Abstract

NEUROVASCULAR UNIT

The neurovascular unit (NVU) consists of different cell types, including (1) vascular cells such as brain endothelial cells, a site of the anatomical BBB in vivo, pericytes, and vascular smooth muscle cells (VSMCs), (2) glial cells such as astrocytes, microglia and oliogodendroglia, and (3) neurons (Fig. 2A; Zlokovic 2008; Guo and Lo 2009; Moskowitz et al. 2010). The close proximity of nonneuronal neighboring cells with each other and with neurons allows for effective cell-to-cell cross-communications that are critical for normal functions in the healthy central nervous system (CNS) and are increasingly recognized as important in the disease process in multiple neurological disorders (Boillee et al. 2006; Zhong et al. 2008; Zlokovic 2008).

The NVU functions in the healthy brain include control of neurovascular coupling and BBB permeability, matrix interactions, inactivation of neurotransmitters, signaling through angioneurins (i.e., growth factors that have both the neurotrophic and vasculotrophic functions) (Zacchigna et al. 2008), and clearance of toxins from brain (Fig. 2B).

The BBB is a highly specialized, continuous endothelial cell membrane that normally prevents the entry of plasma components, red blood cells, and leukocytes into the brain. In addition, through specific transporters in brain endothelium, the BBB regulates delivery of energy metabolites and essential nutrients that are required for proper neuronal and synaptic functions. The BBB is responsible for maintaining the constant “chemical” composition of brain interstitial fluid required for optimal brain function. Under physiological conditions, the BBB and pericytes control entry from blood and promote clearance from brain of various potentially neurotoxic and vasculotoxic macromolecules (Zlokovic 2008).

Alzheimer disease is associated with microvascular dysfunction, defective BBB, and vascular factors (Bailey et al. 2004; Wu et al. 2005; Zlokovic 2005, 2008; Paul et al. 2007; Zipser et al. 2007; Kalaria 2010; Knopman and Roberts 2010; Miyazaki et al. 2011; Neuwelt et al. 2011). Microvascular degeneration diminishes CBF, resulting in shortages in oxygen supply, energy substrates, and nutrients to the brain. On the other hand, microvascular defects compromise clearance of neurotoxic molecules from brain, resulting in accumulation of pathological deposits in brain interstitial fluid, nonneuronal cells, and neurons. According to the recent evidence, microvascular injury ultimately leads to neuronal dysfunction and development of neurodegenerative changes. Vascular damage may also contribute to the development of cerebral β-amyloidosis and cerebral amyloid angiopathy (CAA) caused by accumulation of Aβ in brain and the vessel wall, respectively (Zlokovic 2008).

CEREBRAL BLOOD FLOW DYSREGULATION AND REDUCTIONS

An adequate blood supply is ensured by a tight coupling between local tissue metabolic demands and blood perfusion of the active neuronal site. The link between regional synaptic activity and a CBF increase is known as functional hyperemia. Neurovascular coupling requires intact and effectively innervated pial and intracerebral arteries and normal responsiveness of brain endothelium, VSMCs, and pericytes to vasoactive stimuli (Iadecola 2004; Peppiatt et al. 2006; Bell et al. 2010). In addition to VSMCs, recent studies have shown that pericytes control brain capillary diameter by constricting the vessel wall (Peppiatt et al. 2006), which under ischemic conditions can completely obstructs capillary flow (Yemisci et al. 2009). Astrocytes have also been shown to regulate CBF responses by influencing contractile properties of small penetrating intracerebral arteries (Takano et al. 2007; Kuchibhotla et al. 2009).

Functional hyperemia is the basis for functional magnetic resonance imaging that has revolutionized our understanding of human brain in health and disease (Girouard and Iadecola 2006). Functional neuroimaging studies in AD individuals have shown that neurovascular uncoupling or diminished CBF responses to brain activation may occur prior to neurodegenerative changes (Smith et al. 1999; Bookheimer et al. 2000; Ruitenberg et al. 2005; Knopman and Roberts 2010). In addition, it has been reported that cognitively normal individuals bearing the apolipoprotein E (APOE) ε4 allele, which is known to be the major genetic risk factor for late-onset AD (Bertram et al. 2007; Kim et al. 2009; Verghese et al. 2011), have reduced functional hyperemia response in the absence of brain atrophy or Aβ/amyloid accumulation (Sheline et al. 2010). Diminished resting CBF has been also shown in elderly individuals at risk to develop AD (Iadecola 2004; Knopman and Roberts 2010).

