Selective antihypertensive dihydropyridines lower Aß accumulation by targeting both the production and the clearance of Aß across the blood-brain barrier.

Several large population-based or clinical trial studies have suggested that certain dihydropyridine (DHP) L-type calcium channel blockers (CCBs) used for the treatment of hypertension may confer protection against the development of Alzheimer disease (AD). However, other studies with drugs of the same class have shown no beneficial clinical effects. To determine whether certain DHPs are able to impact underlying disease processes in AD (specifically the accumulation of the Alzheimer Aß peptide), we investigated the effect of several antihypertensive DHPs and non-DHP CCBs on Aß production. Among the antihypertensive DHPs tested, a few, including nilvadipine, nitrendipine and amlodipine inhibited Aß production in vitro, whereas others had no effect or raised Aß levels. In vivo, nilvadipine and nitrendipine acutely reduced brain Aß levels in a transgenic mouse model of AD (Tg PS1/APPsw) and improved Aß clearance across the blood-brain barrier (BBB), whereas amlodipine and nifedipine were ineffective showing that the Aß-lowering activity of the DHPs is independent of their antihypertensive activity. Chronic oral treatment with nilvadipine decreased Aß burden in the brains of Tg APPsw (Tg2576) and Tg PS1/APPsw mice, and also improved learning abilities and spatial memory. Our data suggest that the clinical benefit conferred by certain antihypertensive DHPs against AD is unrelated to their antihypertensive activity, but rely on their ability to lower brain Aß accumulation by affecting both Aß production and Aß clearance across the BBB.

Anti-Tumoral Activity of a Short Decapeptide Fragment of the Alzheimer's Abeta Peptide.

The inhibition of angiogenesis is regarded as a promising avenue for cancer treatment. Although some antiangiogenic compounds are in the process of development and testing, these often prove ineffective in vivo, therefore the search for new inhibitors is critical. We have recently identified a ten amino acid fragment of the Alzheimer Abeta peptide that is anti-angiogenic both in vitro and in vivo. In the present study, we investigated the antitumoral potential of this decapeptide using human MCF-7 breast carcinoma xenografts nude mice. We observed that this decapeptide was able to suppress MCF-7 tumor growth more potently than the antiestrogen tamoxifen. Inhibition of tumor vascularization as determined by PECAM-1 immunostaining and decreased tumor cell proliferation as determined by Ki67 immunostaining were observed following treatment with the Abeta fragment. In vitro, this peptide had no direct impact on MCF-7 tumor cell proliferation and survival suggesting that the inhibition of tumor growth and tumor cell proliferation observed in vivo is related to the antiangiogenic activity of the peptide. Taken together these data suggest that this short Abeta derivative peptide may constitute a new antitumoral agent.

Serum beta-amyloid correlates with neuropsychological impairment.

AIMS: Evidence suggests a relationship between peripheral Abeta and AD. We hypothesized that higher levels of serum Abeta(1-42) would be associated with memory impairment, thought to occur early in the disease, and rises in serum Abeta(1-40), which occur later, would be associated with impairment in non-memory measures. METHODS: Using a cross-sectional design, we examined the relationship of serum Abeta(1-40), Abeta(1-42), and the ratio of Abeta(1-42/1-40) to neuropsychological measures in 40 cognitively normal controls, 13 MCI subjects, and 25 AD patients. RESULTS: Serum Abeta(1-42) and the ratio of Abeta(1-42/1-40) were significantly higher in the MCI group compared to the controls. A significant relationship in the hypothesized direction (poorer scores associated with higher Abeta(1-40) serum levels) was found between Abeta(1-40) and measures of executive functions across the entire cohort of individuals tested and with measures of language and processing speed in the AD group. Regression analysis found that neuropsychological measures accounted for 26% of the variance in serum Abeta(1-40,) in the MCI/AD but not the controls. Furthermore that language and executive measures were significant predictors. CONCLUSIONS: Results provide preliminary data to partially support our hypotheses and suggest that changes in serum Abeta levels may be attributed to pathological changes within the brain.

Diagnostic utility of APOE, soluble CD40, CD40L, and Abeta1-40 levels in plasma in Alzheimer's disease.

