Basement membrane alterarions in kernicteric brain microvasculature and pericyte response to bilirubin

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Kernicterus is a neuropathological condition characterized by deposition of unconjugated bilirubin (UCB) in specific brain regions that can lead to permanent sequelae and death, particularly in premature infants. UCB-induced toxicity has been studied in nerve and glial cells and, more recently, in brain microvascular endothelial cells. However, the effects of UCB on pericytes or on the basement membrane were never reported. We performed in vitro studies to assess apoptotic death, nitrosative stress and inflammatory reaction elicited by human brain vascular pericytes exposed to UCB. We also assessed the basement membrane component, collagen type IV, in brain sections of cortex, basal nuclei,hippocampus and cerebellum, collected at autopsy of a kernicteric preterm newborn. Using the pericyte marker, α-smooth muscle actin, we characterized the cells and confirmed the normal outgrowth towards a typical morphology with long processes. UCB induced an early secretion of interleukin-6, followed by that of vascular endothelial growth factor. mRNA upregulation preceded the secretion and confirmed the precocious profile of IL-6. UCB also caused the release of nitrites, which was maximum at 72 h incubation. The earlier upregulation of endothelial nitric oxide synthase expression confirmed the induction of nitric oxide production by UCB, although not excluding that other isoforms of the enzyme are also involved. Probably as a corolary of all these events, apoptotic cell death occurs in a time- and concentration-dependent manner. Through immunohistochemistry we examined the area occupied and the immunoreactivity of collagen type IV, which were reduced in the kernicterus case as compared with a non-icteric control. These findings are the first to demonstrate the compromise of pericytes and the impairment of collagen IV by hyperbilirubinemia and raise some basis for creation of possible target-directed therapy against pericyte and basement membrane damages as a result of UCB exposure.