Is There a Link Between Alzheimer’s and Atherosclerosis?
PI- Paula Grammas, PhD; $900,000, 01/15/04 - 12/31/07
Alzheimer's disease (AD) is a neurodegenerative disease that affects more than 4 million Americans. We are the first to demonstrate that brain blood vessels release neurotoxic proteins in AD. However, the factors that cause this vessel dysfunction are not known. It is our hypothesis that risk factors involved in the pathogenesis of atherosclerosis are also causally linked to the development of vascular-mediated neuronal cell death in AD. Our studies are timely and important as increasing evidence points to a link between atherosclerosis and AD. Aim 1: To determine the effects of systemic oxidant stress or hyperlipidemia on vascular thrombin release, vascular-mediated neurotoxicity and on the cognitive performance of apoE transgenic mice. Brain blood vessels isolated from apoE knock-out or transgenic mice expressing human E3 or E4 are used to assess the role of apoE isoforms on vascular expression of thrombin. Diet-induced hyperhomocystinemia and hyperlipidemia, are used to assess the role of oxidant stress and lipids, respectively, on vascular thrombin release and vascular-mediated neurotoxicity. Also, these transgenic mice are utilized to evaluate possible apoE isoform-specific effects of oxidant and lipid stress on impairments in learning and memory. Aim 2: To determine if risk factors involved in the pathogenesis of atherosclerosis are also causally linked to the development of vascular-mediated neuronal cell death in AD. Brain microvessels are isolated from AD patients and non-demented patients and analyzed for levels and/or activity of thrombin and other possible neurotoxic proteins, including, matrix metalloproteinases (MMPs), inflammatory cytokines and chemokines, and endothelin-l. Protein levels are determined by ELISA and Western blots and mRNA levels assessed by Northern blots and RT-PCR. The role that apoE isoforms play in regulating these proteins is determined by comparing microvessels isolated from patients with different APOE genotypes. In vitro addition of oxygen species or lipid molecules to isolated brain microvessels is used to assess the effects of oxidative stress and lipids, respectively, on release of thrombin, MMPs, inflammatory proteins, and endothelin-l. Apoptosis and necrosis are measured in cultured neuronal cells exposed to these proteins. These results would, for the first time, identify a mechanistic cascade linking cardiovascular risk factors to vascular-mediated neuronal cell death in AD and identify novel targets for therapeutic intervention.
Vascular Inflammation in the Aging Brain
PI- Paula Grammas, PhD; $1.1 million 08/01/06 - 07/31/10
Despite the fact that many neurodegenerative diseases are age-related disorders, how aging predisposes the CNS to the development of neurodegenerative pathologies has not been adequately addressed. Numerous studies have documented an increase in inflammatory proteins in neurodegenerative diseases. However, whether age-related changes in inflammatory mediators are causally-linked to age-related disease progression is unknown. Our laboratory has shown that in Alzheimer's disease a pro-inflammatory cerebral vasculature releases neurotoxins and likely contributes to neuronal cell death. It is our hypothesis that age-related inflammatory changes in brain blood vessels contribute to age-related pathology in the brain. Because neuronal cell death is a central lesion in age-associated neurodegenerative diseases, we postulate a chain of events linking age-related increases in inflammation with vascular-mediated neuronal cell death. Aim 1- To determine if expression of inflammatory proteins increases in the cerebral vasculature with age. Fischer 344 rats at 6,12, 18, and 24 months of age are used to determine whether aging, in the absence of disease, affects vascular expression of inflammatory cytokines (IL-1β, IL-6, IL-8, TNFα) and chemokines (RANTES, MCP-1, MIP1-α). Inflammatory proteins are determined by ELISA, western and Northern blots, and real-time PCR and RT-PCR in isolated brain microvessels and by immunohistochemistry on tissue sections. Aim 2- To determine if age-related inflammatory changes are causally-linked to vascular-mediated neuronal cell death. Our model predicts that in aging vascular inflammation in the brain contributes to vascular-mediated neuronal cell death after injury. The cerebral microvasculature of Fischer 344 rats at 6,12, 18, and 24 months is exposed to lipid or oxidative stress using both in vivo and in vitro injury models and vascular-mediated neuronal cell death assessed. Administration of anti-inflammatory drugs prior to injury is used to determine whether inhibiting inflammation prevents release of neurotoxic factors. These results would, for the first time, identify a mechanistic cascade linking age-related inflammatory changes to vascular-mediated neuronal cell death. Results from this project could highlight a heretofore-unappreciated aspect of aging and suggest that approaches aimed at minimizing vascular inflammation and maintaining brain vascular function could be important for successful brain aging.
Vascular-mediated Neuronal Death in Alzheimer's
PI- Paula Grammas, PhD; $1.25 million, 09/30/99 - 08/31/05
PI- Paula Grammas, PhD; $1.25 million, 07/01/07 - 04/30/12
Data are emerging to support the idea that factors and processes characteristic of angiogenesis are found in the Alzheimer disease (AD) brain. We have shown that in AD microvessels express or release many inflammatory, proangiogenic proteins. Despite increases in proangiogenic factors in the AD brain, evidence for increased vascularity is lacking. In our model we hypothesize that the angiogenic process does not progress to new vessel growth because an imbalance of pro- and anti-angiogenic factors results in aborted angiogenic signaling. In this project we test the hypothesis that AD microvessels express an angiogenic phenotype and that this abnormal activation of brain endothelial cells is important for the development of AD pathology. Aim 1: To test the hypothesis that in AD brain microvessels become activated but fail to complete angiogenesis because an imbalance of pro- and anti-angiogenic factors results in aborted angiogenic signaling. Brain microvessels are isolated from AD patients, age-matched non-demented controls, and patients with inflammatory and non-inflammatory CNS disease. Isolated vessels are compared for expression of pro- and anti-angiogenic factors including thrombin, VEGF, endothelin-1 TGF-β, nitric oxide, thrombospondin, and amyloid beta (Aβ). The activities of signaling kinases phosphatidylinositol-3 kinase (PI3K)/Akt, p38 kinase, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) are measured. Immunohistochemistry of brain sections is used to assess the spatial correlation of pro- and anti-angiogenic proteins with Aβ deposition and AD pathology. Aim 2: To test the hypothesis that acquisition of the angiogenic phenotype contributes to the pathogenesis of AD pathology and cognitive impairment in animal models of AD. To determine the temporal association between acquisition of the angiogenic phenotype and the onset of disease, markers of angiogenesis are measured in isolated brain microvessels obtained from AD transgenic mice before the onset of cognitive changes and AD pathology and at several ages during disease progression. A causal link between the angiogenic phenotype and disease progression is evaluated using antiangiogenic drugs. Administration of these drugs to animals prior to the onset of behavioral changes and AD pathology will determine whether inhibiting the angiogenic phenotype affects the course of disease. Taken together, data from Aim 1 showing the clinical relevance of angiogenic changes in AD and results from Aim 2 demonstrating a causal link between the angiogenic phenotype and disease progression would argue strongly for a new therapeutic approach in AD. These results could be very exciting because the angiogenic brain endothelial cell is a novel, unexplored therapeutic target, and several antiangiogenic drugs are currently in use in Phase III clinical trials. Thus, clinical studies with angiogenesis inhibitors could be rapidly designed and implemented in AD patients.