A continuous inflammatory state is associated with Alzheimer's disease (AD) evidenced by an increase in proinflammatory cytokines around beta-amyloid (Abeta) deposits. In addition, functional loss of CD40L is shown to result in diminished Amyloid precursor proton (APP) processing and microglial activation, supporting a prominent role of CD40-CD40L in AD etiology. We therefore hypothesize that a peripheral increase in Abeta may result in corresponding increase of sCD40 and sCD40L further contributing to AD pathogenesis. We measured plasma Abeta, sCD40 and sCD40L levels in 73 AD patients and compared to 102 controls matched on general demographics. We demonstrated that Abeta(1-40), levels of sCD40 and sCD40L are increased in AD and declining MMSE scores correlated with increasing sCD40L, which in turn, correlated positively with Abeta(1-42). We then combined sCD40, sCD40L, Abeta and APOE and found that this biomarker panel has high sensitivity and specificity (>90%) as a predictor of clinical AD diagnosis. Given the imminent availability of potentially disease modifying therapies for AD, a great need exists for peripheral diagnostic markers of AD. Thus, we present preliminary evidence for potential usefulness for combination of plasma sCD40, sCD40L along with Abeta(1-40) and APOE epsilon4 in improving the clinical diagnosis of AD.

Potent anti-angiogenic motifs within the Alzheimer beta-amyloid peptide.

Abeta peptides are the major constituents of senile plaques and cerebrovascular deposits in the brains of patients with Alzheimer's disease. We have shown previously that Abeta1-40 and Abeta1-42 peptides are potently anti-angiogenic both in vitro and in vivo. The current study characterizes important sequences within the Abeta peptide that are required to exert its anti-angiogenic activity. We have used human umbilical vein endothelial cells to assess the anti-angiogenic activity of short fragments of Abetain vitro in a Matrigel network assay and in vivo in a rat corneal model of angiogenesis. The anti-angiogenic activity of these short peptide fragments is not related to effects on apoptosis or necrosis. Using normal and mutated peptide fragments, we show that the sequence VHHQKLVFF is sufficient to exhibit potent anti-angiogenic effects. This small peptide may therefore have clinical relevance as an anti-angiogenic agent.

The influence of diagnosis, intra- and inter-person variability on serum and plasma Abeta levels.

Evidence suggests that high peripheral beta-amyloid (Abeta)(1-40) levels and low ratios of Abeta(1-42)/Abeta(1-40) are associated with increased risk for Alzheimer's disease (AD). In this cross-sectional design, serum and plasma samples from 67 AD patients and 146 controls (similar in age and gender) were evaluated using Abeta(1-40) and Abeta(1-42) ELISA. Coefficient of variance was calculated for intra- and inter-person variability of Abeta(1-40) and Abeta(1-42). Abeta(1-40) correlated with age, MMSE and their Abeta(1-42)/Abeta(1-40) ratios (p<0.05). Significantly higher Abeta(1-40) levels were observed in AD patients than controls (p<0.05) but no difference was observed for Abeta(1-42) (p>0.05). Serum Abeta(1-42)/Abeta(1-40) ratios were also significantly lower in AD patients than controls (p<0.05). Lower intra-person than inter-person variability was observed for serum and plasma Abeta(1-40) and Abeta(1-42) and these were higher in controls than in AD patients. The intra-person variability of serum Abeta(1-40) did not influence the group differences observed between AD patients and controls. Significant interaction was observed between diagnosis and intra-person variability for serum Abeta(1-40) levels (p<0.05) and was supported by our finding of higher intra-person variability for serum Abeta(1-40) in controls (26.97%) than in AD patients (18.35%). We confirm the previously observed differences in blood Abeta levels between AD and control groups. In addition, we now report the presence of high intra- and inter-person variability possibly due to factors that influence peripheral Abeta levels and warrant further investigation before the potential use of Abeta as an AD biomarker can be fully exploited.

Inhibition of Abeta production by NF-kappaB inhibitors.

The transcription factor nuclear factor kappaB (NF-kappaB) is widely expressed in the nervous system and increased NF-kappaB immunoreactivity has been observed in Alzheimer's disease (AD) brains in the nuclei of neurons within the vicinity of diffuse beta-amyloid plaques. Beta-amyloid (Abeta) peptides are the main constituent of senile plaques and are known to stimulate NF-kappaB activity. In the present study, we investigated the effect of various NF-kappaB inhibitors on the production of Abeta1-40, Abeta1-42, secreted APP (sAPPbeta and sAPPalpha) and APP C-terminal fragments (APP-CTF) using CHO cells overexpressing the beta-amyloid precursor protein (APP). Our data show that NF-kappaB inhibitors decrease both Abeta1-40 and Abeta1-42 production. In addition, we show that some NF-kappaB inhibitors decrease sAPPbeta and APP-CTFbeta suggesting that they reduce the beta-secretase cleavage of APP. Altogether our data suggest that NF-kappaB inhibitors may be of therapeutic importance for the treatment of AD pathology not only by blocking inflammatory processes but also by directly inhibiting the production of Abeta peptides.