Studies in animal models have indicated that CBF reductions can induce and/or amplify neuronal dysfunction and/or neuropathological changes resembling AD pathology. For example, it has been shown that Aβ constricts cerebral arteries (Thomas et al. 1996), and that endothelium-dependent regulation of cortical microcirculation is diminished in a mouse model of AD before Aβ accumulation (Iadecola et al. 1999). Moreover, in AD mice, mild hypoperfusion increases neuronal Aβ levels and tau phosphophorylation at an epitotope associated with AD-type paired helical filaments (Koike et al. 2010). Brain ischemia in rodents leads to accumulation of hyperphosphorylated tau in neurons and filament formation similar to that present in human AD tauopathy (Gordon-Krajcer et al. 2007). Arterial carotid occlusion in rats leads to memory impairment, neuronal dysfunction, synaptic changes and accumulation of neurotoxic Aβ oligomers (Wang et al. 2010). Mice expressing APP and transforming growth factor β develop neurovascular uncoupling, cholinergic denervation, accelerated Aβ deposition and age-dependent cognitive decline (Ongali et al. 2010).

It has been demonstrated that moderate CBF reductions, comparable to those as seen in the aging brain, are associated with diminished cerebral protein synthesis (Hossmann 1994; Iadecola 2004). CBF reductions >50% impair ATP synthesis and decrease the ability of neurons to fire action potentials. In addition, focal CBF reductions comparable to those as in chronic neurodegenerative disorders such as AD lead to shifts in intracellular pH, water and electrolytes that are attributed to a loss of activity of multiple energy-dependent ion pumps such as sodium/hydrogen exchanger and ATP-dependent sodium pump at the BBB (Zlokovic 2008). CBF reductions may also lead to accumulation of different toxins and glutamate in brain owing to a loss of activity at the BBB of ATP-binding cassette (ABC) efflux transporters (Dutheil et al. 2010; Elali and Hermann 2011) and Na-dependent transporters for the excitatory amino acids (O’Kane et al. 1999; Hardingham 2009), respectively. Severe reductions in CBF (>80%), similar to those found after an ischemic stroke lead to neuronal death. It is of note that changes in the NVU including degeneration of brain capillaries and/or reductions in the resting CBF may be the first sign of the disease process prior to neuronal changes and neurodegeneration.

MICROVASCULAR DEGENERATION

Alzheimer disease individuals and other dementia patients frequently have focal degenerative changes in brain microcirculation including atrophy and reductions in capillary network, a rise in endothelial vacuolization and loss of mitochondria, accumulation of collagen and perlecans in the basement capillary membrane, loss of BBB tight junction proteins (Farkas and Luiten 2001; Bailey et al. 2004; Iadecola 2004; Wu et al. 2005; Zlokovic 2005; Kalaria 2010), and leakage of blood-derived molecules (Paul et al. 2007; Zipser et al. 2007; Kalaria 2010). Aβ accumulation and amyloid deposition in pial and intracerebral arteries lead to CAA, which according to some studies is present in >80% of AD patients (Jellinger 2010; Viswanathan and Greenberg 2011). AD patients with CAA frequently develop atrophy in the VSMC layer of small arteries, causing a rupture of the vessel wall and intracerebral bleeding in about 30% of patients, which in turn aggravates dementia (Ghiso and Frangione 2002; Cordonnier 2011). Patients with hereditary cerebral β-amyloidosis with CAA in leptomeningeal and intracerebral arteries of the Dutch, Iowa, Arctic, Flemish, Italian, and Piedmont L34V type develop massive hemorrhagic strokes and dementia owing to VSMC degeneration in the vessel wall (Fossati et al. 2010). Similar, duplication of the APP gene results in AD dementia with CAA and intracerebral hemorrhage (Rovelet-Lecrux et al. 2006).

BBB DYSFUNCTION IN AD

Changes in the expression of several BBB transporters mediating nutrient transport, ion pumps, ABC transporters, and/or receptors mediating transport of peptides and proteins, including blood-to-brain and brain-to-blood exchanges of Aβ, have been described in AD and AD models (see below). Here we will focus on (1) glucose transporter 1 (GLUT1), which is a BBB-specific transporter that is of special importance because glucose is a key energy source for brain, (2) the receptor for advanced glycation end products (RAGE) that mediates Aβ reentry into the brain from circulation and the neurovascular inflammatory response, and (3) lipoprotein receptor-related protein 1 (LRP), which is a major Aβ clearance receptor at the BBB mediating Aβ efflux from brain and its systemic clearance.

VASCULAR-SPECIFIC GENES

Recent findings suggest that unsuccessful vascular regeneration may lead to degeneration of brain endothelium in AD and AD models. It has been shown that brain endothelial cells in AD express extremely low levels of the mesenchyme homeobox gene 2 (MEOX-2; Wu et al. 2005), a transcription factor that regulates vascular cell differentiation and remodeling, and whose expression in the adult brain is restricted to the vascular system (Gorski and Walsh 2003). Low levels of MEOX-2 expression in AD brain endothelium have been shown to mediate an aberrant angiogenic response to vascular endothelial growth factor (Wu et al. 2005), ultimately resulting in vessel regression associated with reductions in the resting CBF (Fig. 4). Low levels of MEOX-2 also promote proteasomal degradation of LRP in brain endothelium (Wu et al. 2005) that diminishes Aβ clearance at the BBB. On the other hand, accumulation of Aβ on the outer membrane of the blood vessels is anti-angiogenic per se (Paris et al. 2004a,b). Therefore, Aβ may act in concert with low expression of MEOX2 at the BBB to focally reduce brain capillary density in AD models and AD. Importantly, MEOX2 expression is diminished by hypoxia, suggesting that hypoxia may be upstream of MEOX2 depletion seen in AD brain endothelium (Wu et al. 2005).

