Perillyl alcohol modulates activation, permeability and integrity of human brain endothelial cells induced by Plasmodium falciparum

Abstract

Background: Cerebral malaria (CM) is a severe immunovasculopathy caused for Plasmodium falciparum infection, which is characterised by the sequestration of parasitised red blood cells (pRBCs) in brain microvessels. Previous studies have shown that some terpenes, such as perillyl alcohol (POH), exhibit a marked efficacy in preventing cerebrovascular inflammation, breakdown of the brain-blood barrier (BBB) and brain leucocyte accumulation in experimental CM models.

Objective: To analyse the effects of POH on the endothelium using human brain endothelial cell (HBEC) monolayers co-cultured with pRBCs.

Methodology: The loss of tight junction proteins (TJPs) and features of endothelial activation, such as ICAM-1 and VCAM-1 expression were evaluated by quantitative immunofluorescence. Microvesicle (MV) release by HBEC upon stimulation by P. falciparum was evaluated by flow cytometry. Finally, the capacity of POH to revert P. falciparum-induced HBEC monolayer permeability was examined by monitoring trans-endothelial electrical resistance (TEER).

Findings: POH significantly prevented pRBCs-induced endothelial adhesion molecule (ICAM-1, VCAM-1) upregulation and MV release by HBEC, improved their trans-endothelial resistance, and restored their distribution of TJPs such as VE-cadherin, Occludin, and JAM-A.

Conclusions: POH is a potent monoterpene that is efficient in preventing P. falciparum-pRBCs-induced changes in HBEC, namely their activation, increased permeability and alterations of integrity, all parameters of relevance to CM pathogenesis.

Fig. 1:. perillyl alcohol (POH) downmodulates intercellular cell adhesion molecule 1 (ICAM-1) upregulation in human brain endothelial cells (HBEC) caused by Plasmodium falciparum. (A) Unstimulated HBEC showed basal expression of ICAM-1 (green), and nucleus with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). (B) HBEC pre-stimulated with tumour necrosis factor (TNF) for 24 h. (C) HBEC co-cultivated with parasitised red blood cells (pRBCs) during 24 h. (D) HBEC co-cultivated with pRBCs and treated with POH for 24 h. The data are represented as mean ± standard deviation (SD). TCCF: total corrected cellular fluorescence. This graph is representative of three different experiments. **p < 0.01. Scale bar: 50 µm.

Fig. 2:. perillyl alcohol (POH) downmodulates vascular cell adhesion molecule 1 (VCAM-1) upregulation in human brain endothelial cells (HBEC) caused by Plasmodium falciparum. (A) Unstimulated HBEC showed basal expression of VCAM-1 (green), and nucleus with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). (B) HBEC pre-stimulated with tumour necrosis factor (TNF) for 24 h. (C) HBEC co-cultivated with parasitised red blood cells (pRBCs) during 24 h. (D) HBEC co-cultivated with pRBCs and treated with POH for 24 h. The data are represented as mean ± standard deviation (SD). TCCF: total corrected cellular fluorescence. This graph is representative of three different experiments. ***p < 0.001. Scale bar: 50 µm.

Fig. 3:. perillyl alcohol (POH) modulates occludin-1 expression on human brain endothelial cells (HBEC). (A-B) HBEC unstimulated showed a normal ocluddin-1 expression (green) and nucleus with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). (C) HBEC co-cultivated with parasitised red blood cells (pRBCs). (D) HBEC co-cultivated with pRBC and co-treated with 20 µM POH (E-F) HBEC co-cultivated with pRBC and treated with 20 µM of POH, 6 and 12 h after pRBC, respectively. Data are expressed as mean ± standard deviation (SD). TCCF: total corrected cellular fluorescence. This graph is representative of three different experiments. ***p < 0.001; **p < 0.01; NS p >0.05. Scale bar: 40 µm.

Fig. 4:. perillyl alcohol (POH) modulates junctional adhesion molecule A (JAM-A) expression on human brain endothelial cells (HBEC). (A-B) unstimulated HBEC showed a normal JAM-A expression (green) and nucleus with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). (C) HBEC co-cultivated with parasitised red blood cells (pRBCs). (D) HBEC co-cultivated with pRBC and co-treated with 20 µM POH. (E-F) HBEC co-cultivated with pRBC and treated 20 µM of POH, 6 and 12 h after pRBC, respectively. The data are expressed as mean ± standard deviation (SD). TCCF: total corrected cellular fluorescence. This graph is representative of three different experiments. *p < 0.05; NS p > 0.05. Scale bar: 40 µm.

Fig. 5:. perillyl alcohol (POH) prevents vascular endothelial (VE)-cadherin modulation on human brain endothelial cells (HBEC). (A-B) unstimulated HBEC showed a normal VE-cadherin expression (green) and nucleus with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue). (C) HBEC co-cultivated with parasitised red blood cells (pRBCs). (D) HBEC co-cultivated with pRBCs and co-treated with 20 µM POH (E-F) HBEC co-cultivated with pRBCs and treated 20 µM of POH, 6 and 12 h after pRBCs, respectively. The data are expressed as mean ± standard deviation (SD). TCCF: total corrected cellular fluorescence. This graph is representative of three different experiments. ***p < 0.001; NS p > 0.05. Scale bar: 40 µm.

Fig. 6:. perillyl alcohol (POH) increases trans-endothelial electrical resistance (TEER) of Plasmodium falciparum induced human brain endothelial cells (HBEC) TEER was monitored during 72 h using electric cell-substrate impedance sensing (ECIS). HBEC were seeded in 8-well slides and allowed to grow for two to three days, until confluence. Confluent HBEC monolayers were activated with tumour necrosis factor (TNF) (10 ng/mL) for 24 h parasitised red blood cells (pRBCs) were added directly at a ratio of 50 pRBCs/cell and 20 µM of POH was added concomitantly. Histamine (100 mM) was used as a positive control. POH alone on unstimulated cells was used as the control. The grey shaded area represents the period during which pRBCs and other compounds were added to HBEC monolayers. Significant differences relative to pRBC-treated HBEC; ***p < 0.001.

Fig. 7:. perillyl alcohol (POH) reduces the microvesicle (MV) release by Plasmodium falciparum-induced human brain endothelial cells (HBEC). After HBEC and parasitised red blood cells (pRBCs) were co-cultured for 24 h, the supernatant was collected from each well and processed for multiple centrifugations. Pellet was collected and kept at -80ºC until Annexin V-FITC/7-AAD kit (Beckman Coulter) analysis. (A) Histograms of Annexin-V⁺ events from HBEC co-cultivated with pRBC and treated with POH, in the indicated conditions. (B) Quantification of Annexin-V+ events from HBEC treated as in A. Data are represented as mean ± standard deviation (SD). POH alone on unstimulated cells was used as the control. This graph is a combination of five different co-culture experiments. *p < 0.05; NS p > 0,05.