Impact of altered glycaemia on blood-brain barrier endothelium: an in vitro study using the hCMEC/D3 cell line.
BACKGROUND
Cerebrovascular complications involving endothelial dysfunction at the blood-brain barrier (BBB) are central to the pathogenesis of diabetes-related CNS disorders. However, clinical and experimental studies have reported contrasting evidence in relation to the effects of hyperglycemia on BBB permeability and function. Similarly the effect of hypoglycemia on BBB integrity is not well understood. Therefore, we assessed the differential impact of hypo and hyperglycemic conditions on BBB integrity and endothelial function in vitro using hCMEC/D3, a well characterized human brain microvascular endothelial cell line.


METHODS
Parallel monolayers of hCMEC/D3 were exposed to normal, hypo- or hyperglycemic media, containing 5.5, 2.2 or 35 mM D-glucose, respectively. Following 3-24h exposure, the expression and distribution of BBB tight junction (ZO-1 and claudin-5) adherence junction (VE-cadherin) proteins, and glucose transporters as well as inflammatory (VCAM-1) and oxidative stress (Nrf-2) markers were analyzed by immunofluorescence and western blotting. Endothelial release of growth factors and pro-inflammatory cytokines were determined by ELISA. Further, the impact of altered glycemia on BBB permeability was assessed in hCMEC/D3 - astrocyte co-cultures on Transwell supports using fluorescent dextrans (4-70 kDa).


RESULTS
Compared to controls, exposure to hypoglycemia (3 and 24h) down-regulated the expression of claudin-5 and disrupted the ZO-1 localization at cell-cell contacts, while hyperglycemia marginally reduced claudin-5 expression without affecting ZO-1 distribution. Permeability to dextrans (4-10 kDa) and VEGF release at 24h were significantly increased by hypo- and hyperglycemia, although 70 kDa dextran permeability was increased only under hypoglycemic conditions. The expression of SGLT-1 was up-regulated at 24h hypoglycemic exposure while only a modest increase of GLUT-1 expression was observed. In addition, the expression of Nrf-2 and release of interleukin-6 and PDGF-BB, were down-regulated by hypoglycemia (but not hyperglycemia), while both conditions induced a marginal and transient increase in VCAM-1 expression from 3 to 24h, including a significant increase in VE-cadherin expression at 3 h following hyperglycemia.


CONCLUSIONS
In summary, our findings demonstrate a potential impairment of BBB integrity and function by hypo or hyperglycemia, through altered expression/distribution of TJ proteins and nutrient transporters. In addition, hypoglycemic exposure severely affects the expression of oxidative and inflammatory stress markers of BBB endothelium.
Ravi K
RK
Sajja
Shikha
S
Prasad
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S, Coulter Street, Amarillo, TX 79106, USA. luca.cucullo@ttuhsc.edu.
Luca
L
Cucullo