VEGF receptor-1 involvement in pericyte loss induced byEscherichia coliin anin vitromodel of blood brain barrier

Abstract

SummaryThe key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood-brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co-culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co-culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca 2+-independent phospholipase A2 (cPLA2 and iPLA2) enzyme activities. In supernatants of E. coli-stimulated co-cultures, an increase in prostaglandins (PGE2) and VEGF production in comparison with untreated cocultures were found. Incubation with E. coli in presence of AACOCF3 or BEL caused a decrease of PGE2 and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR-1 but not VEGFR-2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR-1, but not VEGFR-2 blockade, or in presence of AACOCF3 or BEL, elevated TEER values, reduced permeability and BRPC loss were found. These data suggest that VEGFR-1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.

Klebsiella pneumoniae Induces an Inflammatory Response in an In Vitro Model of Blood-Retinal Barrier

“…The presence of K. pneumoniae induced no increase in the VEGF secretion by BRPC. These data are in agreement with the results of our previous study showing that, in brain microvascular endothelial cells, PGE 2 action was associated with endothelial VEGF release (31). Moreover, in the untreated cocultures, the amount of VEGF was greater than the predicted sum of that produced in solo cultures.…”

“…Our previous studies have shown the role of PLA 2 s, prostaglandins, and VEGF release in governing the penetration of Escherichia coli into the brain (30,31). Recognizing the importance of retinal endothelial cells and pericytes as a barrier to endogenous infectious agents in human endophthalmitis, in this study, we investigated the effects of K. pneumoniae on these cells.…”

Pathogens Penetrating the Central Nervous System: Infection Pathways and the Cellular and Molecular Mechanisms of Invasion

“…These effects were not observed when the pericyte vascular endothelial growth factor receptor 1 was blocked using a polyclonal antibody. These findings suggest that the vascular endothelial growth factor released by brain endothelial cells may bind to the vascular endothelial growth factor receptor 1 on adjacent pericytes and trigger ablation of the pericytes from the basal membrane, potentially opening a paracellular route (119).…”

“…Primary human, rodent, and bovine brain microvascular endothelial cells have been used in some studies (97,(117)(118)(119); however, the isolation of these cells is technically demanding, and in vitro culture dramatically alters the transcriptome, leading to downregulation of genes involved in BBB function (120,121). Human umbilical vein endothelial cells (HUVECs) (122)(123)(124), Caco-2 cells, and Madin-Darby canine kidney cells (125) have been used as surrogate models of the BBB, although the noncerebral origin of these cells may limit their relevance.…”

“…Furthermore, the activation of host cytosolic phospholipase A 2 by E. coli OmpA and Nlp1 may play a role in reducing the integrity of the BBB. Using cocultures of primary BBMECs and primary bovine retinal pericytes seeded onto Transwell membranes, Salmeri et al demonstrated that the arachidonic acid released following cytosolic phospholipase A 2 activation acts as a substrate for cyclooxygenase to produce prostaglandins, which trigger the synthesis of vascular endothelial growth factor by brain endothelial cells (119). This was associated with a significant decrease in transendothelial electrical resistance and a dramatic detachment of pericytes from the Transwell membrane.…”