Genomic regulation after CD40 stimulation in microglia: relevance to Alzheimer's disease.

Key pathological processes in Alzheimer's disease (AD) include the accumulation of amyloid beta peptide (Abeta) which, in excess, triggers pathological cascades including widespread inflammation, partly reflected by chronic microglial activation. It has previously been suggested that CD40/CD40L interaction promotes AD like pathology in transgenic mice. Thus, amyloid burden, gliosis and hyperphosphorylation of tau are all reduced in transgenic models of AD lacking functional CD40L. We therefore hypothesized that cellular events leading to altered APP metabolism, inflammation and increased tau phosphorylation underlying these observations would be regulated at the genomic level. In the present report, we used the Affymetrix (GeneChip) oligonucleotide microarray U133A to gain insight into the global and simultaneous transcriptomic changes in response to microglia activation after CD40/CD40L ligation. As expected, regulation of elements of the NF-kappaB signaling, chemokine and B cell signaling pathways was observed. Taken together, our data also suggest that CD40 ligation in human microglia specifically perturbs many genes associated with APP processing.

Model of Alzheimer's disease amyloid-beta peptide based on a RNA binding protein.

Although Alzheimer's Abeta peptide has been shown to form beta-sheet structure, a high-resolution molecular structure is still unavailable to date. A search for a sequence neighbor using Abeta(10-42) as the query in the Protein Data-Bank (PDB) revealed that an RNA binding protein, AF-Sm1 from Archaeoglobus fulgidus (PDB entry: 1i4k chain Z), shared 36% identical residues. Using AF-Sm1 as a template, we built a molecular model of Abeta(10-42) by applying comparative modeling methods. The model of Abeta(10-42) contains an antiparallel beta-sheet formed by residues 16-23 and 32-41. Hydrophobic surface constituted by residues 17-20 (LVFF) separates distinctly charged regions. Residues that interact with RNA in the AF-Sm1 crystal structure were found to be conserved in Abeta. Using a native gel we demonstrate for the first time that RNA can interact with Abeta and selectively retard the formation of fibrils or higher-order oligomers. We hypothesize that in a similar fashion to AF-Sm1, RNA interacts with Abeta in the beta-hairpin (beta-turn-beta) structure and prevents fibril formation.

Anti-angiogenic activity of the mutant Dutch A(beta) peptide on human brain microvascular endothelial cells.

Cerebral amyloid angiopathy is a common pathological feature of patients with Alzheimer's disease (AD) and it is also the hallmark of individuals with a rare autosomal dominant disorder known as hereditary cerebral hemorrhage with amyloidosis-Dutch type. We have shown previously that wild type A(beta) peptides are anti-angiogenic both in vitro and in vivo and could contribute to the compromised cerebrovascular architecture observed in AD. In the present study, we investigated the potential anti-angiogenic activity of the Dutch A(beta)(1-40) (E22Q) peptide. We show that compared to wild type A(beta), freshly solubilized Dutch A(beta) peptide more potently inhibits the formation of capillary structures induced by plating human brain microvascular endothelial cells onto a reconstituted basement membrane. Aggregated/fibrillar preparations of wild type A(beta) and Dutch A(beta) do not appear to be anti-angiogenic in this assay. The stronger anti-angiogenic activity of the Dutch A(beta) compared to wild type A(beta) appears to be related to the increased formation of low molecular weight A(beta) oligomers in the culture medium surrounding human brain microvascular endothelial cells. Using oligonucleotide microarray analysis of human brain microvascular endothelial cells, followed by a genome-scale computational analysis with the Ingenuity Pathways Knowledge Base, networks of genes affected by an anti-angiogenic dose of Dutch A(beta) were identified. This analysis highlights that several biological networks involved in angiogenesis, tumorigenesis, atherosclerosis, cellular migration and proliferation are disrupted in human brain microvascular endothelial cells exposed to Dutch A(beta). Altogether, these data provide new molecular clues regarding the pathological activity of Dutch A(beta) peptide in the cerebrovasculature.

Inhibition of angiogenesis and tumor growth by beta and gamma-secretase inhibitors.

