Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells

Abstract

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1-10 µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Keywords: Angiogenesis; Cannabidivarin; Cannabigerol; Cannabinoids; Endoplasmic reticulum stress; Placenta.

Fig. 1

CBDV and CBG impact on cell viability. HTR-8/SVneo cells were treated with different concentrations (1–10 µM) of the compounds for 24 and 48 h. Effects were assessed through MTT assay (A) and LDH release (B). Results are presented as mean ± SEM (n = 4). Significant differences between control and CBDV or CBG-treated cells are denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001)

Fig. 2

CBDV and CBG increase the expression of ER-stress markers. HTR-8/SVneo cells were treated with CBDV and CBG (5 μM) for 48 h, with or without the TRPV1 antagonist CPZ (0.2 µM). A-D Expression of the HSPA5, sXBP1, ATF4 and DDIT3 genes was analyzed through RT-PCR. Results show transcript levels normalized against ACTB. E The protein expression of p-eIF2α and CHOP was evaluated by Western blot. TG (0.1 µM), an ER-stress inducer, was used as positive control. Data are presented as the mean ± SEM (n = 5). Significant differences between control and CBDV or CBG-treated cells are denoted by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001); between CBDV and CBDV + CPZ by ## (p < 0.01); between CBG and CBG + CPZ by §§§ (p < 0.001)

Fig. 3

Effects of CBDV and CBG on caspase activation, ROS/RNS generation and mitochondrial membrane potential. A, B Evaluation of the activity of the initiator caspases-8 and -9 (48 h); C ROS/RNS generation (0 h) and D of Δψm (48 h). CBDV and CBG-treated cells (5 µM) were pre-incubated with CPZ (0.2 µM), 4µ8c (1 µM) or GSK (0.5 µM), to study the dependence of TRPV1 and UPR pathways. CCCP (30 µM) and PMA (50 ng/mL) were used as positive controls for Δψm and ROS generation, respectively. STS (10 µM) was used as inducer of apoptosis. The results are presented as mean ± SEM (n = 3). Significant differences between control and treatments are indicated by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001); between CBDV-treated cells with or without CPZ, 4µ8c or GSK are denoted by ## (p < 0.01) and ### (p < 0.001); between CBG-treated cells with or without CPZ or 4µ8c are identified by § (p < 0.05) and §§ (p < 0.01)

Fig. 4

Analysis of the main biomarkers of apoptotic cell death and the expression of TRB3 and p-AKT proteins, for CBDV and CBG-treated cells. A Evaluation of the activity of effector caspases-3/-7, and of the expression of pro-caspase-3 B and cleaved-PARP C by Western blotting. D Evaluation of the protein expression of TRB3 and of AKT phosphorylation (p-AKT), by Western blotting. CBDV-treated cells were pre-incubated 4µ8c (1 µM) or GSK (0.5 µM), to analyze the role of UPR pathways in the activity of effector caspases. STS (10 µM) was used as inducer of apoptosis. The results are presented as mean ± SEM (n = 5). Significant differences between control and treatments are indicated by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001)

Fig. 5

Impact of CBDV and CBG on tube formation by HTR-8/SVneo cells. Results are showed as the mean ± SEM of total segments’ length (µm) (n = 5). Legend: Red surrounded by blue—junctions; Segments—yellow; Branches—green; Meshes—cyan; Extremities—Orange; Artefactual branches—blue; Nodes—magenta. Significant differences between the control and CBDV and CBG are indicated by *** (p < 0.001)

Fig. 6

CBDV and CBG dysregulate the gene expression of angiogenesis-related factors. (A-D) Analysis of the mRNA levels of VEGFA, PGF, sFLT1 and FLT1, evaluated through RT-PCR. To explore the involvement of TRPV1 and UPR pathways on the changes observed in CBDV and CBG-treated cells (5 µM), they were pre-incubated with CPZ (0.2 µM), 4µ8c (1 µM) or GSK (0.5 µM). TG (0.1 µM) was used as ER stress inducer. The results are presented as mean ± SEM (n = 5). Significant differences between control and treatments are indicated by * (p < 0.05), ** (p < 0.01) and *** (p < 0.001); between CBDV-treated cells with or without CPZ, 4µ8c or GSK are denoted by # (p < 0.05), ## (p < 0.01) and ### (p < 0.001); between CBG-treated cells with or without CPZ or 4µ8c are denoted by §§ (p < 0.01) and §§§ (p < 0.001)