We have shown that in isolated peripheral mammalian artery, freshly solubilized B-amyloid (Ar) peptides stimulate a pro-inflammatory pathway (sPLA2-MAPK-cPLA2-AA-LOX/COX) involving activation of secretory phospholipase A2 (PLA2), mitogen-activated protein kinase (MAPK) kinase (MEKI/2), p38 MAPK, cytosolic PLA2 (cPLA2), and the release of arachidonic acid. Arachidonic acid (AA) is metabolized into pro-inflammatory eicosanoids via the 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) enzymes. We also have data that strongly support the AB-induced activation of this pathway in rodent and bovine cerebrovasculature when challenged by freshly solubilized AB. We have also shown that elements of the pathway are present and have basal activity in microcerebrovessels from autopsied Alzheimer's (AD) brains, which can be reduced by blocking elements of the pathway. Furthermore, challenging these microvessels with AB increases the eicosanoid products of the pathway, an effect that again, can be blocked by inhibiting pathway enzymes. We have also shown that AB stimulates the release of eicosanoids and AA in human cerebrovascular smooth muscle cells, confirming that AB activates PLA2 in these cells. These findings also explain our previous observations that AB can dramatically enhance the endothelin-l (ET-l)-induced vasoconstriction in mammalian vessels via activation of this pathway. Taken together, our data show that soluble AB peptides can trigger a pro-inflammatory reaction in vitro and ex vivo in the vasculature. These findings are consistent with our hypothesis: that Afi can activate this sPLA2-MAPK-cPLA3-AA-LOX/COX pathway In the cerebrovasculature In Al) brains. The purpose of this application is to quantify the extent to which AB can activate this proinflammatory pathway in the cerebrovasculature in life. Taken together Specific aims 1 and 3 will compare basal amounts and activity of elements of the pathway between normal and AD cerebral vessels. To determine whether chronic exposure to AS sensitizes the cerebrovasculature to additional AS stimulation, normal and AD cerebral vessels will be further challenged with AB.
Specific AIm 2 provides for the culture of human cerebral vessel endothelial and smooth muscle cells derived from AD and control cases. Examination of the effects of AS stimulation on the pathway will be determined in these cells.
Specific Aim 4 will examine the relationship between AS and basal amounts/activity of elements of the pathway in the cerebrovasculature of transgenic and control mice with age. The transgenic animals we have chosen (TgAPPsw) are a model of increasing levels of soluble (and insoluble) cerebral and cerebrovascular AS. Finally, in specific aim 5 we wish to examine the effects of stimulation and inhibition of the pathway on BAPP expression and metabolism. The sub-hypothesis tested here wills that the AS-activated pathway further increases AS production in the vasculature. As a body of knowledge, these findings will clarify the importance of AS's ability to stimulate this fundamental inflammatory pathway in the cerebrovasculature in Alzheimer's disease. Furthermore, it is likely to provide valuable insights into the early mechanisms of the pathogenesis of Cerebral Amyloid Angiopathy.
