Robin Altman

The vascular contribution to Alzheimer's disease.

AD (Alzheimers disease) is a progressive neurodegenerative disease of unknown origin. Despite questions as to the underlying cause(s) of this disease, shared risk factors for both AD and atherosclerotic cardiovascular disease indicate that vascular mechanisms may critically contribute to the development and progression of both AD and atherosclerosis. An increased risk of developing AD is linked to the presence of the apoE4 (apolipoprotein E4) allele, which is also strongly associated with increased risk of developing atherosclerotic cardiovascular disease. Recent studies also indicate that cardiovascular risk factors, including elevated blood cholesterol and triacylglycerol (triglyceride), increase the likelihood of AD and vascular dementia. Lipids and lipoproteins in the circulation interact intimately with the cerebrovasculature, and may have important effects on its constituent brain microvascular endothelial cells and the adjoining astrocytes, which are components of the neurovascular unit. The present review will examine the potential mechanisms for understanding the contributions of vascular factors, including lipids, lipoproteins and cerebrovascular Abeta (amyloid beta), to AD, and suggest therapeutic strategies for the attenuation of this devastating disease process. Specifically, we will focus on the actions of apoE, TGRLs (triacylglycerol-rich lipoproteins) and TGRL lipolysis products on injury of the neurovascular unit and increases in blood-brain barrier permeability.

Inhibition of vascular inflammation by dehydroepiandrosterone sulfate in human aortic endothelial cells: Roles of PPARα and NF-κB

Dehydroepiandrosterone sulfate (DHEAS) is a hormone produced by the adrenal gland and is a precursor for both androgens and estrogens. Atherosclerosis is a well characterized inflammatory disease, but little is known about the role of DHEAS in vascular inflammation. We hypothesize that DHEAS can reduce inflammation in vascular endothelial cells and the mechanism involves the peroxisome proliferator-activated receptor alpha (PPARalpha), thereby inhibiting transcription factors involved in endothelial cell inflammation. To test our hypothesis, aortic endothelial cells were pretreated for 48 h with DHEAS, then with TNF-alpha. TNF-alpha-induced upregulation of the expression of inflammatory genes interleukin (IL)-8 and intracellular adhesion molecule (ICAM)-1 was attenuated by incubation with DHEAS. DHEAS inhibited the TNF-alpha-induced surface expression of vascular cell adhesion molecule (VCAM)-1. This effect was abolished by the addition of MK866, a PPARalpha inhibitor, indicating that PPARalpha is involved in the mechanism of this inhibition. The addition of the aromatase inhibitor letrozole had no effect on the inhibition of TNF-alpha-induced VCAM-1 expression by DHEAS. Treatment of endothelial cells with DHEAS dramatically inhibited the TNF-alpha-induced activation of NF-kappaB, an inflammatory transcription factor, and increased protein levels of the NF-kappaB inhibitor, IkappaB-alpha. These results signify the ability of DHEAS to directly inhibit the inflammatory process and show a potential direct effect of DHEAS on vascular inflammation that has implications for the development of atherosclerotic cardiovascular disease.

Binding of Apolipoprotein E Inhibits the Oligomer Growth of Amyloid-β Peptide in Solution as Determined by Fluorescence Cross-correlation Spectroscopy

Background: ApoE is the most significant risk factor for Alzheimer disease, with known effects on Aβ deposition in the brain. Results: ApoE binds to aggregating Aβ peptides and maintains a faster diffusion rate for the Aβ peptide over time. Conclusion: Binding of apoE to Aβ slows the oligomerization of Aβ. Significance: FCCS measurements quantify isoform-dependent differences in apoE binding to Aβ in solution. One of the primary neuropathological hallmarks of Alzheimer disease is the presence of extracellular amyloid plaques resulting from the aggregation of amyloid-β (Aβ) peptides. The intrinsic disorder of the Aβ peptide drives self-association and progressive reordering of the conformation in solution, and this dynamic distribution of Aβ complicates biophysical studies. This property poses a challenge for understanding the interaction of Aβ with apolipoprotein E (apoE). ApoE plays a pivotal role in the aggregation and clearance of Aβ peptides in the brain, and the ϵ4 allele of APOE is the most significant known genetic modulator of Alzheimer risk. Understanding the interaction between apoE and Aβ will provide insight into the mechanism by which different apoE isoforms determine Alzheimer disease risk. Here we applied alternating laser excitation fluorescence cross-correlation spectroscopy to observe the single molecule interaction of Aβ with apoE in the hydrated state. The diffusion time of freely diffusing Aβ in the absence of apoE shows significant self-aggregation, whereas in the presence of apoE, binding of the protein results in a more stable complex. These results show that apoE slows down the oligomerization of Aβ in solution and provide direct insight into the process by which apoE influences the deposition and clearance of Aβ peptides in the brain. Furthermore, by developing an approach to remove signals arising from very large Aβ aggregates, we show that real-time single particle observations provide access to information regarding the fraction of apoE bound and the stoichiometry of apoE and Aβ in the complex.