The involvement of beta-secretase and gamma-secretase in producing the beta-amyloid component of senile plaques found in the brain of Alzheimer's patients has fueled a major research effort to design selective inhibitors of these proteases. Interestingly, gamma-secretase cleaves several proteins including Notch, E-cadherin, CD44 and ErbB-4 (erythroblastic leukemia viral oncogene homolog 4), which are important modulators of angiogenesis. The beta-amyloid precursor protein, which is cleaved by beta-secretase and gamma-secretase to produce beta-amyloid, is highly expressed in the endothelium of neoforming vessels suggesting that it might play a role during angiogenesis. These data prompted us to explore the effects of beta and gamma-secretase inhibitors of different structures on angiogenesis and tumor growth. Both the gamma and beta-secretase inhibitors tested reduce endothelial cell proliferation without inducing cellular toxicity, suppress the formation of capillary structures in vitro and oppose the sprouting of microvessel outgrowths in the rat aortic ring model of angiogenesis. Moreover, they potently inhibit the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumors xenotransplanted into nude mice. Altogether these data suggest that the gamma and beta-secretases play an essential role during angiogenesis and that inhibitors of the beta and gamma-secretases may constitute new classes of anti-angiogenic and anti-tumoral compounds.

Inflammatory cytokine levels correlate with amyloid load in transgenic mouse models of Alzheimer's disease.

BACKGROUND: Inflammation is believed to play an important role in the pathology of Alzheimer's disease (AD) and cytokine production is a key pathologic event in the progression of inflammatory cascades. The current study characterizes the cytokine expression profile in the brain of two transgenic mouse models of AD (TgAPPsw and PS1/APPsw) and explores the correlations between cytokine production and the level of soluble and insoluble forms of Abeta. METHODS: Organotypic brain slice cultures from 15-month-old mice (TgAPPsw, PS1/APPsw and control littermates) were established and multiple cytokine levels were analyzed using the Bio-plex multiple cytokine assay system. Soluble and insoluble forms of Abeta were quantified and Abeta-cytokine relationships were analyzed. RESULTS: Compared to control littermates, transgenic mice showed a significant increase in the following pro-inflammatory cytokines: TNF-alpha, IL-6, IL-12p40, IL-1beta, IL-1alpha and GM-CSF. TNF-alpha, IL-6, IL-1alpha and GM-CSF showed a sequential increase from control to TgAPPsw to PS1/APPsw suggesting that the amplitude of this cytokine response is dependent on brain Abeta levels, since PS1/APPsw mouse brains accumulate more Abeta than TgAPPsw mouse brains. Quantification of Abeta levels in the same slices showed a wide range of Abeta soluble:insoluble ratio values across TgAPPsw and PS1/APPsw brain slices. Abeta-cytokine correlations revealed significant relationships between Abeta1-40, 1-42 (both soluble and insoluble) and all the above cytokines that changed in the brain slices. CONCLUSION: Our data confirm that the brains of transgenic APPsw and PS1/APPsw mice are under an active inflammatory stress, and that the levels of particular cytokines may be directly related to the amount of soluble and insoluble Abeta present in the brain suggesting that pathological accumulation of Abeta is a key driver of the neuroinflammatory response.

Inhibition of angiogenesis by Abeta peptides.

Abeta peptides are naturally occurring peptides forming beta-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimer's disease. Since several peptides adopting a beta-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Abeta on angiogenesis. We show that in vitro, Abeta dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Abeta peptides containing a higher content of beta-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Abeta dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Abeta dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Abeta on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Abeta potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Abeta delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Abeta is an angiogenesis inhibitor.

Impaired angiogenesis in a transgenic mouse model of cerebral amyloidosis.

Abeta peptides are naturally occurring peptides, which are thought to play a key role in the pathophysiology of Alzheimer's disease (AD). In AD cases, levels of soluble and insoluble Abeta peptides increase in the brain as well as in the cerebrovasculature, a phenomenon that does not occur in extra-cranial vessels. There are frequently anomalies in the cerebrovasculature in AD, and despite increases in several pro-angiogenic factors in AD brain, evidence for increased vascularity is lacking; in fact there is evidence to the contrary. It has also been recently shown that Abeta peptides may have profound anti-angiogenic effects in vitro and in vivo. We therefore investigated whether there is evidence for altered angiogenesis in the vasculature in a transgenic mouse model of Abeta amyloidosis (Tg APPsw line 2576). In vitro, the formation of capillary-like structures on a reconstituted extracellular matrix by endothelial cells isolated from Tg APPsw is impaired. Ex vivo, the sprouting of new capillaries from arterial explants (over expressing Abeta) isolated from 9-month-old Tg APPsw is reduced compared to arterial explants isolated from control littermates. In addition, Tg APPsw mice show a reduction in vascular density in the cortex and hippocampus compared to control littermates. Altogether, our data suggest that the over expression of APPsw in the vasculature may oppose angiogenesis.

