5 were 115 bp, 193 bp, 151 bp, 157 bp, and 94 bp, respectively. The plus (+) sign indicates cDNA and negative (-) sign indicates total RNA (without reverse transcriptase) used as the negative control. The lower panel shows Ct values determined by qPCR using HUVEC cDNA corresponding to 1 ng total RNA. (B) Concentration (as indicated)-dependent effect of SST14 on Akt and ERK2 phosphorylation. C: buffer-treated control. Representative Western blots of 3 experiments. The lower panel shows the quantification of the Western blots shown above, the dotted line shows the normalised basal levels of pAkt and pERK2. n = 3, p < 0.05; * vs. control (pAkt), ^# vs. control (pERK2). (C) Time-dependent effect of SST14 (1 nM) on Akt and ERK2 phosphorylation. Representative Western blots of phosphorylation and total Akt and ERK. (D) Concentration-dependent effect of SST14 (1 nM) on FSK-induced cAMP production. ECs were treated with SST14 or buffer (CTR) for 10 min followed by treatment with FSK (10 μM). n = 6, p < 0.05; * vs. control, ^# vs. FSK alone.

Figure 2

Effect of SST on endothelial barrier function. (A) HUVEC permeability: HUVEC monolayers cultured on filter membranes were treated with SST14 (1 nM) or buffer (CTR) followed by treatment with thrombin (Thr; 0.5 U /mL). Flux of albumin across HUVEC monolayers 10 min (maximum effect) after Thr treatment is presented. n = 5; p < 0.05; * vs. control, ^# vs. Thr alone. (B) HUVEC monolayers cultured on glass coverslips were treated with SST14 (1 nM) or buffer (CTR) followed by treatment with thrombin (Thr; 0.5 U /mL). Cells were fixed with ice-cold methanol and VE-cadherin was immuno-stained using a mouse anti-VE-cadherin (human) antibody. Scale bar: 50 μm.

Figure 3

Effect of inhibitors of Akt and MEK/ERK pathways on SST14-mediated HUVEC sensitisation. HUVEC monolayers cultured on filter membranes were treated with SST14 (1 nM) or buffer (CTR) followed by treatment with thrombin (Thr; 0.5 U /mL). In the sets of experiments where Akt and MEK/ERK inhibitors were used, these were added 30 min before adding SST14 or buffer. Flux of albumin across HUVEC monolayers 10 min (maximum effect) after Thr treatment is presented. n = 3; p < 0.05; * vs. control, ^# vs. Thr alone, ^§ vs. SST+Thr.

Figure 4

Effect of SST14 on RhoA/Rock pathways in HUVECs. (A) Schematic presentation of thrombin-mediated activation of RhoA/Rock signalling. (B) HUVECs were treated with thrombin (0.5 IU/mL) in the presence of buffer or SST14 (1 nM). Where indicated, cells were treated with U0126 (5 µM) for 30 min before treating with SST14 and thrombin. Representative Western blots of 3 experiments. (C) Quantification of blots from (B). p < 0.05; * vs. control, ^# vs. Thr alone, ^§ vs. SST+Thr.

Figure 5

Effect of SST14 on cAMP/PKA-mediated inhibition of RhoA/Rock signalling. (A) Schematic presentation of FSK-mediated inhibition of RhoA/Rock signalling and the possible interaction with SSTR14 signalling. (B) HUVECs were treated with FSK (5 μM) in the presence of buffer or SST14 (1 nM). Where indicated, cells were treated with U0126 (5 µM) for 30 min before treating with SST and FSK. Representative Western blots of 3 experiments. (C) Quantification of blots from (B). p < 0.05; * vs. control, ^# vs. FSK alone.

Figure 6

Effect of SST14 on HUVEC proliferation and in vitro angiogenesis (3D Spheroids). (A) Cell density measured by time-lapse live-cell imaging. Cells were treated with 1 nM SST14 or buffer (CTR) under low-growth factor conditions and imaged after every 30 min for a period of 72 h. n = 3; p < 0.05; * vs. control. (B) 3D spheroid assay. HUVECs were treated with SST14 (1 nM) or buffer (CTR) followed by treatment with VEGF (10 ng/mL). n = 6; p < 0.05; * vs. control, ^# vs. VEGF alone.