Postprandial VLDL lipolysis products increase monocyte adhesion and lipid droplet formation via activation of ERK2 and NFκB

Postprandial lipemia is characterized by a transient increase in circulating triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL) and has been shown to activate monocytes in vivo. Lipolysis of VLDL releases remnant particles, phospholipids, monoglycerides, diglycerides, and fatty acids in close proximity to endothelial cells and monocytes. We hypothesized that postprandial VLDL lipolysis products could activate and recruit monocytes by increasing monocyte expression of proinflammatory cytokines and adhesion molecules, and that such activation is related to the development of lipid droplets. Freshly isolated human monocytes were treated with VLDL lipolysis products (2.28 mmol/l triglycerides + 2 U/ml lipoprotein lipase), and monocyte adhesion to a primed endothelial monolayer was observed using a parallel plate flow chamber coupled with a CCD camera. Treated monocytes showed more rolling and adhesion than controls, and an increase in transmigration between endothelial cells. The increased adhesive events were related to elevated expression of key integrin complexes including Mac-1 [α(m)-integrin (CD11b)/β2-integrin (CD18)], CR4 [α(x)-integrin (CD11c)/CD18] and VLA-4 [α4-integrin (CD49d)/β1-integrin (CD29)] on treated monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes with VLDL lipolysis products increased expression of TNFα, IL-1β, and IL-8 over controls, with concurrent activation of NFkB and AP-1. NFκB and AP-1-induced cytokine and integrin expression was dependent on ERK and Akt phosphorylation. Additionally, fatty acids from VLDL lipolysis products induced ERK2-dependent lipid droplet formation in monocytes, suggesting a link to inflammatory signaling pathways. These results provide novel mechanisms for postprandial monocyte activation by VLDL lipolysis products, suggesting new pathways and biomarkers for chronic, intermittent vascular injury.

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide.

Background The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimers disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species. Methodology/Principal Findings To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species. Conclusions Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.

Testosterone-derived estradiol production by male endothelium is robust and dependent on p450 aromatase via estrogen receptor alpha

Vascular endothelium expresses both the estrogen receptors (ERs) α and β, and ERα mediates development of early atherosclerosis in male mice. This process is thought to be testosterone-dependent. We hypothesized that male murine aortic endothelium produces robust levels of estradiol by aromatase conversion of testosterone, and that regulation of this process is mediated by the presence of ERs, primarily ERα. Aortic endothelium was isolated from ERα knockout (ERα -/-) and wild-type (ERα +/+) male mice and treated with testosterone or the 5α reduction product dihydrotestosterone (DHT), with or without the P450 aromatase inhibitor anastrazole, or a non-specific estrogen receptor antagonist. Aromatase gene expression and estradiol production were assayed. Treatment with testosterone, but not DHT, caused increased aromatase expression and estradiol production in ERα +/+ endothelium that was attenuated by disruption of ERα in the ERα -/- group. Anastrazole inhibition of aromatase reduced testosterone-induced aromatase expression and estradiol levels in both ERα -/- and ERα +/+ endothelium. Antagonism of both ERs decreased testosterone-induced aromatase expression in both wild-type and knockout groups. The effects of the receptor antagonist on estradiol production differed between the two groups, however, with a reduction in estradiol release from the ERα +/+ cells and complete abolition of estradiol release from the ERα -/- cells. Thus, estradiol production in vascular endothelium from male mice is robust, depends on the aromatic conversion of testosterone and requires functional ERα to achieve maximal levels of estradiol generation. Local vascular production of aromatase-mediated estradiol in response to circulating testosterone may affect ERα-dependent mechanisms to increase susceptibility to early atheroma formation in male mice. This pathway may have important therapeutic relevance for reducing the risk of atherosclerotic cardiovascular disease in human males.