Nilvadipine antagonizes both Abeta vasoactivity in isolated arteries, and the reduced cerebral blood flow in APPsw transgenic mice.

The development of Alzheimer's disease (AD) is generally thought to correlate with cerebral accumulation of Abeta. It has previously been shown that Abeta peptides enhance vasoconstriction in isolated arteries and oppose certain vasorelaxants. Moreover, exogenous application of Abeta peptides causes cerebral vasoconstriction in rodents and in transgenic mouse models of AD that overexpress Abeta there is reduced cerebral blood flow. In the present study, we investigated the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, on Abeta induced vasoconstriction in isolated arteries and in vivo on cerebral blood flow (CBF) of an AD transgenic mouse model overexpressing Abeta (Tg APPsw line 2576). Nilvadipine completely inhibited the vasoactivity elicited by Abeta in rat aortae and in human middle cerebral arteries. The effect of a short treatment duration (2 weeks) with nilvadipine on regional CBF was investigated in 13-month-old Tg APPsw mice and control littermates using a laser Doppler imager. Additionally, CBF was also measured in 20-month-old Tg APPsw mice and control littermates that were chronically treated with nilvadipine for 7 months. Untreated Tg APPsw mice showed a reduction of regional CBF compared to their untreated control littermates. Nilvadipine restored cortical perfusion levels in Tg APPsw to values similar to those observed in control littermates without notably affecting the CBF of control mice. All together, these data suggest that nilvadipine might be useful for the treatment of oligemia associated with AD.

Increased TNFalpha production and Cox-2 activity in organotypic brain slice cultures from APPsw transgenic mice.

beta-Amyloid (Abeta) peptides are the principal component of senile plaques and vascular deposits in Alzheimer's disease and are derived from the proteolytic cleavage of the beta-amyloid precursor protein (APP). We have previously shown that synthetic Abeta can stimulate cyclooxygenase-2 (Cox-2) activity in brain organotypic slice cultures. In the present study, we used brain slices from transgenic APP Swedish (TgAPPsw) mice and control littermates of different age groups to determine the effect of APP overexpression on the levels of prostaglandin and tumor necrosis factor alpha (TNFalpha) release. The production of eicosanoid and TNFalpha was increased as a function of age in organotypic brain slice culture from TgAPPsw mice compared to age matched control littermates. We also showed that the selective Cox-2 inhibitor NS-398 reduces the production of eicosanoid and TNFalpha in organotypic brain slice cultures of TgAPPsw mice. In conclusion, our data suggest that either activity or expression of Cox-2 is increased in TgAPPsw mice brains as a function of age, contributing to an increased production of pro-inflammatory eicosanoids and TNFalpha.

Vasoactive effects of A beta in isolated human cerebrovessels and in a transgenic mouse model of Alzheimer's disease: role of inflammation.

A beta peptides are the major protein constituents of Alzheimer's disease (AD) senile plaques and also form some deposits in the cerebrovasculature leading to cerebral amyloid angiopathy and hemorrhagic stroke. Functional vascular abnormalities are one of the earlier clinical manifestations in both sporadic and familial forms of AD. Most of the cardiovascular risk factors (for instance, diabetes, hypertension, high cholesterol levels, atherosclerosis and smoking) constitute risk factors for AD as well, suggesting that functional vascular abnormalities may contribute to AD pathology. We studied the effect of A beta on endothelin-1 induced vasoconstriction in isolated human cerebral arteries collected following rapid autopsies. We report that freshly solubilized A beta enhances endothelin-1 induced vasoconstriction in isolated human middle cerebral and basilar arteries. The vasoactive effect of A beta in these large human cerebral arteries is inhibited by NS-398, a selective cyclooxygenase-2 inhibitor and by SB202190, a specific p38 Mitogen Activated Protein Kinase inhibitor suggesting the involvement of a pro-inflammatory pathway. Using a scanner laser Doppler imager, we observed that cerebral blood flow is decreased in the double transgenic APPsw Alzheimer mouse (PS1/APPsw) compared to PS1 littermates and can be improved by chronic treatment with either NS-398 or SB202190. Altogether, our data suggest a link between inflammation and the compromised cerebral hemodynamics in AD.