Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers

Abstract

Background The blood–brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group’s previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC. Methods We used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC’s impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC’s effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5. Results Our data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data. Conclusion In agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

Blood–Brain Barrier Transporters: Opportunities for Therapeutic Development in Ischemic Stroke

“…The release of ROS from smoking that causes inflammation also promotes considerable BBB impairment [235]. Nicotine from tobacco smoke leads to increased BBB permeability through disruption of endothelial cell tight junctions; specifically, this is thought to occur due to a modified distribution of tight junction scaffolding protein zonula occludens-1 (ZO-1) [236][237][238]. The integrity of the cerebral microvasculature can be further compromised if a smoking-induced increase in blood viscosity occurs in tandem with BBB tight junction disruption [239].…”

A Quasi-Physiological Microfluidic Blood-Brain Barrier Model for Brain Permeability Studies

“…The barrier function of some recently published iPSC-based BBB microfluidic models was reported to be comparable to in vivo values [17,18,21,32]. These data were obtained by correlating the permeability coefficients of dextran in these models against rat pial post-capillaries using imaging studies [22].…”

“…We assessed the passage of [ 13 C 12 ] sucrose and [ 13 C 6 ] mannitol, known as one of the ideal paracellular permeability markers [23,32], across iBMEC cell monolayers cultured in single and triculture models under a continuous flow condition (0.15 dyne/cm 2 ) to investigate the difference in barrier properties. Results show that the permeability of [ 13 C 12 ] sucrose and [ 13 C 6 ] mannitol across the single culture of the iBMEC monolayer was 1.883 × 10 −6 ± 3.763 × 10 −7 and 2.380 × 10 −6 ± 5.738 × 10 −7 cm/s (n = 3 biological replicates), respectively, after 2 days in the culture, while the permeability of sucrose and mannitol in the triculture was 4.923 × 10 −7 ± 1.187 × 10 −7 and 6.760 × 10 −7 ± 1.071 × 10 −7 cm/s (n = 3 biological replicates), respectively (see Figure 3A,B).…”

Metformin Preserves VE–Cadherin in Choroid Plexus and Attenuates Hydrocephalus via VEGF/VEGFR2/p-Src in an Intraventricular Hemorrhage Rat Model

“…For example, tight junction proteins, such as ZO-1 and claudin-5 of ChP, may also contribute to BBB permeability. Metformin was reported to decrease BBB permeability via increasing claudin-5 expression and restoring ZO-1 distribution [ 61 ]. Besides, activated by NF-κB dependent inflammatory signal, the potassium cotransporter in ChP, NKCC1, was reported to be upregulated after IVH, accompanied with the CSF hypersecretion, which may also make sense in metformin’s protection in IVH-induced hydrocephalus [ 62 ].…”