Role of hypertention-induced cerebrovascular dysfunction in physiopathology of Alzheimer disease: molecular and cellular mechanisms. – VADAD

To create the first model of dual pathology (hypertension + Alzheimer's disease), we implanted subcutaneously the osmotic mini-pumps infusing hypertensive doses of angiotensin II into control C57BL/6 mice and transgenic mice expressing the mutated human amyloid precursor and presenilin 1proteins. The treatment began at 2 months of age and continued until the end of the experiment (4.5 or 8 months).
Four experimental groups were studied: i) sham operated control mice (WT group); ii) control mice treated with angiotensin II (HT group); iii) sham operated «Alzheimer's« mice (AD group); and iv) «Alzheimer's« mice treated with angiotensin II (AD&HT group). Monitoring of arterial blood pressure made on the vigil mouse (non-invasive method) showed that at two months there was no significant difference in blood pressure between the control mice and transgenic «Alzheimer's« mice. The infusion of angiotensin II induced a significant and sustained raise in systolic blood pressure in both control and transgenic mice (170 mmHg against 110 mmHg in normotensive mice). The effects of this treatment on the brain vasculature, amyloid burden (number of amyloid plaques in different brain regions, levels of soluble amyloid beta in brain extracts and in plasma) and behavior (episodic-like memory test) were analyzed.

Using the episodic-like memory test we showed that in AD&HT mice, cognitive deficit occurs at an earlier stage (4.5 months) than in non-hypertensive APPPS1 model (8 months). In HT&AD mice, this deficit was accompanied by an increase in number of cortical amyloid plaques (by 8%, P<0.05) and doubling the levels of soluble amyloid beta in brain tissue and in the plasma. In addition, HT&AD mice presented cerebrovascular alterations: 25% reduction in microvessel density, 30-40% increase in amyloid deposits around microvessels, and decreased expression of VEGF-A in the brain. In contrast, the normotensive APPPS1 mice exhibited an increased expression of VEGF-A and TGFbeta, and a tendency to increase in microvessel density in the cortex, suggesting the activation of angiogenic process that may represent an adaptive response to probable cerebral hypoxia. Hypertension, however, inhibits this response, thus exacerbating Alzheimer's-like pathology. We also found that the levels of nitric oxide synthase 1 and 3, and the brain nitrites/nitrates levels were reduced in AD&HT mice (by 49%, 34 % and 33 %, respectively, compared to WT mice, P<0.05).
Our results indicate that hypertension accelerates the development of Alzheimer's type structural and functional alterations by reducing the production of nitric oxide and the reduction of the brain vasculature plasticity.

To complete our protocol, we will perform the additional electrophysiological experiments in 4 studied groups of mice. In a second step, we will evaluate the effects of antihypertensive therapy on the development and severity of experimental Alzheimer's disease.

The results of this first part of the work were presented at:
9th International Workshop «Structure and function of the vascular system«, February 6-8, 2014, Paris, France (Merkulova-Rainon T, Henrion D & Lévy BI. Arterial Hypertension and Alzheimer disease: experimental study);
5th Congress of the French Society of Angiogenesis, April 2-4 2014, Chamonix, France (Cifuentes D, Poittevin M, Bonnin P, Hilal R, Pocard M, Kubis N, Merkulova-Rainon T, Lévy BI. Hypertension reduces angiogenesis and increases amyloid load in a mouse model of Alzheimer’s disease).
An article reporting these results is currently under revision in «Hypertension ” (IF=6.87)
Cifuentes D, Poittevin M, Dere E, Broquères-You D, Bonnin P, Pocard M, Mariani J, Kubis N, Merkulova-Rainon T, Lévy BI. Hypertension accelerates the progression of Alzheimer-like pathology in a mouse model of the disease.

Alzheimer disease (AD) is a chronic neurodegenerative disorder accounting for 50-75% of dementia cases and representing the leading cause of dementia in the elderly. Although amyloid beta peptide (Abeta) and abnormally phosphorylated tau protein remain in the center of AD research, the etiology of AD is largely undefined and effective treatments are still not available. Cerebrovascular dysfunction, including cerebral amyloid angiopathy, brain-blood barrier impairment and microvascular abnormalities, is frequently observed in AD patients and currently believed to affect clinical manifestation and severity of neurodegeneration and to contribute to cognitive decline.
Very few AD animal models present the entire spectrum of cerebrovascular dysfunction, close to that found in humans. Accumulating evidence indicates that mid-life history of hypertension (HT), a major vascular risk factor, significantly affects both incidence and course of AD. Our project is thus to establish experimental models of AD associated with HT and to highlight the molecular and cellular mechanisms underlying HT-AD crosstalk.
We will use APPPS1 transgenic mice overexpressing the mutated forms of amyloid precursor protein and presinilin-1. Two separated approaches will be used:
1) implantation of osmotic minipumps infusing hypertensive doses of angiotensin II into APPPS1 mice to induce HT,
2) cross-breeding of APPPS1 and NOS3-/- mice (a spontaneously hypertensive model in relation with the absence of endothelial nitric oxide) to obtain bigenic hypertensive-AD mice (APPPS1/NOS3-/-).
Using immune-histochemical and biochemical analyses, behavioral (episodic-like memory) and electrophysiological (LTP) studies, we will compare the effects of these two approaches on the expected worsening of Alzheimer like disease in experimental models which mimics more fully the human AD pathology.
We expect that HT will aggravate AD in several ways:
1. by inducing an earlier onset of the AD neuropathology,
2. by accelerating and exacerbating inflammation, synaptic deficit, neuronal lost and memory decline,
3. by provoking, in cooperation with AD, more severe cerebral amyloid angiopathy and microvascular disease, and
4. by increasing the damage to the blood brain barrier.

We will further address the molecular and cellular mechanisms underlying these effects. We will particularly evaluate cerebral hypoxia, cerebral angiogenesis, neurovascular unit integrity, brain renin/angiotensin system signaling and the levels and activity of brain proteases implicated in Abeta turnover (in particular, alpha and beta secretases, neprylisine, angiotensin-converting enzyme) in control mice, APPPS1 mice, HT mice and mice with double pathology, at different ages (3, 5 and 8 months).
– Group of control mice (C57Bl6)
– Group of hypertensive mice by infusion of ANGII
– Group of hypertensive mice NOS3-/-
– Group of control APPPS1 mice
– Group of hypertensive APPPS1 mice by infusion of ANGII
– Group of bigenic APPPS1/NOS3-/- mice

These comparative studies would clarify the therapeutic potential of targeting the brain renin angiotensin system, endothelial dysfunction and angiogenesis process in AD and could promote the identification of novel therapeutic targets in AD.
This project is part of a larger research program that investigates the potential therapeutic application to AD of several cell-therapy based approaches developed by the coordinating team. We will use the models of AD associated with the HT, established and characterized within the framework of this project, to assess the efficacy of these therapies in AD.