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Abstract

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix

Several factors have been implicated in Alzheimer's disease (AD) but there is no definite conclusion as to the main pathogenic agents. Mutations in the amyloid precursor protein (APP) that lead to increased production of amyloid β peptide (Aβ) are associated with the early-onset, familial forms of AD. However, in addition to ageing, the most common risk factors for the sporadic, prevalent form of AD are hypertension, hypercholesterolaemia, ischaemic stroke, the ApoE4 allele and diabetes, all characterized by a vascular pathology. In AD, the vascular pathology includes accumulation of Aβ in the vessel wall, vascular fibrosis, and other ultrastructural changes in constituent endothelial and smooth muscle cells. Moreover, the ensuing chronic cerebral hypoperfusion has been proposed as a determinant factor in the accompanying cognitive deficits. In transgenic mice that overexpress mutated forms of the human APP (APP mice), the increased production of Aβ results in vascular oxidative stress and loss of vasodilatory function. The culprit molecule, superoxide, triggers the synthesis of other reactive oxygen species and the sequestration of nitric oxide (NO), thus impairing resting cerebrovascular tone and NO-dependent dilatations. The Aβ-induced cerebrovascular dysfunction can be completely abrogated in aged APP mice with antioxidant therapy. In contrast, in mice that overproduce an active form of the cytokine transforming growth factor-β1 and recapitulate the vascular structural changes seen in AD, antioxidants have no beneficial effect on the accompanying cerebrovascular deficits. This review discusses the beneficial role and limitations of antioxidant therapy in AD cerebrovascular pathology.

In addition to the well-recognized deterioration of cognitive and mnemonic functions, Alzheimer's disease (AD) is characterized, at the neuropathological examination, by: (i) deposition of amyloid β peptide (Aβ) in brain tissue (neuritic plaques) and in the wall of cerebral blood vessels (cerebral amyloid angiopathy or CAA); (ii) glial cell activation; (iii) dystrophic neuronal processes in proximity and within Aβ plaques; (iv) progressive loss of synapses and neurones; and (v) a vascular pathology in which the structure of cerebral blood vessels is severely damaged. The vascular pathology in AD is not limited to the accumulation of Aβ in the vessel wall, but is characterized by atherosclerosis, vascular fibrosis, and structural and inflammatory changes of blood vessels. The vessels display a thickened basal lamina and are surrounded by activated astrocytes and microglial cells (Buée et al. 1994; Vinters et al. 1994; Zarow et al. 1997; Paris et al. 2000). Pericytes, endothelial and smooth muscle cells may also exhibit degenerative changes at a late stage of the disease (Kalaria, 1997). Moreover, cortical microvessels are denervated and, in particular, perivascular cholinergic (ACh) nerve terminals from the basal forebrain, which are an important regulator of cortical cerebral blood flow (CBF, Hamel, 2004), are largely lost in AD (Tong & Hamel, 1999). As a result of these alterations, cortical CBF is severely compromised in AD patients (Iadecola, 2004; Zlokovic, 2005). Since the brain is totally dependent on a constant blood supply because it has no reserve of either glucose or oxygen, such a threat to cerebral perfusion is likely to have dramatic consequences for neuronal functions. Indeed, perfusion deficits in AD often precede the neurodegenerative changes, and it has been suggested that they are not merely a consequence but rather a pathogenic factor in AD (Shi et al. 2000; de la Torre, 2005). It therefore appears important to gain a better understanding of the pathogenic mechanisms involved in the cerebrovascular pathology, since they may represent an interesting therapeutic avenue for patients with increased risk factors of developing AD.