Interestingly, mice with a single allele of Meox2 develop a primary cerebral endothelial hypoplasia with an intact BBB, but a significant brain perfusion deficit (Wu et al. 2005), which has been shown to lead to secondary neurodegenerative changes prior to Aβ accumulation (Bell et al. 2010). Neurodegenerative changes in Meox2^+/− mice were, however, significantly less pronounced than in pericyte-deficient mice, which have a comparable brain hypoperfusion to Meox2^+/− mice but also a compromised BBB (Bell et al. 2010). These data indicate that chronic hypoperfusion alone can cause neuronal injury, but not to the same extent as when combined with BBB breakdown.

Recent studies have also shown that AD patients as well as mouse models with high cerebrovascular levels of serum response factor (SRF) and myocardin (MYOCD), the two transcription factors that control VSMC differentiation, develop a hypercontractile cerebral arterial phenotype, resulting in brain hypoperfusion, diminished functional hyperemia, and CAA (Chow et al. 2007; Bell et al. 2009). MYOCD, a SAF-A/B, Acinus, and PIAS domain family nuclear protein, is a VCMC-specific transcriptional co-activator that binds SRF to induce gene expression (Wang et al. 2001). SRF is a ubiquitously expressed transcription factor that binds to a ten-base pair cis element called a CArG box, which is located in the regulatory region of numerous target genes (Sun et al. 2006). MYOCD and SRF constitute a molecular switch for the VSMC differentiation program (Chen et al. 2002; Li et al. 2003). In addition, it has been shown that increased levels of MYOCD and SRF in AD VSMCs may suppress Aβ clearance and thus exacerbate CAA (Bell et al. 2009). Namely, high levels of MYOCD and SRF in VSMCs lead to directed expression of sterol response element binding protein 2 (SREBP2), which is a major LRP transcriptional suppressor ultimately resulting in LRP depletion, which diminishes LRP-mediated Aβ clearance from the vessel wall (Fig. 4). Hypoxia increases MYOCD levels in VSMCs (Reynolds et al. 2004; Chow et al. 2007; Bell et al. 2009), and it has been shown that it is also upstream of elevated MYOCD/SRF expression in cerebral arterial VSMCs (Bell et al. 2009). More studies are needed, however, to establish the exact role of vascular-specific genes MEOX2 and MYOCD in the development of Alzheimer neurovascular dysfunction.

CONCLUDING REMARKS

Recent clinical observations provide strong evidence for the link between cerebrovascular disease and AD and the role of vascular risk factors in AD. In this chapter, we have briefly reviewed literature on dysregulated and diminished CBF, BBB dysfunction, and impaired vascular clearance of Aβ from brain, supporting an essential role of the neurovascular and BBB mechanisms in AD pathogenesis. Several studies in animal models of AD and more recently in AD patients (Mawuenyega et al. 2010) have demonstrated a diminished Aβ clearance from brain. The recognition of Aβ clearance pathways opens exciting new therapeutic opportunities for AD. It is now established that faulty clearance from brain and across the BBB leads to elevated Aβ levels in brain that in turn have been shown to contribute to the formation of neurotoxic Aβ oligomers (Walsh et al. 2002) and the development of Aβ-mediated brain storage disorder and cerebral β-amyloidosis (Zlokovic 2008).

The activation of neurovascular pathogenic pathways has been shown to compromise synaptic and neuronal functions prior to and/or in parallel with Aβ accumulation and development of intraneuronal tangles, neuronal loss, and dementia. Some early molecular targets within the neurovascular pathway include receptors RAGE and LRP at the BBB and possibly vascular-specific genes MEOX2 and MYOCD.

Focusing on comorbidity, vascular risk factors associated with AD such as hypoperfusion, hypertension, ministrokes, and/or diabetes might generate useful models of human dementia. The proposed neurovascular model of AD raises a set of new important questions that require further study, as recently discussed (Zlokovic 2011). For example, the molecular basis of the neurovascular link with neurodegenerative disorders is still poorly understood as well as the molecular cues underlying the cross talks between different cell types of the NVU, including vascular and glia cells, and how these cellular interactions influence neuronal activity. Addressing these questions will lead to better understanding of the neurovascular link with neurodegeneration process, which will lead to the development of novel neurovascular-based approaches for AD (Zlokovic 2011).

ACKNOWLEDGMENTS

Footnotes

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