Differential effects of sulforaphane in regulation of angiogenesis in a co-culture model of endothelial cells and pericytes

Abstract

Abstract. Aberrant neovascularization supports nutrients and the oxygen microenvironment in tumour growth, invasion and metastasis. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of vascular conditions. Sulforaphane (SFN), an isothiocyanate, has been reported to possess chemopreventive properties. In the present study, the effects of SFN on cell proliferation and tubular formation have been investigated using endothelial cells (ECs) and pericytes in coculture. SFN showed a dosedependent inhibition on the growth of ECs and pericytes with IC 50 values 46.7 and 32.4 µM, respectively. SFN (5-20 µM) inhibited tube formation in a 3D coculture although a lower dose (1.25 µM) promoted 30% more endothelial tube formation than control. Moreover, SFN affected intercellular communication between ECs and pericytes via inhibition of angiogenic factor such as vascular endothelial growth factor (VEGF) expression in pericytes. However, the expression of its receptor (VEGFR-2) was found significantly increased in ECs. These effects were associated with downregulation of prolyl hydroxylase domain-containing protein 1 and 2 (PHD1/2) and activation of hypoxia-inducible factor-1 (HIF) pathway by SFN. Furthermore, thioredoxin reductase-1 was also upregulated by SFN treatment, suggesting that antioxidant and redox regulation are involved in angiogenesis. Taken together, the results of the present study suggest that SFN differentially regulates endothelial cells and pericytes disrupting their interplay through the VEGF-VEGFR signalling pathway. Anti-angiogenesis property of SFN indicates that it has potential role as an anticancer agent. IntroductionAngiogenesis, the growth of new capillary blood vessels, is a normal and vital process in growth, development and wound healing. However, it is also a fundamental step in the growth of tumours. Nutrients and oxygen, supplied by the blood vessels into the tumours, are essential for the growth and progression of malignant tumours beyond the size of 1-2 mm 3 (1). Newly formed blood vessels can facilitate cells escaping through leakage from primary tumour sites to metastasize, the major cause of mortality for cancer patients. Anti-angiogenesis has been recognized as valuable therapy in treatment of various metastatic cancers since the theory was first proposed by Folkman (2,3). Tumour angiogenesis is believed to be regulated by the interactions between pro-angiogenic and anti-angiogenic factors in the tumour microenvironment (4). Angiogenic response is a dynamic process requiring a series of fine-tuned angiogenic signalling and molecular events. Hypoxia is the common inducer of angiogenesis in the core of large tumours stimulating the release of pro-angiogenic factors to promote endothelial cell proliferation, migration, differentiation and self-assembly into vascular-like structures. Subsequently, perivascular cells are recruited to form mature and stable vessels (5).The interaction between endothelial cells (ECs) and pericytes (PVCs) has g...

Sulforaphane Rescues Ethanol-Suppressed Angiogenesis through Oxidative and Endoplasmic Reticulum Stress in Chick Embryos

“…S2). The concentrations of the SFN, which stimulating angiogenesis in chick YSM and CAM, were higher than the ones used in our in vitro experiments [18][19]32 . This might be due to the diversity and absorptivity between in vivo and in vitro approaches, since the in vivo microenvironment of YSM and CAM are certainly more complicated than one at incubated one layer of cells in .…”

“…This might be due to the diversity and absorptivity between in vivo and in vitro approaches, since the in vivo microenvironment of YSM and CAM are certainly more complicated than one at incubated one layer of cells in . However, most studies showed more than 10µM SFN could suppress angiogenesis in vitro (HUVECs and Bovine aortic endothelial cells) [18][19][32][33][34][35][36] and in vivo (Balb/C mice implanted with VEGF-impregnated Matrigel plugs) 34 experiments. Our observations are generally similar to the experimental results in those literatures.…”

Biphasic effect of sulforaphane on angiogenesis in hypoxia via modulation of both Nrf2 and mitochondrial dynamics

“…Similarly, this bell-shaped effect of SFN and its analogues on angiogenesis in normoxia has also been found in our previous studies utilizing tumor cells and 3-D HUVECs and pericyte co-culture models. 17,22,24,64 The stimulating action of SFN on angiogenesis has been evidenced in ethanol-injected chick yolk sac membrane (YSM) and chorioallantoic membrane (CAM) models at 2.5–10 μM co-treatment, 65 and in 2.5 μM-pretreated endothelial colony forming cells (ECFCs). 66 In contrast, anti-angiogenic activities of SFN and some other ITCs have also been reported in HUVECs and rat aortic ring models at a single dose (>10 μM) of intervention in normoxia.…”

“…Underlying the remarkable efficacy are its pleiotropic activities, for example, inducing antioxidant/detoxifying enzymes, anti-inflammation, mitostatic and pro-apoptotic effects, epigenetic regulation of genes, improving metabolism. 6–9 There is also evidence indicating that SFN and its metabolites and analogues possess anti-angiogenic properties, 7,10 and the potential mechanisms, though not fully elucidated, may involve inducing ECs apoptosis, 11,12 inhibiting matrix metalloproteinase-2/9 (MMP-2/9), 13,14 hypoxia-inducible factor-1α (HIF-1α), 13,15,16 vascular endothelial growth factor (VEGF), 13,15,17,18 VEGF receptor-2 (VEGFR-2), 13,18 disrupting endothelial mitotic progression and microtubule polymerization, 19 anti-inflammation, 20 activating FOXO, 21 inhibiting STAT3 22,23 and inducing ROS. 24…”

Sulforaphane: A review of its therapeutic potentials, advances in its nanodelivery, recent patents, and clinical trials

“…study, SFN was found to differentially regulate the EC and pericytes, thereby disturbing their role in the VEGF-VEGFR signaling pathway (Y Wang, Zhou, Wang, Liu, & Bao, 2017)…”