The Postprandial Effects of a Moderately High-Fat Meal on Lipid Profiles and Vascular Inflammation in Alzheimer's Disease Patients: A Pilot Study.

Objective Alzheimer’s disease (AD) is a neurodegenerative disease of aging with unknown causative factors. Accumulating evidence suggests that inflammation and neurovascular dysfunction play important roles in AD. The postprandial period following a moderately high-fat meal is associated with vascular inflammation in young, healthy individuals; however, this relationship has not been investigated in Alzheimer’s patients despite their exaggerated inflammatory state. Methods Patients with AD and age-matched control subjects were recruited through the UC Davis Alzheimer’s Disease Center. All subjects consumed a moderately high-fat breakfast meal. Fasting and postprandial blood samples were collected for lipid, lipoprotein, and oxylipin analyses, as well as assays for cytokine levels and monocyte activation. Results The plasma lipid analyses revealed similar levels of triglycerides and esterified oxylipins between groups, but there was an interaction between postprandial non-esterified fatty acid (NEFA) levels and body mass index in the AD group compared to the control subjects. The AD group also had increased behenic acid and decreased linoleic and oleic acids in the postprandial period; however, these were not significantly different. Inflammatory assays revealed elevated fasting levels of interleukin (IL)-10 and IL-12 p70, but no change in monocyte activation in the AD group. Conclusion The postprandial period following a moderately high-fat meal is not associated with an exaggerated inflammatory state in Alzheimer’s patients, and basal esterified oxylipin profiles do not indicate elevated oxidative stress. However, the baseline inflammatory state during fasting in AD patients includes elevated levels of plasma IL-10 and IL-12 p70, which may indicate a balance between immune responses mediated by these interleukins.

Synthesis and functional survey of new Tacrine analogs modified with nitroxides or their precursors

A series of new Tacrine analogs modified with nitroxides or pre-nitroxides on 9-amino group via methylene or piperazine spacers were synthesized; the nitroxide or its precursors were incorporated into the Tacrine scaffold. The new compounds were tested for their hydroxyl radical and peroxyl radical scavenging ability, acetylcholinesterase inhibitor activity and protection against Aβ-induced cytotoxicity. Based on these assays, we conclude that Tacrine analogs connected to five and six-membered nitroxides via piperazine spacers (9b, 9b/HCl and 12) exhibited the best activity, providing direction for further development of additional candidates with dual functionality (anti Alzheimers and antioxidant).

Protective spin-labeled fluorenes maintain amyloid beta peptide in small oligomers and limit transitions in secondary structure

Alzheimers disease is characterized by the presence of extracellular plaques comprised of amyloid beta (Aβ) peptides. Soluble oligomers of the Aβ peptide underlie a cascade of neuronal loss and dysfunction associated with Alzheimers disease. Single particle analyses of Aβ oligomers in solution by fluorescence correlation spectroscopy (FCS) were used to provide real-time descriptions of how spin-labeled fluorenes (SLFs; bi-functional small molecules that block the toxicity of Aβ) prevent and disrupt oligomeric assemblies of Aβ in solution. Furthermore, the circular dichroism (CD) spectrum of untreated Aβ shows a continuous, progressive change over a 24-hour period, while the spectrum of Aβ treated with SLF remains relatively constant following initial incubation. These findings suggest the conformation of Aβ within the oligomer provides a complementary determinant of Aβ toxicity in addition to oligomer growth and size. Although SLF does not produce a dominant state of secondary structure in Aβ, it does induce a net reduction in beta secondary content compared to untreated samples of Aβ. The FCS results, combined with electron paramagnetic resonance spectroscopy and CD spectroscopy, demonstrate SLFs can inhibit the growth of Aβ oligomers and disrupt existing oligomers, while retaining Aβ as a population of smaller, yet largely disordered oligomers.

Postprandial apoE Isoform and Conformational Changes Associated with VLDL Lipolysis Products Modulate Monocyte Inflammation

Objective Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation. Methods and Results We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4. Conclusion Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.