Animal models of AD cerebrovascular pathology

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix

Two molecules have been implicated in the cerebrovascular pathology of AD: (i) Aβ, which in soluble form directly alters cerebrovascular functions; and (ii) transforming growth factor-β1 (TGF-β1), which promotes the synthesis of extracellular matrix proteins and contributes to vascular remodelling following injury or lesion (Wyss-Coray et al. 2000). In transgenic mice that overexpress mutated forms of the human amyloid precursor protein (APP; APP mice), including our mouse model (hAPPSw, Ind, Mucke et al. 2000), the Aβ-induced oxidative stress impairs cerebrovascular dilatory responses to acetylcholine (ACh), calcitonin gene-related peptide (CGRP) or other endothelium-dependent dilators (Iadecola et al. 1999; Park et al. 2004; Tong et al. 2005), in addition to altering autoregulation (Niwa et al. 2002b) and functional hyperaemia (Niwa et al. 2002a; Park et al. 2004). Moreover, APP mice exhibit senile plaques, neuronal impairments, ACh denervation (Aucoin et al. 2005), cerebral hypometabolism (Niwa et al. 2002a), alterations in synaptic transmission and cognitive deficits (Palop et al. 2003). Transforming growth factor-β1, a cytokine that increases in several diseases with a vascular pathology, is also upregulated in AD and, particularly, in brain vessels of AD patients (Grammas & Ovase, 2002). Interestingly, polymorphisms in the TGF-β1 gene are associated with increased risk factors for AD (Luedecking et al. 2000) or hypertension (Yamada et al. 2002). Furthermore, transgenic mice that overproduce an active form of TGF-β1 (TGF mice) display structural vascular alterations that compare exquisitely well with those seen in AD brains (Wyss-Coray et al. 2000; Tong et al. 2005). They also exhibit deficits in cerebrovascular dilatory responses to ACh and CGRP and, with aging, in contractile responses to endothelin-1 (ET-1; Tong et al. 2005), in addition to cerebral hypoperfusion (Gaertner et al. 2005) and hypometabolism (Galea et al. 2006). These two mouse models thus reproduce different but complementary aspects of the cerebrovascular pathology found in AD.

Antioxidants and Aβ-induced cerebrovascular dysfunctions

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix

It has been shown that APP mice exhibit signs of oxidative/nitrosative stress in brain vessels before these signs appear in neurones, glial cells or Aβ plaques (Park et al. 2004), indicating that the vasculature is an early target of the Aβ peptide. Indeed, topical application of Aβ1–40 reduced the functional hyperaemic response to whisker stimulation, i.e. the increase in cortical CBF induced by increased neuronal activity in the barrel cortex, probably by increasing production of reactive oxygen species (ROS), since the attenuation of CBF was not observed when Aβ was applied in the presence of free radical scavengers that concurrently prevented the Aβ-induced production of ROS (Park et al. 2004). NADPH oxidase was identified as the major source of the free radicals because ROS production and the associated dysfunctions were abrogated by inhibition of NADPH oxidase and were absent in mice lacking the catalytic subunit gp91phox of this enzyme (Park et al. 2005). The primary mechanism involved in the NADPH oxidase-derived free radical-mediated cerebrovascular dysfunctions was related to the reduced bioavailability of NO (Park et al. 2005). This is consistent with earlier studies that showed that superoxide dismutase (SOD) applied topically to the cerebral cortex prevented the loss of endothelial function in APP mice, or that mice expressing both APP and SOD-1 did not display any endothelial deficit (Iadecola et al. 1999). What is more, in vitro incubation of dysfunctional cerebral arteries from old APP mice with antioxidants such as a synthetic SOD or catalase, involved in the dismutation of ·O2 and catabolism of the resulting H2O2 product, respectively (Fig. 1), completely restored cerebrovascular function (Tong et al. 2005). The antioxidants normalized both basal production of NO by the vessel wall and endothelium-dependent dilatations in response to ACh and CGRP. Additionally, the dilatory response to ACh was fully restored in cerebral arterial segments from aged APP mice (> 17 months) treated in vitro with the NADPH oxidase inhibitor apocynin (Fig. 2), further confirming that even at an advanced stage of the pathology, ·O2 remains the primary initiator of the functional dysregulation.

image

Figure 1. Cellular pathways involved in the synthesis and elimination of the free radicals superoxide (·O2), hydrogen peroxide (H2O2) and peroxynitrite (ONOO), and how superoxide dismutase (SOD), Tempol (a SOD mimetic), catalase and N-acetylcysteine (NAC) are involved as antioxidants Abbreviations: GSH, glutathione; and GSSG, glutathione disulphide.

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image

Figure 2. Incubation with the NADPH oxidase inhibitor apocynin (1 mm, 1 h) reversed the deficit in ACh-induced dilatation in cerebral arteries from old (> 17 months) APP mice *P < 0.05, **P < 0.01 and ***P < 0.001 compared with wild-type littermates (WT).

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Moreover, in aged APP mice treated in vivo for 4–6 weeks with antioxidants such as Tempol (a SOD mimetic) or N-acetylcysteine (NAC, a precursor of glutathione required for the catalytic activity of glutathione peroxidase, another enzyme that degrades H2O2 in addition to catalase, Fig. 1), cerebrovascular responses were normalized to levels of wild-type littermates (Nicolakakis et al. 2005, 2006). Taken together, these observations point to oxidative stress as the main underlying deleterious mechanism through which Aβ alters the reactivity of the cerebral vasculature. In fact, not only does ·O2 limit the bioavailability of NO, but the reaction between ·O2 and NO proceeds much faster than the dismutation of ·O2 by SOD, thus favouring the synthesis of the most potent oxidant, peroxynitrite (ONOO), and together these events perturb pathways required for vasomotor function (see Tong et al. 2005).

Antioxidants and TGF-β1-induced cerebrovascular dysfunctions

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix

Transgenic TGF mice were originally described as a model of the cerebrovascular pathology seen in the AD microvasculature, where there is a thickening of the basal lamina corresponding to accumulation of proteins such as fibronectin, perlecan (Wyss-Coray et al. 2000) and collagen (Tong et al. 2005). These structural alterations translate into regional decreases in CBF (Gaertner et al. 2005), compatible with a reported decrease in glucose metabolism in several brain regions (Galea et al. 2006). We further found that the reactivity of cerebral arteries was altered, with decreases in dilatory responses to ACh and CGRP, impaired basal production of NO by the vessel wall and selective reduction in the contractile response to ET-1 while that to serotonin (5-HT) was unaltered in mice up to 21 months of age (Tong et al. 2005). In these mice, in vitro incubation of the cerebral arteries with the antioxidants SOD or catalase did not improve cerebrovascular function (Tong et al. 2005). Moreover, we recently found that in vivo treatment with either Tempol or NAC (for 4–6 weeks) did not result in any improvement in cerebrovascular function, as shown here for the basal NO synthesis evaluated by measuring the passive decrease in diameter induced by the non-selective NOS inhibitor NG-nitro-l-arginine (L-NNA; Fig. 3), nor in any detectable change in vessel wall stiffness (Nicolakakis et al. 2005, 2006). The results suggest that factors other than oxidative stress are involved in the functional deficits that accompany this model of cerebrovascular fibrosis, at least at this late stage of the pathology. Additionally, it is unlikely that vasomotor dysfunctions are a direct consequence of the structural alterations, since the capacity of the vessels to dilate in response to the NO donor sodium nitroprusside or constrict in response to 5-HT, noradrenaline or high K+ concentrations was not altered (Tong et al. 2005; Tong & Hamel, 2007). Together, these findings suggest that the TGF-β1-induced cerebrovascular functional deficit is not a direct consequence of overproduction of ROS but rather involves defects in the synthesis of vasoactive molecules or their signalling pathways, such as different families of mitogen activated protein kinases.

image

Figure 3. Effects of the antioxidant Tempol on the basal vascular synthesis of NO measured in the presence of 10−5m L-NNA over time Tempol did not restore NO synthesis in vessels from treated TGF mice, indicating that ROS are not responsible for the deficit. WT indicates wild-type littermate control mice.

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Conclusions

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix

Increasing evidence indicates that the vascular component of AD, which translates very early into cerebral hypoperfusion, may contribute to the neuronal and cognitive deficits (Benarroch, 2007). It thus appears important to gain a better understanding of the cellular mechanisms that underlie this pathology, since they may represent therapeutic targets that are particularly promising for patients with diseases that increase the risk of developing AD. Based on the results obtained with antioxidant treatments in APP and TGF mice, which represent two different yet complementary aspects of the vascular pathology seen in AD, it appears that these types of drugs will only be effective against the Aβ-induced vascular oxidative stress, while the dysfunctions that occur concurrently with alterations of vascular wall structure will remain unresponsive to these treatments. These data point to the growing importance of identifying molecules with beneficial effects on cerebrovascular dysfunctions induced by both Aβ and TGF-β1 in order to bear any therapeutic relevance for patients affected with or susceptible to developing AD.

References

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Animal models of AD cerebrovascular pathology
  4. Antioxidants and Aβ-induced cerebrovascular dysfunctions
  5. Antioxidants and TGF-β1-induced cerebrovascular dysfunctions
  6. Conclusions
  7. References
  8. Appendix

Acknowledgements

Studies performed by the authors were supported by grants from the Alzheimer Society of Canada, and the Canadian Institutes of Health Research (grant MOP-64194). N.N. is a recipient of a CIHR studentship, B.O. and T.A. of respective fellowships from les Fonds de la recherche en santé du Québec (FRSQ) and the MNI-Jeanne Timmins Costello Foundation.